实验用小型猪细胞色素P450家族新基因克隆及其CYP3A29基因多态研究
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摘要
一、研究目的及背景
由于猪的食性、解剖生理特点、以及药物代谢特点与人类的相似性,以猪进行药物的药理、毒理、药代、药效的研究日渐增多。随着动物保护运动的兴起,“3R”原则逐渐推广,非人灵长类及犬等高等动物使用受到限制,猪等食用动物的应用逐渐增多。因此,猪作为药物评价的实验动物具有良好前景。而小型猪由于体型小的优势,其实验操作方便,易于标准化质量控制,饲养成本低,其应用独具优势,也是目前在医学生物学研究中的常规实验用猪。
作为药物评价模型,要求模型动物的药物代谢反应机制以及药物与机体与之间的相互作用方面与人具有相似性,利用模型动物能够较为准确地模拟药物在人体内的代谢过程。药物在体内的过程大致可以分为吸收、分布、代谢和排泄四个阶段。药物的药代动力学性质及其生物利用度、毒副作用及药物相互作用都由这四个阶段决定。其中药物代谢的主要场所是肝脏。肝脏进行生物转化则依赖于微粒体中的多种酶系,其中最重要的是细胞色素P450混合功能氧化酶系统。由于该酶系广泛分布于肝、肾、脑、皮肤、肺、胃肠道及胎盘等组织器官,因此由P450酶系催化的氧化还原反应可发生在体内许多部位,但仍然以肝脏为主。
P450酶系普遍存在于各种脊椎动物、无脊椎动物、植物、真菌和细菌中。该酶系组成复杂,由基因多样性控制,称为P450基因超家族。到目前为止,根据最新公布的数据,仅动物就存在101个P450家族,2345个基因序列。P450基因家族和人类的个体化用药及药物的毒副作用密切相关,因此人类的P450已经研究得比较深入,目前发现57个P450功能基因。而作为成熟模式生物和人类疾病动物模型的小鼠则已发现102个P450功能基因。猪作为一种具有重要应用前景的药物评价动物及医学生物学实验动物,其P450基因的研究相对缺乏,目前通过国际细胞色素P450命名委员会命名的仅有30个功能基因。
公开数据库中海量增长的序列信息为我们发现新基因提供了充足的资源。EST作为基因的部分cDNA序列,利用其发现和克隆新基因已成为获取新基因并进行功能研究的重要途径。对数据库中大量EST进行分类、整合、组装,可直接获得大片段或全长cDNA序列。用该方法也可以快速、经济地获得基因家族的新成员。猪EST数据库中的序列数已达到60万条以上,这些数据为我们提供了发现猪P450家族基因信息的机会。
细胞色素P450酶的遗传多态性对其药物代谢有重要影响,也是和目前人类个体化用药相关的主要因素之一。不同个体在P450基因多态性的产生,可能会造成对同一药物反应的差异,有时由于这种个体间遗传学上的差别,同一药物在不同个体内的效果和毒副作用的差异可以达到300倍之多。根据人类细胞色素P450命名会员会的资料,仅人的CYP3A4目前就有已经证实的40个多态基因(或叫突变体)。中国大陆人主要突变体是CYP3A4*4,该突变使CYP3A4酶活性降低。研究已经证明,对于人类肝脏含量最高,也是代谢药物最多的CYP3A亚家族,其最合适的动物模型是(小型)猪。猪的肝脏微粒体具有人CYP3A4探针底物的代谢活性和相同的抑制剂特性,并认为猪的CYP3A29可能是人CYP3A4的同源酶。那么猪CYP3A29是否具有同样的多态性呢?如果这样的多态性存在的话,其是否会影响其参与代谢途径的代谢活性呢?如果CYP3A29存在遗传多态性且影响其代谢活性的话,用猪作为人类CYP3A4底物药的代谢模型就必须要考虑其影响效果。
我国具有丰富和优秀的小型猪资源品系。巴马香猪和贵州小型香猪作为其代表品种之一,其来源均为香猪,主要产于我国贵州省从江、三都、和广西环江、巴马等少数民族聚居地区。这些地区多属偏僻山区,交通闭塞,不仅其它猪种的血缘难以进入,而且群众习惯于将猪进行亲子交配,同胞、半同胞交配等高度近交的自繁自养方式。并且由于长期的自然选择,虽近交程度较高,却少见遗传疾病及畸形,从而形成独特小型猪群体。巴马香猪和贵州小型香猪均以小型化为育种目标进行了定向选育。其中巴马香猪近交程度高、被毛呈白色,而且耐近交、遗传稳定、个体表型一致,在医学生物学领域应用中证明是优秀的模型动物,并积累了丰富的解剖、生理生化及基础生物学特性背景资料。对巴马香猪和贵州小型香猪进行其细胞色素P450基因的相关研究,将对其作为药物评价动物模型的应用具有重要意义。
因此,本研究利用公开数据库的序列资源,用生物信息学方法预测猪的细胞色素P450新基因,以此对我国资源小型猪品系巴马香猪进行相关基因的克隆,为我国独特小型猪品系作为药物评价动物模型提供基础依据;同时研究巴马香猪和贵州小型香猪细胞色素P450关键亚型CYP3A29的品系间及品系内多态性,评估其序列多态差异对药物代谢动物模型的影响;对两种小型猪品系进行微卫星遗传背景检测分析,观察动物品系间与品系内微卫星遗传背景差异与其CYP3A29基因多态的相互关系。
二、研究内容和结果
1.猪细胞色素P450基因的生物信息学预测和分析
根据其它哺乳动物的同源P450基因序列,主要利用猪现有的EST数据库,用生物信息学方法对猪P450家族基因进行电子克隆,获得猪细胞色素P450家族新基因;并对猪细胞色素P450基因进行EST电子表达谱、可变剪接等分析。结果:应用生物信息学方法预测获得37个可能的猪细胞色素P450新基因序列,利用EST电子表达分析获得了猪不同组织的P450基因表达数据,发现了猪P450基因的46个可变剪接体,为小型猪药物代谢研究提供了P450基因的基础数据和资料。通过生物信息学方法共得到37个可能的猪细胞色素P450新基因序列,分布于11个家族,19个亚家族,23个亚型。其中9条具有全长编码区序列。序列分析表明预测得到的序列均具有P450结构域或与其它物种相应P450序列高度相似,聚类分析表明其和已知猪的P450基因或人的相应P450基因具有同源性。对已知和预测的共52个猪P450基因进行EST电子表达谱分析,获得了其不同组织的P450基因表达数据,其中在猪的肝脏表达量最高的P450基因依次是CYP2D25(50.26%)、CYP2E1(31.09%)、CYP2C33(3.69%)、CYP2A19(3.11%)、CYP2D21(2.07%),并和人的EST电子表达谱进行了比较。发现了猪P450基因的46个可变剪接体,其中编码区的可变剪接有31个,5’UTR的可变剪接有3个,3’UTR的可变剪接有9个,另有2个可变剪接同时发生在编码区和3’UTR,1个可变剪接同时发生在编码区和5’UTR。
2.巴马香猪预测细胞色素P450基因的克隆和验证
根据对猪细胞色素P450基因序列的预测结果,设计引物通过RT-PCR扩增相应基因序列,进行TA克隆和序列测定,与预测的P450基因序列进行比较。结果:克隆获得了我国资源小型猪品系巴马香猪的18个细胞色素P450新基因序列,均已在GenBank登录,登录号EF625344-EF625358,并已提交国际细胞色素P450命名委员会进行命名,不仅为小型猪药物代谢研究提供了P450基因序列资料,同时表明用生物信息学方法结合实验验证是快速高效获得猪P450新基因的有效途径。对预测的猪细胞色素P450基因19个序列的RT-PCR克隆验证均获得了符合预期片段大小的扩增产物,经序列测定和比对,17个P450基因的克隆序列和生物信息学预测结果基本一致,1个克隆序列可能为猪cyp4f6基因的一个新等位基因。其中1个克隆序列cyp4f2v4和预测序列相差较大,blastx检索NCBI未发现相似P450序列,因此把这条序列不作为猪的P450基因序列。cyp4f3v1的扩增序列和原来差异较大,但和cyp4f6v1序列非常相似,并能和cyp4f6v1*1、cyp4f6v1*2、cyp4f6v1*3其同拼接成一条contig,将这条contig用blastx检索NCBI,未发现相同序列,但和CYP4F6亚家族有高度相似,故其应为猪cyp4f6基因的一个新变体。其余序列均和预测结果基本一致,cyp39a1通过分段扩增,cyp39a1*1和cyp39a1*2可拼接成一个contig,其编码蛋白和预测序列完全相同,应该包含了其编码区全长。所有和预测序列一致的序列均已在GenBank登录,登录号EF625344-EF625358。所有序列已提交国际细胞色素P450命名委员会进行命名。
3.小型猪细胞色素P450基因CYP3A29多态研究
设计引物通过RT-PCR扩增不同个体巴马香猪和贵州小型香猪的CYP3A29基因编码区全长,进行TA克隆和测序,通过序列比对检测和发现其多态性位点,并对其多态位点的分布和影响进行分析。结果:发现了巴马香猪和贵州小型香猪CYP3A29基因的6个SNP位点及1个可变剪接体,其SNP分布具有品系及个体间多态性,在不同品系中可呈不同的SNP类型,其中巴马香猪具有品系内SNP类型的一致性,贵州小型香猪品系内具有SNP类型差异,说明猪P450基因具有和人相似的遗传多态性,在猪作为药物代谢模型的应用中应关注品系及个体间的差异。共获得13头巴马香猪和5头贵州小型香猪的CYP3A29基因序列,比对结果表明,其中具有6个可能的SNP位点,1个可变剪接体,1个可能的猪CYP3A29亚家族新基因。6个可能的SNP位点中,5个均为同义替换,不改变编码的氨基酸类型,对编码的蛋白序列无影响,1个由于碱基缺失引起移码突变和翻译提前终止。CYP3A29*1A、CYP3A29*1B、CYP3A29*1C三个SNP位点,均表现为品系间的差异,即巴马香猪和贵州小型香猪各为一个SNP亚型;位点CYP3A29*1E和CYP3A29*2则表现小型猪品系和其它品系间的序列差异;CYP3A29*1D位点1头贵州小型香猪和其它贵州小型香猪为不同的SNP类型。贵州小型香猪G3a2902发现一个可变剪接,其在499-587位发生片段缺失,并产生移码使编码蛋白被截断。在巴马香猪B3a2914发现一个非特异序列,其可能是猪CYP3A亚家族的一个新成员。
4.实验小型猪群体的微卫星遗传学检测
选用美国猪基因组协作计划推荐用于全球猪遗传多样性研究的21个微卫星标记,采用微卫星荧光标记-半自动基因分型方法,对实验所用的2个小型猪群体-巴马香猪和贵州小型香猪进行遗传学检测和评估,鉴定其遗传背景和遗传关系,分析其个体间相似性和差异程度。结果:巴马香猪和贵州小型香猪具有较好遗传稳定性,群体内具有良好遗传多样性,巴马香猪的个体均一性要好于贵州小型香猪,两群体间有较大遗传差异,其遗传稳定性说明两种小型猪是适宜医学生物学研究的封闭群实验动物,与人群的遗传状况相似;同时其品系间及品系内的遗传差异与其P450基因的SNP多态分布相符,进一步说明在猪作为药物代谢模型的应用中应关注品系及个体间的差异。两种小型猪及对照荣昌猪和大白猪在所检21个微卫星位点均具有良好遗传多样性,共检测到等位基因281个,每位点7-22个,巴马香猪、贵州小型香猪、荣昌猪、大白猪分别为132、125、174和119个,巴马香猪检测到的等位基因数要大于贵州小型香猪,但巴马香猪的遗传多样性指数Shannon信息指数和多态信息含量均低于贵州小型香猪,其遗传杂合度低于贵州小型香猪,纯合度高于后者,提示巴马香猪个体间的相似程度要大于贵州小型香猪。两种小型猪间的遗传分化指数Fst为0.2270,Nei标准遗传距离为0.9134,提示其群体间遗传分化较大,具有较大遗传差异。Hardy-Weinberg遗传平衡检测表明,两种小型猪的21个位点中均有7个偏离平衡,荣昌猪和大白猪则分别有10和11个偏离平衡,表明两种小型猪在繁育过程中能够较好地保持遗传稳定性。
三、研究结论
1.应用生物信息学方法预测获得37个可能的猪细胞色素P450新基因序列,分布于11个家族,19个亚家族,23个亚型;利用EST电子表达分析获得了猪不同组织的P450基因表达数据,发现了猪P450基因的46个可变剪接体。为小型猪药物代谢研究提供了P450基因的基础数据和资料。
2.克隆获得了我国资源小型猪品系巴马香猪的18个细胞色素P450新基因序列,均已在GenBank登录,登录号EF625344-EF625358,并已提交国际细胞色素P450命名委员会进行命名,不仅为小型猪药物代谢研究提供了P450基因序列资料,同时表明用生物信息学方法结合实验验证是快速高效获得猪P450新基因的有效途径。
3.发现了巴马香猪和贵州小型香猪CYP3A29基因的6个SNP位点及1个可变剪接体,其SNP分布具有品系及个体间多态性,在不同品系中可呈不同的SNP类型,其中巴马香猪具有品系内SNP类型的一致性,贵州小型香猪品系内具有SNP类型差异,说明猪P450基因具有和人相似的遗传多态性,在猪作为药物代谢模型的应用中应关注品系及个体间的差异。
4.巴马香猪和贵州小型香猪具有较好遗传稳定性,群体内具有良好遗传多样性,巴马香猪的个体均一性要好于贵州小型香猪,两群体间有较大遗传差异,其遗传稳定性说明两种小型猪是适宜医学生物学研究的封闭群实验动物,与人群的遗传状况相似;同时其品系间及品系内的遗传差异与其P450基因的SNP多态分布相符,进一步说明在猪作为药物代谢模型的应用中应关注品系及个体间的差异。
1 Research objective and background
Because of the similarity of food habit, anatomy and physiology characteristics and drug metabolism characteristics of pigs and those of human, there are more and more researches about the pharmacology, toxicology, pharmacokinetics and pharmacodynamics of pigs. Accompanying the animal protection movement, 3R principle is becoming increasingly popularized, usage of higher animals such as non-human primates and dogs is restricted, and usage of edible animals such as pigs is gradually increasing. Therefore, pigs have a good prospect as experimental animals of drug evaluation. Because miniature pigs have the advantage of small body size, the experimental operation of them is convenient, the standardized quality control of them is easy, and the cost of feed is low. Therefore the application of them has a special advantage and they are routine experimental pigs in biomedicine at present.
As drug evaluation models, the animals are required to have similarity compared with human in drug reaction mechanism and the interaction between the drug and the body. Thus the metabolic process of the drug in human body can be more exactly simulated through utilizing model animals. The metabolic process of drugs roughly includes four phases which are absorption, distribution, metabolism and excretion. The pharmacokinetics characters, bioavailability, toxic and side effects and the interaction of drugs are determined by the four phases. Drug metabolism is mainly in the liver. The biological transformation in the liver depends on multiple enzyme systems in microsomes, of which cytochrome P450 mixed-function oxidise system is the most important. Because the enzyme system is universally distributed in tissues and organs such as liver, kidney, brain, skin, lung, gastrointestinal tract and placenta, oxidation-reduction reaction catalyzed by P450 enzyme system can take place in many locations in vivo, of which liver is still the most important.
P450 enzyme system is universally distributed in various vertebrate animals, invertebrate animals, plants, fungi and bacteria. The enzyme system which is called P450 gene superfamily has a very complex composition and is controlled by gene diversity. To this day, there are 101 P450 families and 2345 genes in animals according to the newest published data. Because P450 gene families are closely related with human personalized medicine and toxic and side effects, P450 gene families of human are relatively deeply studied, at present there are 57 functional genes having been discovered. While in mouse which is an mature animal model of organism and human diseases, there are 102 P450 functional genes having been discovered. Although pig is an drug evaluation and biomedical animal model having a good prospect, researches of P450 gene of pigs are relatively deficient, there are only 29 cytochrome P450 functional genes which have been named by Cytochrome P450(CYP) Allele Nomenclture Committee.
The great magnanimity of increased sequences in the public database provide us sufficient resources for discovering new genes. As a part of cDNA sequence, EST is used to discover and clone new genes, which is an important route of acquiring new genes and carrying out function study. Large fragment or total length cDNA sequence can be directly acquired through classification, integration and composition of the great magnanimity of EST in the database. New members of gene families can be acquired fast and economically with this method. There are already more than 600,000 sequences of EST in the pig EST database. These data provide us an opportunity to find more information about P450 gene family.
Genetic polymorphism of cytochrome P450 is very important to drug metabolism and it is one of the most important factors related with human personalized medicine at present. Genetic polymorphism of cytochrome P450 of different individuals may cause different drug reactions to the same drug. Sometimes because of the genetic difference of individuals, the difference of effects and toxic and side effects of the same drug in different individuals can achieve 300 fold. According to the information of Cytochrome P450(CYP) Allele Nomenclture Committee, there are 40 polymorphic alleles (or mutants) of CYP3A4 of human having been confirmed. The main mutant of human in China Mainland is CYP3A4*4, which decreases the enzymatic activity of CYP3A4. Researches have demonstrated that for CYP3A subfamily, which is with the highest content in liver and can metabolism maximum drugs, the most suitable animal model is (mini) pig. Microsomes in pig’s liver have the same metabolic activity as human CYP3A4 probe substrate and the same characteristics of inhibitors as that of human, it is considered that CYP3A29 of pigs may be a homologous enzyme of CYP3A4 of human. Is CYP3A29 of pigs has a same polymorphism as that of human? If this polymorphism does exist, will it influence the metabolic activity of the metabolic pathway it participates? If this polymorphism does exist and dose influence the metabolic activity of the metabolic pathway, the influence effects of using pigs as metabolic models of human CYP3A4 substrate must be considered.
There are plentiful and superior miniature pig strains. As two of the represent strains, Bama miniature pigs and Guizhou miniature pigs, whose source are both Xiang pigs, are mainly produced in habitation of minority ethnic groups such as Congjiang, Guizhou, Sandou, Guizhou, Huanjiang, Guangxi, and Bama, Guangxi. Most of these areas are remote mountain areas which have very poor traffic. Other pig strains can’t enter, what’s more, the crowd there are used to making high inbred self-breeding and self-cultivating of pigs through parent-offspring mating, sibling mating and half-sib mating of pigs. Because of long-term natural selection, although there is a high extent of inbreeding, there are few genetic diseases and deformities. Thus there is a unique group of miniature pigs. Bama miniature pigs and Guizhou miniature pigs are both oriently selected with a breeding object of miniaturization. Bama miniature pigs have a higher extent of inbreeding and have white hair, can bear inbreeding, have stable heredity and a consistent phenotype. They have been proved to be superior animal models. Also, there are much background information about anatomy, physiology, biochemistry and basic biological characteristics. Related researches of Cytochrome P450 of Bama miniature pigs and Guizhou miniature pigs will have a significant meaning on the application of animal models of drug evaluation.
Therefore, this study makes use of the sequences in the public databse to predict new genes of Cytochrome P450 with a bioinformatics method. On the basis of this, related gene cloning of Bama miniature pig is carried out, which provides a foundation for taking Chinese miniature pig strain as an drug evaluation animal model. At the same time, the inter-strain and intra-strain polymorphism of the key subtype of Cytochrome P450 of Bama miniature pig and Guizhou miniature pig, which is CYP3A29, is studied, and the influence of the difference of polymorphism on the drug metabolism animal model. Detection and analysis of the microsatellite genetic background of the two miniature pigs is carried out to observe the mutual relationship between the difference of the inter-strain and intra-strain microsatellite genetic background and the gene polymorphism of CYP3A29.
2 Content and results of the research
2.1 Bioinformatics prediction and analysis of Cytochrome P450 gene of pigs
On the basis of homologous P450 gene sequences of other mammals and mainly taking advantage of the current EST database, silico cloning of Cytochrome P450 gene families of pigs was done with bioinformatics methods to acquire new genes of Cytochrome P450 gene families of pigs. Analysis of silico gene expression profile and alternative splicings of Cytochrome P450 of pigs were also done. Results: 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods by prediction, gene expression data of gene expression in different tissues of pigs were acquired with EST silicon expression analysis, and 46 alternative splicings of Cytochrome P450 of pigs were discovered, which provided basic data and document of P450 gene for drug metabolism research of miniature pigs. 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods. These sequences distributed in 11 families, 19 subfamilies and had 23 genes. Of which 9 sequences had full-length coding regions. Sequence analysis indicated that the sequences acquired by prediction all had a P450 structural domain or were highly similar to the corresponding P450 sequences of other species. Cluster analysis indicated that these sequences were homologous to the already-known P450 genes of pigs or corresponding P450 genes of human. 52 P450 gene expression data were acquired with EST silicon expression analysis on the already-known or predicted P450 genes. P450 genes which had the highest expression quantity in the liver of pigs were CYP2D25(50.26%), CYP2E1(31.09%), CYP2C33(3.69%), CYP2A19(3.11%) and CYP2D21(2.07%)in order, and these P450 gene expression data were compared with EST silicon expression of human. 46 alternative splicings of cytochrome P450 of pigs were discovered, of which there were 31 alternative splicings in coding regions, 3 alternative splicings in 5’UTR, 9 alternative splicings in 3’UTR, another two alternative splicings in coding regions and 3’UTR at the same time, and the other one alternative splicing in coding regions and 5’UTR at the same time.
2.2 Clone and verification of predictive cytochrome P450 gene of Bama Miniature Pig
One the basis of the prediction results of Cytochrome P450 gene sequences of pigs, corresponding gene sequences were amplificated with RT-PCR using designed primers, and then TA cloning and sequencing were done, results of which were compared to the predicted Cytochrome P450 gene sequences. Results: 18 Cytochrome P450 new gene sequences of Bama miniature pig were acquired by cloning. All the new gene sequences were registered in GeneBank, registration number was EF625344-EF625358. And the new gene sequences were presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. The research not only provided information about P450 gene sequences for drug metabolism study of miniature pigs, but also indicated that bioinformatics method combining experimental testing was a fast and effective route to acquire P450 new gene sequences. RT-PCR and TA cloning verification of the predicted 19 sequences of Cytochrome P450 gene sequences of pigs all acquired amplification products consistent with the expected fragment sizes. By sequencing and comparing, 17 cloned sequences of P450 gene were consistent with the predicted sequences by bioinformatics method, and 1 cloned sequence might be a neomorph of cyp4f6 gene of pig. 1 cloned sequence, cyp4f2v4 had a great difference compared with the predicted sequence. NCBI retrieval by Blastx didn’t discover similar P450 sequences, so that this sequence was not regarded as a P450 sequence of pigs. Amplification sequence of cyp4f3v1 had a greater difference with the original sequence, but it was very similar to the cyp4f6v1 sequence, one contig could be attained when it was spliced with cyp4f6v1*1, cyp4f6v1*2 and cyp4f6v1*3. Although NCBI retrieval of this contig by Blastx didn’t find an identical sequence, it highly resembled CYP4F6 subfamily, therefore it might be a new variant of the cyp4f6 gene of pigs. The other sequences were consistent with the predicted results on the whole. Amplification of Cyp39a1 was performed in 2 segment, cyp39a1*1 and cyp39a1*2 could be merged to form a contig, whose encoded protein was completely identical to the predicted results, and the protein should contain full-length of the coding region. All the sequences which were consistent with the predicted sequences were all registered in GeneBank, and the registration number is EF625344-EF625358. All the sequences have been presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names.
2.3 Polymorphism of CYP3A29 of miniature pigs
Full length coding region of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were amplificated with RT-PCR using designed primers, and then TA cloning and sequencing were done, polymorphism sites were acquired by sequence comparing and then their distribution and influence were analyzed. Results: 6 SNP sites and 1 alternative splicing of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were discovered. Distribution of SNP had strain and individual polymorphism. SNP could be of different types in different strains. SNP of Bama miniature pig had intra-strain consistent type, and SNP of Guizhou miniature pig had intra-strain different types. This demonstrated that P450 gene sequences of pigs had similar genetic polymorphism as those of human, therefore while using pigs as metabolic models, inter-strain and individual difference should be noted. CYP3A29 gene sequences of 13 Bama miniature pigs and 5 Guizhou miniature pigs were acquired. Contrasting results showed that there were 6 possible SNP sites, 1 alternative splicing, 1 possible new gene of CYP3A29 subfamily. Of the 6 possible SNP sites, 5 of them were samesense mutation, amino acids types weren’t changed, and encoded protein sequences weren’t influenced. Frameshift mutation and premature termination of translation caused by base deletion happened to one of them. Three SNP sites, CYP3A29*1A, CYP3A29*1B and CYP3A29*1C all displayed inter-strain difference, that was to say, there were different SNP subtypes in Bama miniature pig and Guizhou miniature pig. CYP3A29*1E and CYP3A29*2 sites displayed difference of miniature pig strain and other strain. For CYP3A29*1D site, there was 1 Guizhou miniature pig had a SNP type different from the others. There was one alternative splicing of G3a2902 in Guizhou miniature pig, 499-587 fragment deletion was found, at which site there was frame shift resulting interrupted protein. A non-specific sequence was found in one Bama miniature pig, it might be a new member of CYP3A subfamily of pigs.
2.4 Microsatellite genetics detection in experimental miniature pig populations
With the selected 21 microsatellite marks recommended by USA Pig Genome Cooperation Project for genetic diversity research of pigs worldwide and a fluorescence-based semi-automated genotyping method, genetics detection and evaluation was done to 2 miniature pig populations, which were Bama miniature pigs and Guizhou miniature pigs, to identify its genetic background and genetic relationship and analyze individual similarities and differences. Results: There was good genetic stability and genetic variations in both Bama miniature pig and Guizhou miniature pig. Individual homogenicity of Bama miniature pig was better than that of Guizhou miniature pig, there was a great genetic differences between the two groups. The genetic stability demonstrated that two strains of pigs were closed colonies suitable for biomecical research, having similar heredity character as human. At the same time genetic diversity inter-population and intra-population was consisitent with SNP polymorphism of P450 gene, which further demonstrated that inter-population and individual difference should be noted in application of pigs as drug metabolism models. There was good genetic diversity in the detected 21 microsatellite sites in the two populations of miniature pigs and Rong Chang pigs and large white pigs. Together 281 alleles were detected, and there were 7-22 alleles each locus. Those of Bama miniature pigs, Guizhou miniature pigs, Rong Chang pigs and large white pigs were 132, 125, 174 and 119, respectively. Alleles detected in Bama miniature pigs were more than detected in Guizhou miniature pigs, however, Shannon information index and polymorphism information content of Bama Miniature pigs were both lower than Guizhou miniature pigs, genetic heterozygosis of the former was lower than the latter and homozygosis of the former was higher than the latter, which indicated resemblance extent between individuals of Bama miniature pigs was higher than that between Guizhou miniature pigs. Genetic differentiation index between the two strains of miniature pigs, Fst was 0.2270. Nei standard genetic distance was 0.9134, which suggested there was great genetic differentiation between the two strains. Hardy-Weinberg genetic equilibrium detection suggested among the 21 sites of the two strains of minature pigs, there were 7 diverging from equilibrium. While in Rong Chang pigs and large white pigs, there were 10 and 11 sites diverging from equilibrium, respectively, which suggested they could maintain good genetic stability during breeding.
3 conclusion
3.1 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods by prediction, gene expression data of gene expression in different tissues of pigs were acquired with EST silicon expression analysis, and 46 alternative splicings of Cytochrome P450 of pigs were discovered, which provided basic data and document of P450 gene for drug metabolism research of miniature pigs.
3.2 18 Cytochrome P450 new gene sequences of Bama miniature pig were acquired by cloning. All the new gene sequences were registered in GeneBank, registration number was EF625344-EF625358. And the new gene sequences were presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. The research not only provided information about P450 gene sequences for drug metabolism study of miniature pigs, but also indicated that bioinformatics method combining experimental testing was a fast and effective route to acquire P450 new gene sequences.
3.3 6 SNP sites and 1 alternative splicing of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were discovered. Distribution of SNP had strain and individual polymorphism. SNP could be of different types in different strains. SNP of Bama miniature pig had intra-strain consistent type, and SNP of Guizhou miniature pig had intra-strain different types. This demonstrated that P450 gene sequences of pigs had similar genetic polymorphism as those of human, therefore while using pigs as metabolic models, inter-strain and individual difference should be noted.
3.4 There was good genetic stability and genetic variations in both Bama miniature pig and Guizhou miniature pig. Individual homogenicity of Bama miniature pig was better than that of Guizhou miniature pig, there was a great genetic differences between the two groups. The genetic stability demonstrated that two strains of pigs were closed colonies suitable for biomecical research, having similar heredity character as human. At the same time genetic diversity inter-population and intra-population was consisitent with SNP polymorphism of P450 gene, which further demonstrated that inter-population and individual difference should be noted in application of pigs as drug metabolism models.
由于猪的食性、解剖生理特点、以及药物代谢特点与人类的相似性,以猪进行药物的药理、毒理、药代、药效的研究日渐增多。随着动物保护运动的兴起,“3R”原则逐渐推广,非人灵长类及犬等高等动物使用受到限制,猪等食用动物的应用逐渐增多。因此,猪作为药物评价的实验动物具有良好前景。而小型猪由于体型小的优势,其实验操作方便,易于标准化质量控制,饲养成本低,其应用独具优势,也是目前在医学生物学研究中的常规实验用猪。
作为药物评价模型,要求模型动物的药物代谢反应机制以及药物与机体与之间的相互作用方面与人具有相似性,利用模型动物能够较为准确地模拟药物在人体内的代谢过程。药物在体内的过程大致可以分为吸收、分布、代谢和排泄四个阶段。药物的药代动力学性质及其生物利用度、毒副作用及药物相互作用都由这四个阶段决定。其中药物代谢的主要场所是肝脏。肝脏进行生物转化则依赖于微粒体中的多种酶系,其中最重要的是细胞色素P450混合功能氧化酶系统。由于该酶系广泛分布于肝、肾、脑、皮肤、肺、胃肠道及胎盘等组织器官,因此由P450酶系催化的氧化还原反应可发生在体内许多部位,但仍然以肝脏为主。
P450酶系普遍存在于各种脊椎动物、无脊椎动物、植物、真菌和细菌中。该酶系组成复杂,由基因多样性控制,称为P450基因超家族。到目前为止,根据最新公布的数据,仅动物就存在101个P450家族,2345个基因序列。P450基因家族和人类的个体化用药及药物的毒副作用密切相关,因此人类的P450已经研究得比较深入,目前发现57个P450功能基因。而作为成熟模式生物和人类疾病动物模型的小鼠则已发现102个P450功能基因。猪作为一种具有重要应用前景的药物评价动物及医学生物学实验动物,其P450基因的研究相对缺乏,目前通过国际细胞色素P450命名委员会命名的仅有30个功能基因。
公开数据库中海量增长的序列信息为我们发现新基因提供了充足的资源。EST作为基因的部分cDNA序列,利用其发现和克隆新基因已成为获取新基因并进行功能研究的重要途径。对数据库中大量EST进行分类、整合、组装,可直接获得大片段或全长cDNA序列。用该方法也可以快速、经济地获得基因家族的新成员。猪EST数据库中的序列数已达到60万条以上,这些数据为我们提供了发现猪P450家族基因信息的机会。
细胞色素P450酶的遗传多态性对其药物代谢有重要影响,也是和目前人类个体化用药相关的主要因素之一。不同个体在P450基因多态性的产生,可能会造成对同一药物反应的差异,有时由于这种个体间遗传学上的差别,同一药物在不同个体内的效果和毒副作用的差异可以达到300倍之多。根据人类细胞色素P450命名会员会的资料,仅人的CYP3A4目前就有已经证实的40个多态基因(或叫突变体)。中国大陆人主要突变体是CYP3A4*4,该突变使CYP3A4酶活性降低。研究已经证明,对于人类肝脏含量最高,也是代谢药物最多的CYP3A亚家族,其最合适的动物模型是(小型)猪。猪的肝脏微粒体具有人CYP3A4探针底物的代谢活性和相同的抑制剂特性,并认为猪的CYP3A29可能是人CYP3A4的同源酶。那么猪CYP3A29是否具有同样的多态性呢?如果这样的多态性存在的话,其是否会影响其参与代谢途径的代谢活性呢?如果CYP3A29存在遗传多态性且影响其代谢活性的话,用猪作为人类CYP3A4底物药的代谢模型就必须要考虑其影响效果。
我国具有丰富和优秀的小型猪资源品系。巴马香猪和贵州小型香猪作为其代表品种之一,其来源均为香猪,主要产于我国贵州省从江、三都、和广西环江、巴马等少数民族聚居地区。这些地区多属偏僻山区,交通闭塞,不仅其它猪种的血缘难以进入,而且群众习惯于将猪进行亲子交配,同胞、半同胞交配等高度近交的自繁自养方式。并且由于长期的自然选择,虽近交程度较高,却少见遗传疾病及畸形,从而形成独特小型猪群体。巴马香猪和贵州小型香猪均以小型化为育种目标进行了定向选育。其中巴马香猪近交程度高、被毛呈白色,而且耐近交、遗传稳定、个体表型一致,在医学生物学领域应用中证明是优秀的模型动物,并积累了丰富的解剖、生理生化及基础生物学特性背景资料。对巴马香猪和贵州小型香猪进行其细胞色素P450基因的相关研究,将对其作为药物评价动物模型的应用具有重要意义。
因此,本研究利用公开数据库的序列资源,用生物信息学方法预测猪的细胞色素P450新基因,以此对我国资源小型猪品系巴马香猪进行相关基因的克隆,为我国独特小型猪品系作为药物评价动物模型提供基础依据;同时研究巴马香猪和贵州小型香猪细胞色素P450关键亚型CYP3A29的品系间及品系内多态性,评估其序列多态差异对药物代谢动物模型的影响;对两种小型猪品系进行微卫星遗传背景检测分析,观察动物品系间与品系内微卫星遗传背景差异与其CYP3A29基因多态的相互关系。
二、研究内容和结果
1.猪细胞色素P450基因的生物信息学预测和分析
根据其它哺乳动物的同源P450基因序列,主要利用猪现有的EST数据库,用生物信息学方法对猪P450家族基因进行电子克隆,获得猪细胞色素P450家族新基因;并对猪细胞色素P450基因进行EST电子表达谱、可变剪接等分析。结果:应用生物信息学方法预测获得37个可能的猪细胞色素P450新基因序列,利用EST电子表达分析获得了猪不同组织的P450基因表达数据,发现了猪P450基因的46个可变剪接体,为小型猪药物代谢研究提供了P450基因的基础数据和资料。通过生物信息学方法共得到37个可能的猪细胞色素P450新基因序列,分布于11个家族,19个亚家族,23个亚型。其中9条具有全长编码区序列。序列分析表明预测得到的序列均具有P450结构域或与其它物种相应P450序列高度相似,聚类分析表明其和已知猪的P450基因或人的相应P450基因具有同源性。对已知和预测的共52个猪P450基因进行EST电子表达谱分析,获得了其不同组织的P450基因表达数据,其中在猪的肝脏表达量最高的P450基因依次是CYP2D25(50.26%)、CYP2E1(31.09%)、CYP2C33(3.69%)、CYP2A19(3.11%)、CYP2D21(2.07%),并和人的EST电子表达谱进行了比较。发现了猪P450基因的46个可变剪接体,其中编码区的可变剪接有31个,5’UTR的可变剪接有3个,3’UTR的可变剪接有9个,另有2个可变剪接同时发生在编码区和3’UTR,1个可变剪接同时发生在编码区和5’UTR。
2.巴马香猪预测细胞色素P450基因的克隆和验证
根据对猪细胞色素P450基因序列的预测结果,设计引物通过RT-PCR扩增相应基因序列,进行TA克隆和序列测定,与预测的P450基因序列进行比较。结果:克隆获得了我国资源小型猪品系巴马香猪的18个细胞色素P450新基因序列,均已在GenBank登录,登录号EF625344-EF625358,并已提交国际细胞色素P450命名委员会进行命名,不仅为小型猪药物代谢研究提供了P450基因序列资料,同时表明用生物信息学方法结合实验验证是快速高效获得猪P450新基因的有效途径。对预测的猪细胞色素P450基因19个序列的RT-PCR克隆验证均获得了符合预期片段大小的扩增产物,经序列测定和比对,17个P450基因的克隆序列和生物信息学预测结果基本一致,1个克隆序列可能为猪cyp4f6基因的一个新等位基因。其中1个克隆序列cyp4f2v4和预测序列相差较大,blastx检索NCBI未发现相似P450序列,因此把这条序列不作为猪的P450基因序列。cyp4f3v1的扩增序列和原来差异较大,但和cyp4f6v1序列非常相似,并能和cyp4f6v1*1、cyp4f6v1*2、cyp4f6v1*3其同拼接成一条contig,将这条contig用blastx检索NCBI,未发现相同序列,但和CYP4F6亚家族有高度相似,故其应为猪cyp4f6基因的一个新变体。其余序列均和预测结果基本一致,cyp39a1通过分段扩增,cyp39a1*1和cyp39a1*2可拼接成一个contig,其编码蛋白和预测序列完全相同,应该包含了其编码区全长。所有和预测序列一致的序列均已在GenBank登录,登录号EF625344-EF625358。所有序列已提交国际细胞色素P450命名委员会进行命名。
3.小型猪细胞色素P450基因CYP3A29多态研究
设计引物通过RT-PCR扩增不同个体巴马香猪和贵州小型香猪的CYP3A29基因编码区全长,进行TA克隆和测序,通过序列比对检测和发现其多态性位点,并对其多态位点的分布和影响进行分析。结果:发现了巴马香猪和贵州小型香猪CYP3A29基因的6个SNP位点及1个可变剪接体,其SNP分布具有品系及个体间多态性,在不同品系中可呈不同的SNP类型,其中巴马香猪具有品系内SNP类型的一致性,贵州小型香猪品系内具有SNP类型差异,说明猪P450基因具有和人相似的遗传多态性,在猪作为药物代谢模型的应用中应关注品系及个体间的差异。共获得13头巴马香猪和5头贵州小型香猪的CYP3A29基因序列,比对结果表明,其中具有6个可能的SNP位点,1个可变剪接体,1个可能的猪CYP3A29亚家族新基因。6个可能的SNP位点中,5个均为同义替换,不改变编码的氨基酸类型,对编码的蛋白序列无影响,1个由于碱基缺失引起移码突变和翻译提前终止。CYP3A29*1A、CYP3A29*1B、CYP3A29*1C三个SNP位点,均表现为品系间的差异,即巴马香猪和贵州小型香猪各为一个SNP亚型;位点CYP3A29*1E和CYP3A29*2则表现小型猪品系和其它品系间的序列差异;CYP3A29*1D位点1头贵州小型香猪和其它贵州小型香猪为不同的SNP类型。贵州小型香猪G3a2902发现一个可变剪接,其在499-587位发生片段缺失,并产生移码使编码蛋白被截断。在巴马香猪B3a2914发现一个非特异序列,其可能是猪CYP3A亚家族的一个新成员。
4.实验小型猪群体的微卫星遗传学检测
选用美国猪基因组协作计划推荐用于全球猪遗传多样性研究的21个微卫星标记,采用微卫星荧光标记-半自动基因分型方法,对实验所用的2个小型猪群体-巴马香猪和贵州小型香猪进行遗传学检测和评估,鉴定其遗传背景和遗传关系,分析其个体间相似性和差异程度。结果:巴马香猪和贵州小型香猪具有较好遗传稳定性,群体内具有良好遗传多样性,巴马香猪的个体均一性要好于贵州小型香猪,两群体间有较大遗传差异,其遗传稳定性说明两种小型猪是适宜医学生物学研究的封闭群实验动物,与人群的遗传状况相似;同时其品系间及品系内的遗传差异与其P450基因的SNP多态分布相符,进一步说明在猪作为药物代谢模型的应用中应关注品系及个体间的差异。两种小型猪及对照荣昌猪和大白猪在所检21个微卫星位点均具有良好遗传多样性,共检测到等位基因281个,每位点7-22个,巴马香猪、贵州小型香猪、荣昌猪、大白猪分别为132、125、174和119个,巴马香猪检测到的等位基因数要大于贵州小型香猪,但巴马香猪的遗传多样性指数Shannon信息指数和多态信息含量均低于贵州小型香猪,其遗传杂合度低于贵州小型香猪,纯合度高于后者,提示巴马香猪个体间的相似程度要大于贵州小型香猪。两种小型猪间的遗传分化指数Fst为0.2270,Nei标准遗传距离为0.9134,提示其群体间遗传分化较大,具有较大遗传差异。Hardy-Weinberg遗传平衡检测表明,两种小型猪的21个位点中均有7个偏离平衡,荣昌猪和大白猪则分别有10和11个偏离平衡,表明两种小型猪在繁育过程中能够较好地保持遗传稳定性。
三、研究结论
1.应用生物信息学方法预测获得37个可能的猪细胞色素P450新基因序列,分布于11个家族,19个亚家族,23个亚型;利用EST电子表达分析获得了猪不同组织的P450基因表达数据,发现了猪P450基因的46个可变剪接体。为小型猪药物代谢研究提供了P450基因的基础数据和资料。
2.克隆获得了我国资源小型猪品系巴马香猪的18个细胞色素P450新基因序列,均已在GenBank登录,登录号EF625344-EF625358,并已提交国际细胞色素P450命名委员会进行命名,不仅为小型猪药物代谢研究提供了P450基因序列资料,同时表明用生物信息学方法结合实验验证是快速高效获得猪P450新基因的有效途径。
3.发现了巴马香猪和贵州小型香猪CYP3A29基因的6个SNP位点及1个可变剪接体,其SNP分布具有品系及个体间多态性,在不同品系中可呈不同的SNP类型,其中巴马香猪具有品系内SNP类型的一致性,贵州小型香猪品系内具有SNP类型差异,说明猪P450基因具有和人相似的遗传多态性,在猪作为药物代谢模型的应用中应关注品系及个体间的差异。
4.巴马香猪和贵州小型香猪具有较好遗传稳定性,群体内具有良好遗传多样性,巴马香猪的个体均一性要好于贵州小型香猪,两群体间有较大遗传差异,其遗传稳定性说明两种小型猪是适宜医学生物学研究的封闭群实验动物,与人群的遗传状况相似;同时其品系间及品系内的遗传差异与其P450基因的SNP多态分布相符,进一步说明在猪作为药物代谢模型的应用中应关注品系及个体间的差异。
1 Research objective and background
Because of the similarity of food habit, anatomy and physiology characteristics and drug metabolism characteristics of pigs and those of human, there are more and more researches about the pharmacology, toxicology, pharmacokinetics and pharmacodynamics of pigs. Accompanying the animal protection movement, 3R principle is becoming increasingly popularized, usage of higher animals such as non-human primates and dogs is restricted, and usage of edible animals such as pigs is gradually increasing. Therefore, pigs have a good prospect as experimental animals of drug evaluation. Because miniature pigs have the advantage of small body size, the experimental operation of them is convenient, the standardized quality control of them is easy, and the cost of feed is low. Therefore the application of them has a special advantage and they are routine experimental pigs in biomedicine at present.
As drug evaluation models, the animals are required to have similarity compared with human in drug reaction mechanism and the interaction between the drug and the body. Thus the metabolic process of the drug in human body can be more exactly simulated through utilizing model animals. The metabolic process of drugs roughly includes four phases which are absorption, distribution, metabolism and excretion. The pharmacokinetics characters, bioavailability, toxic and side effects and the interaction of drugs are determined by the four phases. Drug metabolism is mainly in the liver. The biological transformation in the liver depends on multiple enzyme systems in microsomes, of which cytochrome P450 mixed-function oxidise system is the most important. Because the enzyme system is universally distributed in tissues and organs such as liver, kidney, brain, skin, lung, gastrointestinal tract and placenta, oxidation-reduction reaction catalyzed by P450 enzyme system can take place in many locations in vivo, of which liver is still the most important.
P450 enzyme system is universally distributed in various vertebrate animals, invertebrate animals, plants, fungi and bacteria. The enzyme system which is called P450 gene superfamily has a very complex composition and is controlled by gene diversity. To this day, there are 101 P450 families and 2345 genes in animals according to the newest published data. Because P450 gene families are closely related with human personalized medicine and toxic and side effects, P450 gene families of human are relatively deeply studied, at present there are 57 functional genes having been discovered. While in mouse which is an mature animal model of organism and human diseases, there are 102 P450 functional genes having been discovered. Although pig is an drug evaluation and biomedical animal model having a good prospect, researches of P450 gene of pigs are relatively deficient, there are only 29 cytochrome P450 functional genes which have been named by Cytochrome P450(CYP) Allele Nomenclture Committee.
The great magnanimity of increased sequences in the public database provide us sufficient resources for discovering new genes. As a part of cDNA sequence, EST is used to discover and clone new genes, which is an important route of acquiring new genes and carrying out function study. Large fragment or total length cDNA sequence can be directly acquired through classification, integration and composition of the great magnanimity of EST in the database. New members of gene families can be acquired fast and economically with this method. There are already more than 600,000 sequences of EST in the pig EST database. These data provide us an opportunity to find more information about P450 gene family.
Genetic polymorphism of cytochrome P450 is very important to drug metabolism and it is one of the most important factors related with human personalized medicine at present. Genetic polymorphism of cytochrome P450 of different individuals may cause different drug reactions to the same drug. Sometimes because of the genetic difference of individuals, the difference of effects and toxic and side effects of the same drug in different individuals can achieve 300 fold. According to the information of Cytochrome P450(CYP) Allele Nomenclture Committee, there are 40 polymorphic alleles (or mutants) of CYP3A4 of human having been confirmed. The main mutant of human in China Mainland is CYP3A4*4, which decreases the enzymatic activity of CYP3A4. Researches have demonstrated that for CYP3A subfamily, which is with the highest content in liver and can metabolism maximum drugs, the most suitable animal model is (mini) pig. Microsomes in pig’s liver have the same metabolic activity as human CYP3A4 probe substrate and the same characteristics of inhibitors as that of human, it is considered that CYP3A29 of pigs may be a homologous enzyme of CYP3A4 of human. Is CYP3A29 of pigs has a same polymorphism as that of human? If this polymorphism does exist, will it influence the metabolic activity of the metabolic pathway it participates? If this polymorphism does exist and dose influence the metabolic activity of the metabolic pathway, the influence effects of using pigs as metabolic models of human CYP3A4 substrate must be considered.
There are plentiful and superior miniature pig strains. As two of the represent strains, Bama miniature pigs and Guizhou miniature pigs, whose source are both Xiang pigs, are mainly produced in habitation of minority ethnic groups such as Congjiang, Guizhou, Sandou, Guizhou, Huanjiang, Guangxi, and Bama, Guangxi. Most of these areas are remote mountain areas which have very poor traffic. Other pig strains can’t enter, what’s more, the crowd there are used to making high inbred self-breeding and self-cultivating of pigs through parent-offspring mating, sibling mating and half-sib mating of pigs. Because of long-term natural selection, although there is a high extent of inbreeding, there are few genetic diseases and deformities. Thus there is a unique group of miniature pigs. Bama miniature pigs and Guizhou miniature pigs are both oriently selected with a breeding object of miniaturization. Bama miniature pigs have a higher extent of inbreeding and have white hair, can bear inbreeding, have stable heredity and a consistent phenotype. They have been proved to be superior animal models. Also, there are much background information about anatomy, physiology, biochemistry and basic biological characteristics. Related researches of Cytochrome P450 of Bama miniature pigs and Guizhou miniature pigs will have a significant meaning on the application of animal models of drug evaluation.
Therefore, this study makes use of the sequences in the public databse to predict new genes of Cytochrome P450 with a bioinformatics method. On the basis of this, related gene cloning of Bama miniature pig is carried out, which provides a foundation for taking Chinese miniature pig strain as an drug evaluation animal model. At the same time, the inter-strain and intra-strain polymorphism of the key subtype of Cytochrome P450 of Bama miniature pig and Guizhou miniature pig, which is CYP3A29, is studied, and the influence of the difference of polymorphism on the drug metabolism animal model. Detection and analysis of the microsatellite genetic background of the two miniature pigs is carried out to observe the mutual relationship between the difference of the inter-strain and intra-strain microsatellite genetic background and the gene polymorphism of CYP3A29.
2 Content and results of the research
2.1 Bioinformatics prediction and analysis of Cytochrome P450 gene of pigs
On the basis of homologous P450 gene sequences of other mammals and mainly taking advantage of the current EST database, silico cloning of Cytochrome P450 gene families of pigs was done with bioinformatics methods to acquire new genes of Cytochrome P450 gene families of pigs. Analysis of silico gene expression profile and alternative splicings of Cytochrome P450 of pigs were also done. Results: 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods by prediction, gene expression data of gene expression in different tissues of pigs were acquired with EST silicon expression analysis, and 46 alternative splicings of Cytochrome P450 of pigs were discovered, which provided basic data and document of P450 gene for drug metabolism research of miniature pigs. 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods. These sequences distributed in 11 families, 19 subfamilies and had 23 genes. Of which 9 sequences had full-length coding regions. Sequence analysis indicated that the sequences acquired by prediction all had a P450 structural domain or were highly similar to the corresponding P450 sequences of other species. Cluster analysis indicated that these sequences were homologous to the already-known P450 genes of pigs or corresponding P450 genes of human. 52 P450 gene expression data were acquired with EST silicon expression analysis on the already-known or predicted P450 genes. P450 genes which had the highest expression quantity in the liver of pigs were CYP2D25(50.26%), CYP2E1(31.09%), CYP2C33(3.69%), CYP2A19(3.11%) and CYP2D21(2.07%)in order, and these P450 gene expression data were compared with EST silicon expression of human. 46 alternative splicings of cytochrome P450 of pigs were discovered, of which there were 31 alternative splicings in coding regions, 3 alternative splicings in 5’UTR, 9 alternative splicings in 3’UTR, another two alternative splicings in coding regions and 3’UTR at the same time, and the other one alternative splicing in coding regions and 5’UTR at the same time.
2.2 Clone and verification of predictive cytochrome P450 gene of Bama Miniature Pig
One the basis of the prediction results of Cytochrome P450 gene sequences of pigs, corresponding gene sequences were amplificated with RT-PCR using designed primers, and then TA cloning and sequencing were done, results of which were compared to the predicted Cytochrome P450 gene sequences. Results: 18 Cytochrome P450 new gene sequences of Bama miniature pig were acquired by cloning. All the new gene sequences were registered in GeneBank, registration number was EF625344-EF625358. And the new gene sequences were presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. The research not only provided information about P450 gene sequences for drug metabolism study of miniature pigs, but also indicated that bioinformatics method combining experimental testing was a fast and effective route to acquire P450 new gene sequences. RT-PCR and TA cloning verification of the predicted 19 sequences of Cytochrome P450 gene sequences of pigs all acquired amplification products consistent with the expected fragment sizes. By sequencing and comparing, 17 cloned sequences of P450 gene were consistent with the predicted sequences by bioinformatics method, and 1 cloned sequence might be a neomorph of cyp4f6 gene of pig. 1 cloned sequence, cyp4f2v4 had a great difference compared with the predicted sequence. NCBI retrieval by Blastx didn’t discover similar P450 sequences, so that this sequence was not regarded as a P450 sequence of pigs. Amplification sequence of cyp4f3v1 had a greater difference with the original sequence, but it was very similar to the cyp4f6v1 sequence, one contig could be attained when it was spliced with cyp4f6v1*1, cyp4f6v1*2 and cyp4f6v1*3. Although NCBI retrieval of this contig by Blastx didn’t find an identical sequence, it highly resembled CYP4F6 subfamily, therefore it might be a new variant of the cyp4f6 gene of pigs. The other sequences were consistent with the predicted results on the whole. Amplification of Cyp39a1 was performed in 2 segment, cyp39a1*1 and cyp39a1*2 could be merged to form a contig, whose encoded protein was completely identical to the predicted results, and the protein should contain full-length of the coding region. All the sequences which were consistent with the predicted sequences were all registered in GeneBank, and the registration number is EF625344-EF625358. All the sequences have been presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names.
2.3 Polymorphism of CYP3A29 of miniature pigs
Full length coding region of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were amplificated with RT-PCR using designed primers, and then TA cloning and sequencing were done, polymorphism sites were acquired by sequence comparing and then their distribution and influence were analyzed. Results: 6 SNP sites and 1 alternative splicing of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were discovered. Distribution of SNP had strain and individual polymorphism. SNP could be of different types in different strains. SNP of Bama miniature pig had intra-strain consistent type, and SNP of Guizhou miniature pig had intra-strain different types. This demonstrated that P450 gene sequences of pigs had similar genetic polymorphism as those of human, therefore while using pigs as metabolic models, inter-strain and individual difference should be noted. CYP3A29 gene sequences of 13 Bama miniature pigs and 5 Guizhou miniature pigs were acquired. Contrasting results showed that there were 6 possible SNP sites, 1 alternative splicing, 1 possible new gene of CYP3A29 subfamily. Of the 6 possible SNP sites, 5 of them were samesense mutation, amino acids types weren’t changed, and encoded protein sequences weren’t influenced. Frameshift mutation and premature termination of translation caused by base deletion happened to one of them. Three SNP sites, CYP3A29*1A, CYP3A29*1B and CYP3A29*1C all displayed inter-strain difference, that was to say, there were different SNP subtypes in Bama miniature pig and Guizhou miniature pig. CYP3A29*1E and CYP3A29*2 sites displayed difference of miniature pig strain and other strain. For CYP3A29*1D site, there was 1 Guizhou miniature pig had a SNP type different from the others. There was one alternative splicing of G3a2902 in Guizhou miniature pig, 499-587 fragment deletion was found, at which site there was frame shift resulting interrupted protein. A non-specific sequence was found in one Bama miniature pig, it might be a new member of CYP3A subfamily of pigs.
2.4 Microsatellite genetics detection in experimental miniature pig populations
With the selected 21 microsatellite marks recommended by USA Pig Genome Cooperation Project for genetic diversity research of pigs worldwide and a fluorescence-based semi-automated genotyping method, genetics detection and evaluation was done to 2 miniature pig populations, which were Bama miniature pigs and Guizhou miniature pigs, to identify its genetic background and genetic relationship and analyze individual similarities and differences. Results: There was good genetic stability and genetic variations in both Bama miniature pig and Guizhou miniature pig. Individual homogenicity of Bama miniature pig was better than that of Guizhou miniature pig, there was a great genetic differences between the two groups. The genetic stability demonstrated that two strains of pigs were closed colonies suitable for biomecical research, having similar heredity character as human. At the same time genetic diversity inter-population and intra-population was consisitent with SNP polymorphism of P450 gene, which further demonstrated that inter-population and individual difference should be noted in application of pigs as drug metabolism models. There was good genetic diversity in the detected 21 microsatellite sites in the two populations of miniature pigs and Rong Chang pigs and large white pigs. Together 281 alleles were detected, and there were 7-22 alleles each locus. Those of Bama miniature pigs, Guizhou miniature pigs, Rong Chang pigs and large white pigs were 132, 125, 174 and 119, respectively. Alleles detected in Bama miniature pigs were more than detected in Guizhou miniature pigs, however, Shannon information index and polymorphism information content of Bama Miniature pigs were both lower than Guizhou miniature pigs, genetic heterozygosis of the former was lower than the latter and homozygosis of the former was higher than the latter, which indicated resemblance extent between individuals of Bama miniature pigs was higher than that between Guizhou miniature pigs. Genetic differentiation index between the two strains of miniature pigs, Fst was 0.2270. Nei standard genetic distance was 0.9134, which suggested there was great genetic differentiation between the two strains. Hardy-Weinberg genetic equilibrium detection suggested among the 21 sites of the two strains of minature pigs, there were 7 diverging from equilibrium. While in Rong Chang pigs and large white pigs, there were 10 and 11 sites diverging from equilibrium, respectively, which suggested they could maintain good genetic stability during breeding.
3 conclusion
3.1 37 possible new gene sequences of Cytochrome P450 of pigs were acquired with bioinformatics methods by prediction, gene expression data of gene expression in different tissues of pigs were acquired with EST silicon expression analysis, and 46 alternative splicings of Cytochrome P450 of pigs were discovered, which provided basic data and document of P450 gene for drug metabolism research of miniature pigs.
3.2 18 Cytochrome P450 new gene sequences of Bama miniature pig were acquired by cloning. All the new gene sequences were registered in GeneBank, registration number was EF625344-EF625358. And the new gene sequences were presented to Cytochrome P450(CYP) Allele Nomenclture Committee to be assigned names. The research not only provided information about P450 gene sequences for drug metabolism study of miniature pigs, but also indicated that bioinformatics method combining experimental testing was a fast and effective route to acquire P450 new gene sequences.
3.3 6 SNP sites and 1 alternative splicing of CYP3A29 gene of Bama miniature pig and Guizhou miniature pig were discovered. Distribution of SNP had strain and individual polymorphism. SNP could be of different types in different strains. SNP of Bama miniature pig had intra-strain consistent type, and SNP of Guizhou miniature pig had intra-strain different types. This demonstrated that P450 gene sequences of pigs had similar genetic polymorphism as those of human, therefore while using pigs as metabolic models, inter-strain and individual difference should be noted.
3.4 There was good genetic stability and genetic variations in both Bama miniature pig and Guizhou miniature pig. Individual homogenicity of Bama miniature pig was better than that of Guizhou miniature pig, there was a great genetic differences between the two groups. The genetic stability demonstrated that two strains of pigs were closed colonies suitable for biomecical research, having similar heredity character as human. At the same time genetic diversity inter-population and intra-population was consisitent with SNP polymorphism of P450 gene, which further demonstrated that inter-population and individual difference should be noted in application of pigs as drug metabolism models.
引文
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