东方田鼠抗日本血吸虫抗性相关基因Mf-HSP90α的克隆筛选及其功能研究
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摘要
流行病学调查及人工感染实验研究证明东方田鼠具有天然的日本血吸虫抗性,这种抗性能够稳定遗传。现代分子遗传学理论认为,生物体对于任何疾病的易感性和抗性都可能是由相应基因所决定的。进一步研究发现东方田鼠体内不同组织及组分体外抗日本血吸虫抗性最强的是血清,被动转移血清的小鼠同样获得日本血吸虫抗性。从东方田鼠血清入手,筛选分离抗日本血吸虫抗性物质成为有效防治日本血吸虫病新的突破口。本研究从东方田鼠骨髓基因表达文库入手,应用表达克隆法逐级筛选,最终获得日本血吸虫抗性基因Mf-HSP90α,生物信息学分析其结构和功能后,制备条件培养基,验证基因表达产物的体外杀伤日本血吸虫童虫效果,并使用逆转录病毒载体pLXSN,构建重组pLXSN-HSP90α质粒,包装重组基因病毒,导入日本血吸虫感染小鼠体内,验证其体内抗虫效果。
一、东方田鼠抗日本血吸虫抗性相关基因gC14.75的克隆筛选
本研究构建了东方田鼠骨髓基因表达文库,根据表达产物的体外杀日本血吸虫童虫活性应用表达克隆法对文库进行筛选：基因表达文库随机分成8个基因池,相应质粒瞬时转染293T细胞,收集48h转染上清(条件培养基),进行体外杀日本血吸虫童虫实验,设立阴性对照：pcDNA1.1/Amp空载体转染293T细胞培养上清；阳性对照：20%东方田鼠血清。条件培养基杀虫率最高的基因池进入下一轮筛选,三轮筛选后获得杀虫效果最好的次级亚基因池gC14。gCl4质粒铺琼脂糖平板,随机挑取单个克隆,根据单克隆插入片段大小的不同,挑选32个进行表达产物杀虫实验,最终获得体外杀虫效果最好的单个克隆gC14.75。基因池条件培养基体外杀童虫率最高一组为gC,童虫死亡率为10.9%,与对照组相比P<0.05,差异具有统计学意义。亚基因池gC1、次级亚基因池gC14条件培养基的杀虫率分别为11.5%、15.9%,与对照组相比P<0.05,差异具有统计学意义。单克隆筛选中体外杀虫率最高的克隆是75号,杀虫率为11.0%。将克隆质粒DNA送Invitrogen公司测序获得抗性基因EST,长度为331 bp,将其命名为gC14.75。通过与GenBank数据库中序列进行同源性比较,发现gC14.75为HSP90α的同源序列。
二、gC14.75基因全长的克隆及其功能分析
HSP90是一个高度保守的基因家族。以gC14.75序列及与其同源性最高的中国仓鼠全长HSP90αcDNA (L33676)序列为模板,分别设计引物,RT-PCR扩增获得gC14.75全长序列。将全长序列测序后,用NCBI核苷酸数据库BLASTN进行序列比对分析,结果显示该基因与HSP90α同源,故将其命名为Mf-HSP90α。进而通过生物信息学方法分析该基因结构及功能。比对分析几种不同物种HSP90α的核苷酸及氨基酸序列,发现氨基酸、核苷酸序列差异很小。为了寻找可能存在的抗日本血吸虫功能差异的依据,我们用3D-JIGSAW (version 2.0)分析比较了东方田鼠与小鼠氨基酸序列的立体结构,结果显示二者在立体结构上存在显著差异。分别将Mf-HSP90α及小鼠HSP90α(Ms-HSP90α)重组到真核表达载体pcDNA1.1/Amp,制备相应条件培养基,进行体外杀日本血吸虫童虫实验。扣除空白对照组童虫死亡率,Mf-HSP90α条件培养基杀虫率为20.3%,与对照组相比较杀虫效果显著,而Ms-HSP90α条件培养基杀虫率仅为4.4%,与Mf-HSP90α相比较存在显著差异。为了进一步探讨东方田鼠与小鼠HSP90α抗虫功能差异的机制。我们进一步从mRNA、蛋白质水平分别用RT-PCR、Western-blot方法比较分析了HSP90α在东方田鼠及小鼠体内不同组织的表达情况,发现同一物种体内不同组织间HSP90α的表达存在显著差异,同一种组织在东方田鼠与小鼠间的表达情况也明显不同。
三、小鼠体内Mf-HSP90α的抗日本血吸虫作用及表达情况分析
为了进一步确认Mf-HSP90α的抗日本血吸虫活性,我们采用了基因转移技术,将该基因导入血吸虫易感动物(小鼠)体内,通过对小鼠模型的抗日本血吸虫效果的考察评估Mf-HSP90α的体内抗虫作用。本实验应用逆转录病毒载体pLXSN,构建pLXSN-HSP90α重组逆转录病毒载体系统,通过小鼠尾静脉注射将重组病毒导入小鼠体内,检测重组病毒治疗后感染小鼠的减虫率、肝脏减卵率、血吸虫虫体改变及小鼠肝脏肉芽肿减少情况。重组病毒载体经PA317细胞包装后,大量收集病毒悬液,经生物学方法检测病毒滴度：pLXSN病毒滴度为1.6×107cfu/ml pLXSN-HSP90α病毒滴度为4×106cfu/ml。分别用pLXSN、pLXSN-HSP90α病毒各200μl／次,共3次经尾静脉注射治疗日本血吸虫感染小鼠,空白对照组注射等体积DMEM。pLXSN- HSP90α重组病毒处理组检获成虫相比DMEM及pLXSN病毒组均要短小,检获成虫数也明显少于两对照组,减虫率与DMEM及pLXSN病毒组相比较分别是40.8%和32.3%。选取具有代表性肝脏制成石蜡切片,进行HE染色,镜下可以观察到pLXSN-HSP90α重组病毒处理组虫卵肉芽肿明显少于两对照组,与肉眼观察肝脏表面结果相同。pLXSN-HSP90α重组病毒处理组LEPG与DMEM及pLXSN病毒组相比,pLXSN-HSP90α重组病毒处理组的减卵率分别是57.9%和49.2%。减虫率、减卵率DMEM与pLXSN病毒组相比P>0.05,差异无统计学意义,pLXSN-HSP90α重组逆转录病毒组与DMEM及pLXSN病毒相比P<0.05,差异有统计学意义。为了探讨Mf-HSP90α抗日本血吸虫功能的机制,我们用RT-PCR的方法分析比较了感染尾蚴7天DMEM, pLXSN-HSP90α重组逆转录病毒不同处理组小鼠体内几种组织HSP90α表达情况,通过对RT-PCR条带灰度扫描,统计发现DMEM处理组小鼠肌肉HSP90α的表达丰度很低,其他组织表达丰度适中,组织间差异不大。pLXSN-HSP90α重组逆转录病毒处理组小鼠肺、脑、骨髓3种组织HSP90α的表达丰度相比其他几种组织明显要高,也明显高于与DMEM处理组肺、脑及骨髓的表达丰度也明显要高。
综上所述,本研究应用表达克隆法从东方田鼠骨髓基因表达文库筛选到一个日本血吸虫抗性相关基因Mf-HSP90α。东方田鼠与小鼠HSP90α氨基酸三维结构存在显著差异,东方田鼠体内不同组织间HSP90α表达丰度存在明显差异。与小鼠相比,同种组织间HSP90α的表达情况也完全不同。体外、体内实验结果显示该基因有着显著的抗日本血吸虫活性。为进一步研究其抗日本血吸虫机制奠定了良好的基础。
Microtus fortis has been proved to be a naturally resistant vertebrate host of Schistosoma japonicum by preventing completion of the parasite's life cycle both through epidemiological investigation and artificial Infection in lab. M. fortis sera were found to have anti-schistosome characteristic in vitro and transfered into mice. We have found that there was no significant difference between the tissues homogenate supernatant of M. fortis and those of the mice in schistosomula-killing effect in vitro. Then we constructed a cDNA library of M. fortis marrow and screened a gene fragment with anti-schistosomula function in some extent by expression cloning. In this study, a 331-bp clone gC 14.75 was screened from M. fortis marrow cDNA library by expression cloning. After searching with BLASTN at the NCBI database, we found cDNA gC 14.75 was homologous with HSP90a. Then we amplified the full-length of M. fortis HSP90a (Mf-HSP90a) and cloned it into the eukaryotic expression vector pcDNA1.1/Amp to test the schistosomula-killing effect of eukaryotic expression products in vitro. We transferred Mf-HSP90a by retroviral expression vector into mice to investigate its function in vivo. Results showed that it had significant effect of killing S. japonicum both in vitro and in vivo.
Chapter 1 Screening of resistance-associated gene against Schistosma japonicum gC14.75 from Microtus fortis
The cDNA expression library of M. fortis marrow was initially screened by testing the effects of conditioned media from transfected 293T cells for their capacity of killing schistosomula in vitro. Conditioned media (50%) of gene pool were added to schistosomula cultured with DMEM in vitro, observed the schistosomula in 96 h. The number of the schistosomula was counted in 96 h and compared with control group, calculated death rate of each group of the experiment. The highest death rate of the 8 gene pools (from gA to gH) conditioned media is gC (10.9%) which was significant different from the negative control group (1.7%). Then further the screen as gene pool. Gene pool C was separated into 8 subpools (from gCl to gC8), in which gC1 has the highest schistosomula-killing rate of 11.5%. Then divided gC1 into 8 secondary subpools (from gC11 to gC18), tested the schistosomula-killing effect of their conditioned media, we screened the highest schistosomula-killing secondary subpool gC14 with killing rate of 15.9% compared to 0.3% of the negative control. Thirty-two clones with different inserted fragments were selected from the gC14 with the highest schistosomula death rate, and then tested the schistosomula-killing capability in vitro of their conditioned media. A clone (gC 14.75) with the most significant schistosomula-killing effect was selected and sequenced by standard techniques.
Chapter 2 Cloning and functional analysis of the full-length of gene gC14.75
After searching with BLASTN at the NCBI database, we found cDNA gC 14.75 was homologous with HSP90α. It had 94% identities with Cricetulus griseus HSP90α(L33676), the identities lied between 1042-1373 neucleotides of C. griseus HSP90α. HSP90s are the most phylogenetically conserved proteins present in all prokaryotes and eukaryotes. So we designed primers according to gC 14.75 sequence and C. griseus HSP90α, tried to amplify the full-length of gC14.75. The amplication product was cloned into pGEM-T easy and sequenced it to ascertain its sequence. Searching with BLASTN at NCBI database indicated that it was a homologue of HSP90α, we named it Mf-HSP90α. Bioinformatics analysis results showed there were no significant difference of HSP90αbetween M. fortis and other species in nucleotide and amino acid sequence. The stereochemical structure of HSP90αbetween M. fortis and mouse was significant different. We cloned HSP90αof M. fortis (Mf-HSP90α) and mouse (Ms-HSP90α) into eukaryotic expression vector pcDNA1.1/Amp to test their schistosomula-killing effect of the eukaryotic expression products in vitro, respectively. The schistosomula-killing rate of Mf-HSP90αwas 20.3% while the schistosomula-killing rate of Ms-HSP90αwas 4.4%. There was significant difference between them. To identify the anti-schistomosome mechanism of Mf-HSP90α, we analyzed the expression abundance of the tissues by RT-PCR and Western-blot in M. fortis and mouse. The expression levels were different among tissues in each kind of animal and there was difference between two kinds of species, too.
Chapter 3 Anti-schistosome effects of Mf-HSP90αon mice infected with Schistosoma japonicum and analysis of the expression levels
To confirm the anti-schistosome function of Mf-HSP90α, we transfered it into mice infected with Schistosoma japonicum by retroviral expression vector pLXSN. Cloned Mf-HSP90αinto pLXSN, and packaged recombinant virus with PA317 cells. Infecting NIH/3T3 cells cultured with polybrene in six 10-fold serial dilutions and selected by G418 to determine virus titers. The titers of pLXSN and pLXSN-HSP90a retrovirus were 1.6×107 cfu/ml and 4×106 cfu/ml respectively. Mice infected with Schistosoma japonicum were administrated systemicly with DMEM, pLXSN and pLXSN-HSP90αretrovirus respectively. In all treatments, adult worms were quantified after perfusion of mice 42 d after infection. Worms recovered from pLXSN-HSP90αretrovirus treatment were stunted relative to worms from DMEM and pLXSN retrovirus treatment. Worm burden and egg reduction of pLXSN-HSP90αretrovirus treatment were statistically different from those of DMEM and pLXSN retrovirus treatment (P<0.05). The expression levels of HSP90αwere different among tissues and between DMEM and pLXSN retrovirus treatment in mice.
In summary, we screened the Mf-HSP90oαgene against Schistosoma japonicum from Microtus fortis by expression cloning, analyzed its bioinformatical structure and function, and compared the expreeion levels among tissues and between Microtus fortis and mouse in vivo. And then we testified its anti-schistosome function in vitro and in vivo. All the results suggest Mf-HSP90αas a novel anti-schistosome molecule.
一、东方田鼠抗日本血吸虫抗性相关基因gC14.75的克隆筛选
本研究构建了东方田鼠骨髓基因表达文库,根据表达产物的体外杀日本血吸虫童虫活性应用表达克隆法对文库进行筛选：基因表达文库随机分成8个基因池,相应质粒瞬时转染293T细胞,收集48h转染上清(条件培养基),进行体外杀日本血吸虫童虫实验,设立阴性对照：pcDNA1.1/Amp空载体转染293T细胞培养上清；阳性对照：20%东方田鼠血清。条件培养基杀虫率最高的基因池进入下一轮筛选,三轮筛选后获得杀虫效果最好的次级亚基因池gC14。gCl4质粒铺琼脂糖平板,随机挑取单个克隆,根据单克隆插入片段大小的不同,挑选32个进行表达产物杀虫实验,最终获得体外杀虫效果最好的单个克隆gC14.75。基因池条件培养基体外杀童虫率最高一组为gC,童虫死亡率为10.9%,与对照组相比P<0.05,差异具有统计学意义。亚基因池gC1、次级亚基因池gC14条件培养基的杀虫率分别为11.5%、15.9%,与对照组相比P<0.05,差异具有统计学意义。单克隆筛选中体外杀虫率最高的克隆是75号,杀虫率为11.0%。将克隆质粒DNA送Invitrogen公司测序获得抗性基因EST,长度为331 bp,将其命名为gC14.75。通过与GenBank数据库中序列进行同源性比较,发现gC14.75为HSP90α的同源序列。
二、gC14.75基因全长的克隆及其功能分析
HSP90是一个高度保守的基因家族。以gC14.75序列及与其同源性最高的中国仓鼠全长HSP90αcDNA (L33676)序列为模板,分别设计引物,RT-PCR扩增获得gC14.75全长序列。将全长序列测序后,用NCBI核苷酸数据库BLASTN进行序列比对分析,结果显示该基因与HSP90α同源,故将其命名为Mf-HSP90α。进而通过生物信息学方法分析该基因结构及功能。比对分析几种不同物种HSP90α的核苷酸及氨基酸序列,发现氨基酸、核苷酸序列差异很小。为了寻找可能存在的抗日本血吸虫功能差异的依据,我们用3D-JIGSAW (version 2.0)分析比较了东方田鼠与小鼠氨基酸序列的立体结构,结果显示二者在立体结构上存在显著差异。分别将Mf-HSP90α及小鼠HSP90α(Ms-HSP90α)重组到真核表达载体pcDNA1.1/Amp,制备相应条件培养基,进行体外杀日本血吸虫童虫实验。扣除空白对照组童虫死亡率,Mf-HSP90α条件培养基杀虫率为20.3%,与对照组相比较杀虫效果显著,而Ms-HSP90α条件培养基杀虫率仅为4.4%,与Mf-HSP90α相比较存在显著差异。为了进一步探讨东方田鼠与小鼠HSP90α抗虫功能差异的机制。我们进一步从mRNA、蛋白质水平分别用RT-PCR、Western-blot方法比较分析了HSP90α在东方田鼠及小鼠体内不同组织的表达情况,发现同一物种体内不同组织间HSP90α的表达存在显著差异,同一种组织在东方田鼠与小鼠间的表达情况也明显不同。
三、小鼠体内Mf-HSP90α的抗日本血吸虫作用及表达情况分析
为了进一步确认Mf-HSP90α的抗日本血吸虫活性,我们采用了基因转移技术,将该基因导入血吸虫易感动物(小鼠)体内,通过对小鼠模型的抗日本血吸虫效果的考察评估Mf-HSP90α的体内抗虫作用。本实验应用逆转录病毒载体pLXSN,构建pLXSN-HSP90α重组逆转录病毒载体系统,通过小鼠尾静脉注射将重组病毒导入小鼠体内,检测重组病毒治疗后感染小鼠的减虫率、肝脏减卵率、血吸虫虫体改变及小鼠肝脏肉芽肿减少情况。重组病毒载体经PA317细胞包装后,大量收集病毒悬液,经生物学方法检测病毒滴度：pLXSN病毒滴度为1.6×107cfu/ml pLXSN-HSP90α病毒滴度为4×106cfu/ml。分别用pLXSN、pLXSN-HSP90α病毒各200μl／次,共3次经尾静脉注射治疗日本血吸虫感染小鼠,空白对照组注射等体积DMEM。pLXSN- HSP90α重组病毒处理组检获成虫相比DMEM及pLXSN病毒组均要短小,检获成虫数也明显少于两对照组,减虫率与DMEM及pLXSN病毒组相比较分别是40.8%和32.3%。选取具有代表性肝脏制成石蜡切片,进行HE染色,镜下可以观察到pLXSN-HSP90α重组病毒处理组虫卵肉芽肿明显少于两对照组,与肉眼观察肝脏表面结果相同。pLXSN-HSP90α重组病毒处理组LEPG与DMEM及pLXSN病毒组相比,pLXSN-HSP90α重组病毒处理组的减卵率分别是57.9%和49.2%。减虫率、减卵率DMEM与pLXSN病毒组相比P>0.05,差异无统计学意义,pLXSN-HSP90α重组逆转录病毒组与DMEM及pLXSN病毒相比P<0.05,差异有统计学意义。为了探讨Mf-HSP90α抗日本血吸虫功能的机制,我们用RT-PCR的方法分析比较了感染尾蚴7天DMEM, pLXSN-HSP90α重组逆转录病毒不同处理组小鼠体内几种组织HSP90α表达情况,通过对RT-PCR条带灰度扫描,统计发现DMEM处理组小鼠肌肉HSP90α的表达丰度很低,其他组织表达丰度适中,组织间差异不大。pLXSN-HSP90α重组逆转录病毒处理组小鼠肺、脑、骨髓3种组织HSP90α的表达丰度相比其他几种组织明显要高,也明显高于与DMEM处理组肺、脑及骨髓的表达丰度也明显要高。
综上所述,本研究应用表达克隆法从东方田鼠骨髓基因表达文库筛选到一个日本血吸虫抗性相关基因Mf-HSP90α。东方田鼠与小鼠HSP90α氨基酸三维结构存在显著差异,东方田鼠体内不同组织间HSP90α表达丰度存在明显差异。与小鼠相比,同种组织间HSP90α的表达情况也完全不同。体外、体内实验结果显示该基因有着显著的抗日本血吸虫活性。为进一步研究其抗日本血吸虫机制奠定了良好的基础。
Microtus fortis has been proved to be a naturally resistant vertebrate host of Schistosoma japonicum by preventing completion of the parasite's life cycle both through epidemiological investigation and artificial Infection in lab. M. fortis sera were found to have anti-schistosome characteristic in vitro and transfered into mice. We have found that there was no significant difference between the tissues homogenate supernatant of M. fortis and those of the mice in schistosomula-killing effect in vitro. Then we constructed a cDNA library of M. fortis marrow and screened a gene fragment with anti-schistosomula function in some extent by expression cloning. In this study, a 331-bp clone gC 14.75 was screened from M. fortis marrow cDNA library by expression cloning. After searching with BLASTN at the NCBI database, we found cDNA gC 14.75 was homologous with HSP90a. Then we amplified the full-length of M. fortis HSP90a (Mf-HSP90a) and cloned it into the eukaryotic expression vector pcDNA1.1/Amp to test the schistosomula-killing effect of eukaryotic expression products in vitro. We transferred Mf-HSP90a by retroviral expression vector into mice to investigate its function in vivo. Results showed that it had significant effect of killing S. japonicum both in vitro and in vivo.
Chapter 1 Screening of resistance-associated gene against Schistosma japonicum gC14.75 from Microtus fortis
The cDNA expression library of M. fortis marrow was initially screened by testing the effects of conditioned media from transfected 293T cells for their capacity of killing schistosomula in vitro. Conditioned media (50%) of gene pool were added to schistosomula cultured with DMEM in vitro, observed the schistosomula in 96 h. The number of the schistosomula was counted in 96 h and compared with control group, calculated death rate of each group of the experiment. The highest death rate of the 8 gene pools (from gA to gH) conditioned media is gC (10.9%) which was significant different from the negative control group (1.7%). Then further the screen as gene pool. Gene pool C was separated into 8 subpools (from gCl to gC8), in which gC1 has the highest schistosomula-killing rate of 11.5%. Then divided gC1 into 8 secondary subpools (from gC11 to gC18), tested the schistosomula-killing effect of their conditioned media, we screened the highest schistosomula-killing secondary subpool gC14 with killing rate of 15.9% compared to 0.3% of the negative control. Thirty-two clones with different inserted fragments were selected from the gC14 with the highest schistosomula death rate, and then tested the schistosomula-killing capability in vitro of their conditioned media. A clone (gC 14.75) with the most significant schistosomula-killing effect was selected and sequenced by standard techniques.
Chapter 2 Cloning and functional analysis of the full-length of gene gC14.75
After searching with BLASTN at the NCBI database, we found cDNA gC 14.75 was homologous with HSP90α. It had 94% identities with Cricetulus griseus HSP90α(L33676), the identities lied between 1042-1373 neucleotides of C. griseus HSP90α. HSP90s are the most phylogenetically conserved proteins present in all prokaryotes and eukaryotes. So we designed primers according to gC 14.75 sequence and C. griseus HSP90α, tried to amplify the full-length of gC14.75. The amplication product was cloned into pGEM-T easy and sequenced it to ascertain its sequence. Searching with BLASTN at NCBI database indicated that it was a homologue of HSP90α, we named it Mf-HSP90α. Bioinformatics analysis results showed there were no significant difference of HSP90αbetween M. fortis and other species in nucleotide and amino acid sequence. The stereochemical structure of HSP90αbetween M. fortis and mouse was significant different. We cloned HSP90αof M. fortis (Mf-HSP90α) and mouse (Ms-HSP90α) into eukaryotic expression vector pcDNA1.1/Amp to test their schistosomula-killing effect of the eukaryotic expression products in vitro, respectively. The schistosomula-killing rate of Mf-HSP90αwas 20.3% while the schistosomula-killing rate of Ms-HSP90αwas 4.4%. There was significant difference between them. To identify the anti-schistomosome mechanism of Mf-HSP90α, we analyzed the expression abundance of the tissues by RT-PCR and Western-blot in M. fortis and mouse. The expression levels were different among tissues in each kind of animal and there was difference between two kinds of species, too.
Chapter 3 Anti-schistosome effects of Mf-HSP90αon mice infected with Schistosoma japonicum and analysis of the expression levels
To confirm the anti-schistosome function of Mf-HSP90α, we transfered it into mice infected with Schistosoma japonicum by retroviral expression vector pLXSN. Cloned Mf-HSP90αinto pLXSN, and packaged recombinant virus with PA317 cells. Infecting NIH/3T3 cells cultured with polybrene in six 10-fold serial dilutions and selected by G418 to determine virus titers. The titers of pLXSN and pLXSN-HSP90a retrovirus were 1.6×107 cfu/ml and 4×106 cfu/ml respectively. Mice infected with Schistosoma japonicum were administrated systemicly with DMEM, pLXSN and pLXSN-HSP90αretrovirus respectively. In all treatments, adult worms were quantified after perfusion of mice 42 d after infection. Worms recovered from pLXSN-HSP90αretrovirus treatment were stunted relative to worms from DMEM and pLXSN retrovirus treatment. Worm burden and egg reduction of pLXSN-HSP90αretrovirus treatment were statistically different from those of DMEM and pLXSN retrovirus treatment (P<0.05). The expression levels of HSP90αwere different among tissues and between DMEM and pLXSN retrovirus treatment in mice.
In summary, we screened the Mf-HSP90oαgene against Schistosoma japonicum from Microtus fortis by expression cloning, analyzed its bioinformatical structure and function, and compared the expreeion levels among tissues and between Microtus fortis and mouse in vivo. And then we testified its anti-schistosome function in vitro and in vivo. All the results suggest Mf-HSP90αas a novel anti-schistosome molecule.
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