鸭瘟病毒蛋白质组二维电泳特性和UL24基因的发现、原核表达及应用研究
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摘要
鸭瘟(Duck Plague,DP)又称鸭病毒性肠炎(Duck Viral Enteritis,DVE),是由鸭瘟病毒(Duck Plague Virus,DPV)引起的常见于鸭、鹅等雁行目禽类的一种急性败血性的接触性传染病,具有较高的发病率和死亡率,是目前水禽养殖业的重要传染病之一。本文通过超速离心获得高纯度的鸭瘟病毒粒子免疫动物制备高免血清,特异与二维电泳分离的病毒蛋白质组印迹而获得单一病毒蛋白,辅以高效液相色谱——四极杆串联电喷雾质谱解析多肽氨基酸序列,以此设计兼并引物扩增鸭瘟病毒基因组,扩增片段进一步杂交验证、分析为鸭瘟病毒UL24(DPV-UL24)基因。通过构建DPV-UL24原核表达质粒并诱导表达,从而对表达蛋白免疫原性、表达蛋白抗体介导的免疫组化和免疫荧光检测人工感染DPV后UL24基因在体内的表达时相与蛋白定位分析、表达蛋白捕获DPV抗体和表达蛋白抗体捕获抗原ELISA方法的研究以及建立了基于DPV-UL24基因检测DPV的PCR方法,结果如下:
1.鸭瘟病毒超微结构研究结果表明:病毒粒子具有疱疹病毒典型形态结构,剖面六角外观轮廓清楚,成熟病毒粒子直径约150～266nm,病毒囊膜、核衣壳和核心清晰可见;囊膜外层较内层着色略深,且可见尚未形成完整囊膜的柄状拖尾结构;多数病毒粒子以单核衣壳为主,一定数量的病毒粒子具有双核衣壳,偶见三核衣壳,核衣壳直径为100～150nm,呈现致密圆形、半圆形或马蹄形等类型;在核衣壳外和囊膜之间可见明显的亮晕;核心DNA电子染色较深,集中分布,直径40～65nm。并且以纯净的病毒粒子免疫成年鸭获得了抗鸭瘟病毒高免血清。
2.鸭瘟病毒蛋白质组研究显示:样品经丙酮处理、30mMDTT水化浓度、pH5-8 IPG固相胶条、混合两性载体电解质、0.8mg的上样量,蛋白分离效果好,获1253个银染蛋白点或388个考染蛋白点,不同时期2-DE分离胶蛋白点匹配率达88%,建立鸭瘟病毒蛋白质组二维电泳模型重复性好、稳定、可靠。以此进行蛋白免疫印迹,结果表明至少可以检测到32种免疫印迹蛋白多肽点,其中有5种非特异性蛋白多肽,在27种DPV蛋白多肽中,相对分子量主要集中在20KD～120KD之间,等电点主要位于pH5.30—pH7.50范围。对选择两个病毒蛋白点高效液相色谱——四极杆串联电喷雾质谱鉴定、解析多肽序列,与病毒蛋白库比对获得了七段可信氨基酸肽段序列。
3.鸭瘟病毒UL24基因的发现、原核表达、产物纯化及其抗体制备:以鸭瘟病毒基因组为模板,氨基酸肽段设计的兼并引物能够扩增出两条(420bp、730bp)片段,其中730bp片段能够与不同限制性内切酶酶切DPV基因组电泳片段杂交,形成大小不等杂交条带,从而验证了该片段为鸭瘟病毒基因组片段;生物信息学分析,该片段与疱疹病毒基因序列同源性最高,表明了扩增基因片段为鸭瘟病毒UL24基因;进一步研究还表明该基因全序列为具有1230bp开放阅读框核酸序列,与26株同属疱疹病毒氨基酸序列具有较高的相似性,与MDV-1亲缘性最近,该基因编码409个氨基酸残基,有16个抗原决定簇,不含有信号肽,在第56位、284位、294位、325位和383位存在5个潜在的N-糖基化位点,跨膜螺旋预测为外膜蛋白。
根据DPV-UL24序列,设计一对特异性引物,从鸭瘟病毒基因组中扩增目标基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭瘟病毒UL24基因正向插入原核表达载体pET-32a(+)的EcoRⅠ和XhoI位点间,成功构建了重组表达质粒pET32a(+)/DPV-UL24。重组表达质粒pET32a(+)/DPV-UL24转化表达宿主菌BL21(DE3),用IPTG诱导,能表达出了大小约为38kD的UL24重组蛋白,与预期表达蛋白分子量大小相符;经对不同诱导时间及诱导剂IPTG浓度等条件进行优化,确定重组质粒pET32a(+)/DPV-UL24的最佳诱导条件为0.2mmol/L IPTG、37℃条件下诱导5h。表达产物用镍柱亲和层析纯化、梯度透析复性后与等量弗氏佐剂混合制备UL24重组蛋白疫苗,四次免疫家兔,获得的兔抗UL24高免血清经辛酸-硫酸铵粗提后,High Q阴离子交换柱层析纯化抗体IgG。
4.鸭瘟病毒UL24基因原核表达产物免疫原性研究:制备的UL24重组蛋白疫苗免疫7日龄雏鸭,并在免疫后3、5、7、10、14、21、28、35、42、49、56、70、84、98d分别血清检测ELISA效价和中和效价,并于免疫后21d将其中一半进行保护试验。结果显示:在免疫后28d重组蛋白ELISA抗体效价OD_(450nm)达到最高1:5120,弱毒疫苗组达1:10240,显著高于对照组(P≤0.05),此后两个月抗体效价均维持较高水平,其消长规律一致。中和试验也表明在免疫后21～28d,重组蛋白组和弱毒免疫组中和效价分别可达1:128、1:256以上,其抗体消长规律与ELISA检测结果相似。重组蛋白免疫保护组发病率50%、死亡率30%,较对照组鸭的发病率100%、死亡率100%明显减少,与弱毒疫苗免疫组(发病率30%、死亡率10%)比较差异不显著。表明DPV-UL24重组蛋白对鸭瘟病毒强毒感染具有一定的保护能力。
5.UL24基因在人工感染鸭瘟病毒鸭组织中的表达时相与定位:采用鸭瘟病毒强毒CHv株(DPV-CHv)人工方法感染28日龄鸭,攻毒后于不同时间采集脾脏、胰腺、哈氏腺、法氏囊、肝脏、肠道、肾脏、肺脏、心、脑等组织或器官,经建立的DPV-UL24基因表达蛋白抗体间接免疫组化方法,检测DPV-UL24基因在感染鸭体内的表达时相、侵染过程与定位分布。结果显示:感染后2h可在脾脏、胸腺、肝脏、十二指肠中检测到DPV-UL24基因编码蛋白抗原:攻毒4h时,哈氏腺、腺胃可检测到DPV-UL24基因表达蛋白,8h时在回肠、肺脏,12h在法氏囊和肾脏,24h时在胰腺和肠道各段均呈现阳性反应。通过对鸭瘟病毒蛋白与UL24基因编码蛋白的侵染过程与定位比较发现,免疫器官与肠道黏膜既是病毒,也是UL24基因编码蛋白的靶器官或靶组织,但UL24基因编码蛋白在脑组织嗜性低,分析表明该蛋白可能是膜蛋白,具有运输核浆与引导病毒组分进入核内参与病毒复制的功能。
同时,建立的DPV-UL24基因表达蛋白抗体介导的间接免疫荧光方法,也对UL24基因在鸭体组织中的表达时相检测,并对该基因表达蛋白侵染与定位分布进行分析。结果显示:感染鸭瘟病毒强毒2h时,UL24基因即可在脾脏、胸腺、法氏囊、哈德氏腺等免疫器官和肝脏、肺脏、腺胃及大部分肠段编码蛋白,4h时,在脑、心肌和胰腺也检测出UL24基因编码蛋白,8h时在食道粘膜层表达蛋白荧光阳性反应。研究表明:脾脏、胸腺、法氏囊、哈氏腺免疫器官和肠道粘膜也是UL24基因编码蛋白主要的靶器官或靶组织,且在靶器官的侵染与分布具有一定选择性,对粘膜上皮细胞损害严重。26份鸭瘟组织检验,检出25份阳性,阳性率为96.1%(25/26),表明免疫荧光检测石蜡切片中DPV-UL24基因编码蛋白的方法灵敏、特异性强。
6.抗鸭瘟病毒UL24基因原核表达蛋白抗体介导的ELISA检测DPV抗原:通过包被兔抗鸭瘟病毒UL24基因原核表达蛋白抗体,以鸭抗DPV-UL24抗体作为夹心抗体,建立并优化DPV抗原捕获ELISA方法,结果表明:兔抗DPV-UL24抗体浓度为5.0μg/100μL,鸭抗DPV-UL24抗体浓度为9.0μg/100μL,酶标抗体浓度为1:2000时获得最适比例稀释度;对乙型肝炎病毒(DHBV)等非鸭瘟病毒感染其它鸭源病原体阳性血清检验均呈现阴性反应,酶标板板间、板内检测变异系数均小于10%,且可检测出46ng纯化的鸭瘟病毒含量,表明了AC-ELISA具有良好的特异、敏感、稳定性。对人工感染DPV强毒后,病毒在雏鸭肝、脾脏、脑、食道、肺、肾等各组织器官的分布与增殖检测与免疫组化、免疫荧光检测UL24基因编码蛋白结果相似。
7.抗鸭瘟病毒UL24基因原核表达蛋白ELISA检测DP抗体:以包被重组DPV-UL24蛋白作为抗原检测DPV抗体研究表明:重组表达蛋白包被浓度为1:80(2.5μg/100μL),被检血清稀稀释倍数为1:320,酶标二抗1:2000时为最适稀释比例,建立的间接ELISA方法检测鸭乙肝、鸭疫里默氏菌病等阳性血清均为阴性,板内或板间重复试验显示变异系数均小于7%,能检出经1:2560倍稀释的鸭瘟阳性血清,对127份鸭临床疫病血清检测,阳性检出率为77.2%,与包被全病毒检测DPV试剂盒相比,符合率为92.5%。
8.基于鸭瘟病毒UL24基因PCR方法建立和应用:根据本研究获得的DPV-UL24基因序列设计一对引物(P1/P2),对DPV基因组DNA进行扩增。结果显示:能扩增出与预期(585bp)大小一致的片段,对鸭病毒性肝炎病毒、鸭乙型肝炎病毒等病原体核酸提取物均不能扩增出任何条带;且DPV-UL24基因引物P1/P2对DPV基因组DNA检测灵敏度可达到1pg;对14份DPV强毒感染鸭病料进行检测,均扩增出大小一致的特异片段,5份未感染DPV鸭病料扩增结果均为阴性。结果表明,鸭瘟病毒UL24基因PCR方法可靠、灵敏,可应用于临床鸭瘟的诊断与监测。
通过对建立的基于鸭瘟病毒UL24基因间接免疫组化、免疫荧光、AC-ELISA和PCR方法的特异性、敏感性和对鸭瘟病料检测结果的比较分析发现,四种方法对鸭瘟阳性病料检测为阳性,对未感染鸭瘟病毒病料、空白对照、山羊血清和鸭源性其它病原体检测均为阴性,说明了四种检测病毒抗原方法均具有良好的特异性;人工感染DPV后分别对鸭组织检测显示,感染4h在脾脏、胸腺、肝脏、十二指肠等部位均呈现阳性反应,而免疫组化方法在感染24h时几乎在全身组织中均有UL24基因蛋白表达,免疫荧光与PCR方法则需12h就呈现阳性反应;AC-ELISA在感染24h对所检测的11份样本均能检出,临床鸭瘟病料检测显示,免疫组化阳性检测率88.4%、免疫荧光阳性检测率96.1%、抗原捕获ELISA阳性检测率为84.6%,PCR阳性率则为100%。
Duck plague(DP),alternatively known as duck virus enteritis(DVE),is an acute, contagious and lethal septicemic disease of waterfowls such as ducks and geese in the group of Anseriformes,which can cause high morbidities and mortalities in birds and poses an important threat to waterfowl product industry.
Certain DPV protein was obtained by means of two-dimensional electrophoresis and was verified by immunoblotting with the anti-DPV hyperimmune antiserum produced with the highly purified DPV preparation obtained by ultracentrifugation.The polypeptide sequences of this protein were analyzed by means of high performance liquid chromatography(HPLC) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry.Duck plague virus UL24(DPV-UL24) gene was isolated from the DPV genome by degenerative PCR with primers designed according the polypeptide sequence of the protein and verified by hybridization test.DPV-UL24 gene prokaryotic expression recombinant vector was constructed and expressed after induction.Based on the above research,the following studies were undertaken:lmmunogenicity test of the expressed DPV-UL24 protein;UL24 gene dynamic distribution and localization in DPV infected ducks by the expressed DPV-UL24 protein mediated immunohistochemistry and immunofluorescence test;Antigen capture enzyme-linked immunoabsorbent assay(ELISA) for DPV specific antibody and DPV-UL24 protein antibody capture ELISA for DPV specific antigen;Establishment of PCR detection method by DPV UL24 gene.These studies are summarized as follows:
1.Ultrastructure studies showed that DEV-CHv virions have the typical morphological characteristics of herpesvirus.The cross sections of the virions were hexagonal and the mature virus with the structure of envelope,nucleocapsid and nucleus were spherical in shape with diameter of 150nm-266nm.The outer part of the envelope was somewhat densely stained than the inner part and some virions with envelop not fully formed were also observed.Most of the DEV virions have one nuclocapsids,some DEV virions have two or three nucleocapsids.The nucleocapsid of DEV was round in shape with diameter of 100nm-150nm,some densely electron-stained vires-related structures which were circle-shaped,semicircle-shaped,or U-shaped could be observed.Between the nucleocapsid and the envelop was certain electron slightly stained part;The DNA in the nucleus which was 40nm-65nm in diameter was slightly electron stained and was in cluster in appearance.Hyperimmune antiserum against DPV was produced by hyperimmunization of the mature ducks with the highly purified DPV particles.
2.Research on duck plague virus proteinomics demonstrated that the following conditions favored the experiment:protein sample extracted by acetone,pH 5-8 gel strip, 30mM DTT reducing agent,carder ampholytes mixturation of loading sampling buffer,0.8 mg protein sample;1253 or 388 protein spots could be obtained respectively by coomassie brilliant blue staining and silver staining.The mean matching rate of the protein spots could reach 88.%each different time.The established model of two-dimenshional electrophoresis was stable,reliable and has a good reproducibility.At least 32 polypeptide spots could be detected by immunoblotting test and five of 32 polypeptide spots were nonspecific.The molecular weights of the 27 DPV polypeptides range from 20KD to 120 KD and the isoelectfic points range from pHS.30 to pH7.50.Two viral protein spots were selected for polypeptide sequence analysis by means of high performance liquid chromatography(HPLC) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry and seven reliable segments sequence were selected after aligning with the virus protein database.
3.The discovery,prokaryotic expression,purification of products and antibody preparation of DPV UL24 gene:Two PCR products of 500 bp and 730 bp were obtained after amplifying the DPV genome with degenerative primers designed according to the polypeptide sequence.The 730 bp PCR product was verified to be duck genome specific by hybridization with DPV genome fragments processed with different restriction enzyme and forming the hybridization bands of different length.Biostatistics analysis showed that the 730 bp fragment has a maximum homology with herpesvirus genome sequence and proved that the amplified product was the DPV UL24 gene.Further analysis showed that the UL24 gene has a 1230 bp open reading flame(ORF) and is highly homologous with 11 strains of herpesvirus and is closest with Mark's virus(MDV).The UL24 protein is a conserved transmembrane protein relatively hydrophobic,with theα-helix and random coil secondary structure contents both being 46.94%.The UL24 gene with its maximum amino acids content being alanine and arginine(8.3%) encodes 409 amino acids,has 16 antigenic determinants,19 phosphorylation sites and does not have signal peptide sequence.Five potential N-glycosylation sites were spotted at the sites of 56,284,294,325 and 383 respectively.The UL24 protein is highly immunogenic,with its encoding gene amino acids codes frequency Nc value being 56.9.Twelve amino acids including alanine are of highly codon usage bias.
DPV-UL24 gene was isolated from DPV genome by PCR with the specific primers designed according to the DPV-UL24 gene sequence.DPV-UL24 gene was subcloned into the vector of pMD18-T and after verification by means of PCR,restriction endonuclease restriction analysis and gene sequencing it was then cloned into the site between the restriction sites of EcoRⅠand XhoI of the pET-32a(+) prokaryotic expression vector and thus the recombinant expression plasmid of pET32a(+)/DPV-UL24 was constructed.The recombinant expression plasmid of pET32a(+)/DPV-UL24 was transformed into the host E.coli of BL21(DE3) and the approximately 38-kD UL24 recombinant protein was obtained after induction with isopropylβ- D -1-thiogalactopyranoside(IPTG),whose molecular weight basically conformed to what was predicted.Through optimization test it was found that the optimal condition was 0.2mmol/L 1PTG with an induction duration of 5 hours.The expressed protein was purified and concentrated by Ni-column affinity chromatography and after renaturation was emulsified with an equal volume of Freund's adjuvant.This product was used as vaccine to prepare hyperimmune sera with rabbits by immunization for four times.Purification of IgG fractions from antiserum was carried out by ion exchange chromatography on Econo-Pac high Q Cartridge after extraction through caprylic acid and equilibrium ammonium sulfate.
4.The study on immunogenicity of prokaryotic expression product of DPV UL24 gene: The prepared UL24 recombinant vaccine was used to vaccinate 7 day old duckling and the serum collected respectively at 3,5,7,10,14,21,28,35,42,49,56,70,84 and 98 days postvaccination underwent ELISA titer and serum neutralization titer evaluation.Half of the ducks underwent challenge test 21 days postimmunization.The result demonstrated that 28 days after immunization,the greatest ELISA titer of 1:5120 at OD_(450nm) could be obtained in the group of recombinant protein group and the greatest ELISA titer of 1:10240 at OD_(450nm) could be obtained in the group of avirulent DPV vaccine group,which are both prominently higher than that of the control group(P≤0.05).Relatively high antibody levels were maintained in the two groups with a same dynamic change of decline. The antibody neutralization test showed that in the recombinant protein group and in the avirulent DPV group,neutralization antibody titer over 1:128 and 1:256 could be obtained 21 to 28 days postvaccination.The neutralization antibody and the ELISA antibody underwent a similar dynamic change in the two group.Analysis indicated that between the recombinant protein group(with its morbidity rate and mortality rate being 50%and 30% respectively) and the avirulent DPV vaccine group(with its morbidity rate and mortality rate being 30%and 10%respectively) no differences of morbidity rate and mortality rate were obtained but between the recombinant protein group and the control group(with its morbidity rate and mortality rate both being 100%) distinct differences of morbidity rate and mortality rate were obtained.All these studies demonstrated that the DPV-UL24 recombinant protein could confer duckling certain protect effects after immunization.
5.The phase and location of UL24 gene express in artificial infected ducklings:The dynamic expression,infection process and distribution of DPV-UL24 gene in infected ducklings were studied by the established DPV-UL24 recombinant protein indirect immunohistochemistry method with the specimens of spleen,pancreas,harderian glands, bursa of Fabricius,liver,intestine,kidney,lung,heart,encephalon of the ducklings challenged at 28 days old with duck plague virulent DPV-CHv strain.The results demonstrated that DPV-UL24 encoding protein antigens could be detected in spleen, thymus,liver and duodenum 2 hour after challenge,in harderian glands and glandular stomach 4 hours after challenge,in ileum and lung 8 hours after challenge,in bursa of Fabricius and kidney 12 hours after challenge,in pancreas each part of intestine 24 hours after challenge.All these results demonstrated that organs of the immune system and the intestinal mucosa are the target organ and target tissue of DPV-UL24 encoding protein. DPV-UL24 encoding protein brain tissue tropism is very low,indicating that this protein is probably transmembrane protein and its function mainly related with the transportation and leading of virus components into nucleus for virion assembly during the virus proliferation.
The dynamic expression,proliferation and distribution of DPV-UL24 encoding protein in infected ducklings were studied by the established DPV-UL24 recombinant protein immunofluroscence detection method in ducks challenged with duck plague virus. The results demonstrated that DPV-UL24 encoding protein antigen could be detected in spleen,thymus,harderian glands,bursa of Fabricius,liver,lung,kidney and most parts of intestine 2 hour after challenge,in pancreas,encephalon and heart 4 hours after challenge, in esophagus 8 hours after challenge.All these results demonstrated that the immune organs of spleen,thymus,harderian glands,bursa of Fabricius and the intestinal mucosa are the target organ and target tissue of DPV-UL24 encoding protein.DPV-UL24 encoding protein was selective in the target organs in the process of virus invasion and distribution, causing serious damage to mucosal epithelium.Of the 26 specimens for inspection by this method,25 specimens were positive and positive rate was 96.1%,indicating that this method for DPV-UL24 encoding protein detection is sensitive and specific.
6.Research on Duck Plague Virus was Detected by Antigen Capture-ELISA with Anti-Prokaryotic Expression Protein of DPV UL24:Double antibody sandwich DPV antigen captured ELISA assay method is established and optimized with the rabbit anti-DPV UL24 prokaryotic expression protein antiserum and the duck anti- DPV UL24 prokaryotic expression protein antiserum.The results demonstrated that the optimized evaluation could be obtained when the rabbit anti DPV-UL24 antibody concentration is 5.0ug/100ul,the duck anti DPV-UL24 antibody concentration is 9.0ug/1O0ul and the enzyme linked antibody dilution is 1:2000.Other duck infected pathogens like duck hepatitis virus B(DHBV) was employed as negative controls and showed negative results. The coefficient s of variation of intra-assay and inter-assay was less than 10%and the minimum DPV content detection is 46 ng DPV purified virus,showing the specific, sensitive and stable characteristics of the established ELISA method.The dynamic proliferation and distribution of DPV-UL24 encoding protein in the specimens of liver, spleen,encephalon,esophagus,lung,and kidney of the infected ducklings were studied by this established ELISA method and the results were similar with that obtained by the established immunohistochemistry and immunoflurescence method.
7.Research on Duck Plague Virus Antibody was Detected by ELISA Using Coating Prokaryotic Expression Protein of DPV UL24:Double antibody sandwich DPV antigen captured ELISA assay method is established and optimized with DPV UL24 prokaryotic expression protein as antigen.The results demonstrated that the optimized evaluation could be obtained when the DPV-UL24 recombinant protein is 80-fold diluted with its concentration being 2.5ug/100ul,the dilution of the examined serum is 1:320 and the enzyme linked antibody dilution is 1:2000.Other duck infected pathogen specific positive antiserum like duck hepatitis virus B(DHBV) and duck Riemirella Anatipestifer(RA) were employed as negative controls and showed negative results.The coefficient s of variation of intra-assay and inter-assay was less than 7%and could detect DPV positive antiserum with a dilution of 1:2560.127 specimens from the clinically infected ducklings were studied by this established ELISA method with a positive rate of 77.2%and similar results were obtained with that obtained by the whole DPV antigen as coated antigen ELISA method.
8.Development and Application of PCR based on UL24 gene for the Detection of Duck plague virus:One pair of primer(P1/P2) was designed according to the DPV-UL24 genes sequence for amplification of DPV genome DNA.The results demonstrated that one PCR product of 585bp was obtained with this primers for DPV genome amplification but no products could be detected with this primer for genome amplification of other pathogen such as duck hepatitis virus and duck hepatitis virus B.The sensitivity of the primer of P1/P2 is lpg and the uniform PCR product fragment could be obtained for amplification of the 14 the specimens collected from DPV virulent virus infected ducks whereas no PCR product fragment could be obtained for amplification of the 5 the specimens collected from ducks not infected with DPV.These results demonstrated that the established UL24 PCR detection method is reliable,sensitivity and could be used in 1 the clinical diagnosis and surveillance of duck plague infection.
Comparisons and analysis of the established indirect immunohistochemistry, immunofluorescent,AC-ELISA and PCR detection methods indicates that positive results could be obtained by the four methods for examination of the DPV positive specimens, specimens not infected with DPV,whereas negative results could be obtained by the four methods for examination of blank control,goat serum and other duck pathogens.These examinations indicated that these four methods are well specific.The examinations of the specimens from the artificially infected ducks indicated that positive results could be obtained in the specimens of spleen,thymus,liver and duodenum collected 4 hours postinfection.Immunohistochemistry examination indicated that UL24 gene encoding protein could be detected in almost all the tissues 24 hours postinfection,whereas UL24 gene encoding protein could be detected by immunofluorescent and PCR detection methods 12 hours postinfection.UL24 gene encoding protein could be detected in the 11 specimens by AC-ELISA methods 24 hours postinfection.The examination of the clinically infected DPV infected specimens indicated that 88.4%positive results could be obtained by immunohistochemistry method,96.1%positive results could be obtained by immunofluorescent method,85.2%positive results could be obtained by AC-ELISA methods,100%positive results could be obtained by PCR methods.
1.鸭瘟病毒超微结构研究结果表明:病毒粒子具有疱疹病毒典型形态结构,剖面六角外观轮廓清楚,成熟病毒粒子直径约150～266nm,病毒囊膜、核衣壳和核心清晰可见;囊膜外层较内层着色略深,且可见尚未形成完整囊膜的柄状拖尾结构;多数病毒粒子以单核衣壳为主,一定数量的病毒粒子具有双核衣壳,偶见三核衣壳,核衣壳直径为100～150nm,呈现致密圆形、半圆形或马蹄形等类型;在核衣壳外和囊膜之间可见明显的亮晕;核心DNA电子染色较深,集中分布,直径40～65nm。并且以纯净的病毒粒子免疫成年鸭获得了抗鸭瘟病毒高免血清。
2.鸭瘟病毒蛋白质组研究显示:样品经丙酮处理、30mMDTT水化浓度、pH5-8 IPG固相胶条、混合两性载体电解质、0.8mg的上样量,蛋白分离效果好,获1253个银染蛋白点或388个考染蛋白点,不同时期2-DE分离胶蛋白点匹配率达88%,建立鸭瘟病毒蛋白质组二维电泳模型重复性好、稳定、可靠。以此进行蛋白免疫印迹,结果表明至少可以检测到32种免疫印迹蛋白多肽点,其中有5种非特异性蛋白多肽,在27种DPV蛋白多肽中,相对分子量主要集中在20KD～120KD之间,等电点主要位于pH5.30—pH7.50范围。对选择两个病毒蛋白点高效液相色谱——四极杆串联电喷雾质谱鉴定、解析多肽序列,与病毒蛋白库比对获得了七段可信氨基酸肽段序列。
3.鸭瘟病毒UL24基因的发现、原核表达、产物纯化及其抗体制备:以鸭瘟病毒基因组为模板,氨基酸肽段设计的兼并引物能够扩增出两条(420bp、730bp)片段,其中730bp片段能够与不同限制性内切酶酶切DPV基因组电泳片段杂交,形成大小不等杂交条带,从而验证了该片段为鸭瘟病毒基因组片段;生物信息学分析,该片段与疱疹病毒基因序列同源性最高,表明了扩增基因片段为鸭瘟病毒UL24基因;进一步研究还表明该基因全序列为具有1230bp开放阅读框核酸序列,与26株同属疱疹病毒氨基酸序列具有较高的相似性,与MDV-1亲缘性最近,该基因编码409个氨基酸残基,有16个抗原决定簇,不含有信号肽,在第56位、284位、294位、325位和383位存在5个潜在的N-糖基化位点,跨膜螺旋预测为外膜蛋白。
根据DPV-UL24序列,设计一对特异性引物,从鸭瘟病毒基因组中扩增目标基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将鸭瘟病毒UL24基因正向插入原核表达载体pET-32a(+)的EcoRⅠ和XhoI位点间,成功构建了重组表达质粒pET32a(+)/DPV-UL24。重组表达质粒pET32a(+)/DPV-UL24转化表达宿主菌BL21(DE3),用IPTG诱导,能表达出了大小约为38kD的UL24重组蛋白,与预期表达蛋白分子量大小相符;经对不同诱导时间及诱导剂IPTG浓度等条件进行优化,确定重组质粒pET32a(+)/DPV-UL24的最佳诱导条件为0.2mmol/L IPTG、37℃条件下诱导5h。表达产物用镍柱亲和层析纯化、梯度透析复性后与等量弗氏佐剂混合制备UL24重组蛋白疫苗,四次免疫家兔,获得的兔抗UL24高免血清经辛酸-硫酸铵粗提后,High Q阴离子交换柱层析纯化抗体IgG。
4.鸭瘟病毒UL24基因原核表达产物免疫原性研究:制备的UL24重组蛋白疫苗免疫7日龄雏鸭,并在免疫后3、5、7、10、14、21、28、35、42、49、56、70、84、98d分别血清检测ELISA效价和中和效价,并于免疫后21d将其中一半进行保护试验。结果显示:在免疫后28d重组蛋白ELISA抗体效价OD_(450nm)达到最高1:5120,弱毒疫苗组达1:10240,显著高于对照组(P≤0.05),此后两个月抗体效价均维持较高水平,其消长规律一致。中和试验也表明在免疫后21～28d,重组蛋白组和弱毒免疫组中和效价分别可达1:128、1:256以上,其抗体消长规律与ELISA检测结果相似。重组蛋白免疫保护组发病率50%、死亡率30%,较对照组鸭的发病率100%、死亡率100%明显减少,与弱毒疫苗免疫组(发病率30%、死亡率10%)比较差异不显著。表明DPV-UL24重组蛋白对鸭瘟病毒强毒感染具有一定的保护能力。
5.UL24基因在人工感染鸭瘟病毒鸭组织中的表达时相与定位:采用鸭瘟病毒强毒CHv株(DPV-CHv)人工方法感染28日龄鸭,攻毒后于不同时间采集脾脏、胰腺、哈氏腺、法氏囊、肝脏、肠道、肾脏、肺脏、心、脑等组织或器官,经建立的DPV-UL24基因表达蛋白抗体间接免疫组化方法,检测DPV-UL24基因在感染鸭体内的表达时相、侵染过程与定位分布。结果显示:感染后2h可在脾脏、胸腺、肝脏、十二指肠中检测到DPV-UL24基因编码蛋白抗原:攻毒4h时,哈氏腺、腺胃可检测到DPV-UL24基因表达蛋白,8h时在回肠、肺脏,12h在法氏囊和肾脏,24h时在胰腺和肠道各段均呈现阳性反应。通过对鸭瘟病毒蛋白与UL24基因编码蛋白的侵染过程与定位比较发现,免疫器官与肠道黏膜既是病毒,也是UL24基因编码蛋白的靶器官或靶组织,但UL24基因编码蛋白在脑组织嗜性低,分析表明该蛋白可能是膜蛋白,具有运输核浆与引导病毒组分进入核内参与病毒复制的功能。
同时,建立的DPV-UL24基因表达蛋白抗体介导的间接免疫荧光方法,也对UL24基因在鸭体组织中的表达时相检测,并对该基因表达蛋白侵染与定位分布进行分析。结果显示:感染鸭瘟病毒强毒2h时,UL24基因即可在脾脏、胸腺、法氏囊、哈德氏腺等免疫器官和肝脏、肺脏、腺胃及大部分肠段编码蛋白,4h时,在脑、心肌和胰腺也检测出UL24基因编码蛋白,8h时在食道粘膜层表达蛋白荧光阳性反应。研究表明:脾脏、胸腺、法氏囊、哈氏腺免疫器官和肠道粘膜也是UL24基因编码蛋白主要的靶器官或靶组织,且在靶器官的侵染与分布具有一定选择性,对粘膜上皮细胞损害严重。26份鸭瘟组织检验,检出25份阳性,阳性率为96.1%(25/26),表明免疫荧光检测石蜡切片中DPV-UL24基因编码蛋白的方法灵敏、特异性强。
6.抗鸭瘟病毒UL24基因原核表达蛋白抗体介导的ELISA检测DPV抗原:通过包被兔抗鸭瘟病毒UL24基因原核表达蛋白抗体,以鸭抗DPV-UL24抗体作为夹心抗体,建立并优化DPV抗原捕获ELISA方法,结果表明:兔抗DPV-UL24抗体浓度为5.0μg/100μL,鸭抗DPV-UL24抗体浓度为9.0μg/100μL,酶标抗体浓度为1:2000时获得最适比例稀释度;对乙型肝炎病毒(DHBV)等非鸭瘟病毒感染其它鸭源病原体阳性血清检验均呈现阴性反应,酶标板板间、板内检测变异系数均小于10%,且可检测出46ng纯化的鸭瘟病毒含量,表明了AC-ELISA具有良好的特异、敏感、稳定性。对人工感染DPV强毒后,病毒在雏鸭肝、脾脏、脑、食道、肺、肾等各组织器官的分布与增殖检测与免疫组化、免疫荧光检测UL24基因编码蛋白结果相似。
7.抗鸭瘟病毒UL24基因原核表达蛋白ELISA检测DP抗体:以包被重组DPV-UL24蛋白作为抗原检测DPV抗体研究表明:重组表达蛋白包被浓度为1:80(2.5μg/100μL),被检血清稀稀释倍数为1:320,酶标二抗1:2000时为最适稀释比例,建立的间接ELISA方法检测鸭乙肝、鸭疫里默氏菌病等阳性血清均为阴性,板内或板间重复试验显示变异系数均小于7%,能检出经1:2560倍稀释的鸭瘟阳性血清,对127份鸭临床疫病血清检测,阳性检出率为77.2%,与包被全病毒检测DPV试剂盒相比,符合率为92.5%。
8.基于鸭瘟病毒UL24基因PCR方法建立和应用:根据本研究获得的DPV-UL24基因序列设计一对引物(P1/P2),对DPV基因组DNA进行扩增。结果显示:能扩增出与预期(585bp)大小一致的片段,对鸭病毒性肝炎病毒、鸭乙型肝炎病毒等病原体核酸提取物均不能扩增出任何条带;且DPV-UL24基因引物P1/P2对DPV基因组DNA检测灵敏度可达到1pg;对14份DPV强毒感染鸭病料进行检测,均扩增出大小一致的特异片段,5份未感染DPV鸭病料扩增结果均为阴性。结果表明,鸭瘟病毒UL24基因PCR方法可靠、灵敏,可应用于临床鸭瘟的诊断与监测。
通过对建立的基于鸭瘟病毒UL24基因间接免疫组化、免疫荧光、AC-ELISA和PCR方法的特异性、敏感性和对鸭瘟病料检测结果的比较分析发现,四种方法对鸭瘟阳性病料检测为阳性,对未感染鸭瘟病毒病料、空白对照、山羊血清和鸭源性其它病原体检测均为阴性,说明了四种检测病毒抗原方法均具有良好的特异性;人工感染DPV后分别对鸭组织检测显示,感染4h在脾脏、胸腺、肝脏、十二指肠等部位均呈现阳性反应,而免疫组化方法在感染24h时几乎在全身组织中均有UL24基因蛋白表达,免疫荧光与PCR方法则需12h就呈现阳性反应;AC-ELISA在感染24h对所检测的11份样本均能检出,临床鸭瘟病料检测显示,免疫组化阳性检测率88.4%、免疫荧光阳性检测率96.1%、抗原捕获ELISA阳性检测率为84.6%,PCR阳性率则为100%。
Duck plague(DP),alternatively known as duck virus enteritis(DVE),is an acute, contagious and lethal septicemic disease of waterfowls such as ducks and geese in the group of Anseriformes,which can cause high morbidities and mortalities in birds and poses an important threat to waterfowl product industry.
Certain DPV protein was obtained by means of two-dimensional electrophoresis and was verified by immunoblotting with the anti-DPV hyperimmune antiserum produced with the highly purified DPV preparation obtained by ultracentrifugation.The polypeptide sequences of this protein were analyzed by means of high performance liquid chromatography(HPLC) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry.Duck plague virus UL24(DPV-UL24) gene was isolated from the DPV genome by degenerative PCR with primers designed according the polypeptide sequence of the protein and verified by hybridization test.DPV-UL24 gene prokaryotic expression recombinant vector was constructed and expressed after induction.Based on the above research,the following studies were undertaken:lmmunogenicity test of the expressed DPV-UL24 protein;UL24 gene dynamic distribution and localization in DPV infected ducks by the expressed DPV-UL24 protein mediated immunohistochemistry and immunofluorescence test;Antigen capture enzyme-linked immunoabsorbent assay(ELISA) for DPV specific antibody and DPV-UL24 protein antibody capture ELISA for DPV specific antigen;Establishment of PCR detection method by DPV UL24 gene.These studies are summarized as follows:
1.Ultrastructure studies showed that DEV-CHv virions have the typical morphological characteristics of herpesvirus.The cross sections of the virions were hexagonal and the mature virus with the structure of envelope,nucleocapsid and nucleus were spherical in shape with diameter of 150nm-266nm.The outer part of the envelope was somewhat densely stained than the inner part and some virions with envelop not fully formed were also observed.Most of the DEV virions have one nuclocapsids,some DEV virions have two or three nucleocapsids.The nucleocapsid of DEV was round in shape with diameter of 100nm-150nm,some densely electron-stained vires-related structures which were circle-shaped,semicircle-shaped,or U-shaped could be observed.Between the nucleocapsid and the envelop was certain electron slightly stained part;The DNA in the nucleus which was 40nm-65nm in diameter was slightly electron stained and was in cluster in appearance.Hyperimmune antiserum against DPV was produced by hyperimmunization of the mature ducks with the highly purified DPV particles.
2.Research on duck plague virus proteinomics demonstrated that the following conditions favored the experiment:protein sample extracted by acetone,pH 5-8 gel strip, 30mM DTT reducing agent,carder ampholytes mixturation of loading sampling buffer,0.8 mg protein sample;1253 or 388 protein spots could be obtained respectively by coomassie brilliant blue staining and silver staining.The mean matching rate of the protein spots could reach 88.%each different time.The established model of two-dimenshional electrophoresis was stable,reliable and has a good reproducibility.At least 32 polypeptide spots could be detected by immunoblotting test and five of 32 polypeptide spots were nonspecific.The molecular weights of the 27 DPV polypeptides range from 20KD to 120 KD and the isoelectfic points range from pHS.30 to pH7.50.Two viral protein spots were selected for polypeptide sequence analysis by means of high performance liquid chromatography(HPLC) and electrospray ionization quadrupole time-of-flight tandem mass spectrometry and seven reliable segments sequence were selected after aligning with the virus protein database.
3.The discovery,prokaryotic expression,purification of products and antibody preparation of DPV UL24 gene:Two PCR products of 500 bp and 730 bp were obtained after amplifying the DPV genome with degenerative primers designed according to the polypeptide sequence.The 730 bp PCR product was verified to be duck genome specific by hybridization with DPV genome fragments processed with different restriction enzyme and forming the hybridization bands of different length.Biostatistics analysis showed that the 730 bp fragment has a maximum homology with herpesvirus genome sequence and proved that the amplified product was the DPV UL24 gene.Further analysis showed that the UL24 gene has a 1230 bp open reading flame(ORF) and is highly homologous with 11 strains of herpesvirus and is closest with Mark's virus(MDV).The UL24 protein is a conserved transmembrane protein relatively hydrophobic,with theα-helix and random coil secondary structure contents both being 46.94%.The UL24 gene with its maximum amino acids content being alanine and arginine(8.3%) encodes 409 amino acids,has 16 antigenic determinants,19 phosphorylation sites and does not have signal peptide sequence.Five potential N-glycosylation sites were spotted at the sites of 56,284,294,325 and 383 respectively.The UL24 protein is highly immunogenic,with its encoding gene amino acids codes frequency Nc value being 56.9.Twelve amino acids including alanine are of highly codon usage bias.
DPV-UL24 gene was isolated from DPV genome by PCR with the specific primers designed according to the DPV-UL24 gene sequence.DPV-UL24 gene was subcloned into the vector of pMD18-T and after verification by means of PCR,restriction endonuclease restriction analysis and gene sequencing it was then cloned into the site between the restriction sites of EcoRⅠand XhoI of the pET-32a(+) prokaryotic expression vector and thus the recombinant expression plasmid of pET32a(+)/DPV-UL24 was constructed.The recombinant expression plasmid of pET32a(+)/DPV-UL24 was transformed into the host E.coli of BL21(DE3) and the approximately 38-kD UL24 recombinant protein was obtained after induction with isopropylβ- D -1-thiogalactopyranoside(IPTG),whose molecular weight basically conformed to what was predicted.Through optimization test it was found that the optimal condition was 0.2mmol/L 1PTG with an induction duration of 5 hours.The expressed protein was purified and concentrated by Ni-column affinity chromatography and after renaturation was emulsified with an equal volume of Freund's adjuvant.This product was used as vaccine to prepare hyperimmune sera with rabbits by immunization for four times.Purification of IgG fractions from antiserum was carried out by ion exchange chromatography on Econo-Pac high Q Cartridge after extraction through caprylic acid and equilibrium ammonium sulfate.
4.The study on immunogenicity of prokaryotic expression product of DPV UL24 gene: The prepared UL24 recombinant vaccine was used to vaccinate 7 day old duckling and the serum collected respectively at 3,5,7,10,14,21,28,35,42,49,56,70,84 and 98 days postvaccination underwent ELISA titer and serum neutralization titer evaluation.Half of the ducks underwent challenge test 21 days postimmunization.The result demonstrated that 28 days after immunization,the greatest ELISA titer of 1:5120 at OD_(450nm) could be obtained in the group of recombinant protein group and the greatest ELISA titer of 1:10240 at OD_(450nm) could be obtained in the group of avirulent DPV vaccine group,which are both prominently higher than that of the control group(P≤0.05).Relatively high antibody levels were maintained in the two groups with a same dynamic change of decline. The antibody neutralization test showed that in the recombinant protein group and in the avirulent DPV group,neutralization antibody titer over 1:128 and 1:256 could be obtained 21 to 28 days postvaccination.The neutralization antibody and the ELISA antibody underwent a similar dynamic change in the two group.Analysis indicated that between the recombinant protein group(with its morbidity rate and mortality rate being 50%and 30% respectively) and the avirulent DPV vaccine group(with its morbidity rate and mortality rate being 30%and 10%respectively) no differences of morbidity rate and mortality rate were obtained but between the recombinant protein group and the control group(with its morbidity rate and mortality rate both being 100%) distinct differences of morbidity rate and mortality rate were obtained.All these studies demonstrated that the DPV-UL24 recombinant protein could confer duckling certain protect effects after immunization.
5.The phase and location of UL24 gene express in artificial infected ducklings:The dynamic expression,infection process and distribution of DPV-UL24 gene in infected ducklings were studied by the established DPV-UL24 recombinant protein indirect immunohistochemistry method with the specimens of spleen,pancreas,harderian glands, bursa of Fabricius,liver,intestine,kidney,lung,heart,encephalon of the ducklings challenged at 28 days old with duck plague virulent DPV-CHv strain.The results demonstrated that DPV-UL24 encoding protein antigens could be detected in spleen, thymus,liver and duodenum 2 hour after challenge,in harderian glands and glandular stomach 4 hours after challenge,in ileum and lung 8 hours after challenge,in bursa of Fabricius and kidney 12 hours after challenge,in pancreas each part of intestine 24 hours after challenge.All these results demonstrated that organs of the immune system and the intestinal mucosa are the target organ and target tissue of DPV-UL24 encoding protein. DPV-UL24 encoding protein brain tissue tropism is very low,indicating that this protein is probably transmembrane protein and its function mainly related with the transportation and leading of virus components into nucleus for virion assembly during the virus proliferation.
The dynamic expression,proliferation and distribution of DPV-UL24 encoding protein in infected ducklings were studied by the established DPV-UL24 recombinant protein immunofluroscence detection method in ducks challenged with duck plague virus. The results demonstrated that DPV-UL24 encoding protein antigen could be detected in spleen,thymus,harderian glands,bursa of Fabricius,liver,lung,kidney and most parts of intestine 2 hour after challenge,in pancreas,encephalon and heart 4 hours after challenge, in esophagus 8 hours after challenge.All these results demonstrated that the immune organs of spleen,thymus,harderian glands,bursa of Fabricius and the intestinal mucosa are the target organ and target tissue of DPV-UL24 encoding protein.DPV-UL24 encoding protein was selective in the target organs in the process of virus invasion and distribution, causing serious damage to mucosal epithelium.Of the 26 specimens for inspection by this method,25 specimens were positive and positive rate was 96.1%,indicating that this method for DPV-UL24 encoding protein detection is sensitive and specific.
6.Research on Duck Plague Virus was Detected by Antigen Capture-ELISA with Anti-Prokaryotic Expression Protein of DPV UL24:Double antibody sandwich DPV antigen captured ELISA assay method is established and optimized with the rabbit anti-DPV UL24 prokaryotic expression protein antiserum and the duck anti- DPV UL24 prokaryotic expression protein antiserum.The results demonstrated that the optimized evaluation could be obtained when the rabbit anti DPV-UL24 antibody concentration is 5.0ug/100ul,the duck anti DPV-UL24 antibody concentration is 9.0ug/1O0ul and the enzyme linked antibody dilution is 1:2000.Other duck infected pathogens like duck hepatitis virus B(DHBV) was employed as negative controls and showed negative results. The coefficient s of variation of intra-assay and inter-assay was less than 10%and the minimum DPV content detection is 46 ng DPV purified virus,showing the specific, sensitive and stable characteristics of the established ELISA method.The dynamic proliferation and distribution of DPV-UL24 encoding protein in the specimens of liver, spleen,encephalon,esophagus,lung,and kidney of the infected ducklings were studied by this established ELISA method and the results were similar with that obtained by the established immunohistochemistry and immunoflurescence method.
7.Research on Duck Plague Virus Antibody was Detected by ELISA Using Coating Prokaryotic Expression Protein of DPV UL24:Double antibody sandwich DPV antigen captured ELISA assay method is established and optimized with DPV UL24 prokaryotic expression protein as antigen.The results demonstrated that the optimized evaluation could be obtained when the DPV-UL24 recombinant protein is 80-fold diluted with its concentration being 2.5ug/100ul,the dilution of the examined serum is 1:320 and the enzyme linked antibody dilution is 1:2000.Other duck infected pathogen specific positive antiserum like duck hepatitis virus B(DHBV) and duck Riemirella Anatipestifer(RA) were employed as negative controls and showed negative results.The coefficient s of variation of intra-assay and inter-assay was less than 7%and could detect DPV positive antiserum with a dilution of 1:2560.127 specimens from the clinically infected ducklings were studied by this established ELISA method with a positive rate of 77.2%and similar results were obtained with that obtained by the whole DPV antigen as coated antigen ELISA method.
8.Development and Application of PCR based on UL24 gene for the Detection of Duck plague virus:One pair of primer(P1/P2) was designed according to the DPV-UL24 genes sequence for amplification of DPV genome DNA.The results demonstrated that one PCR product of 585bp was obtained with this primers for DPV genome amplification but no products could be detected with this primer for genome amplification of other pathogen such as duck hepatitis virus and duck hepatitis virus B.The sensitivity of the primer of P1/P2 is lpg and the uniform PCR product fragment could be obtained for amplification of the 14 the specimens collected from DPV virulent virus infected ducks whereas no PCR product fragment could be obtained for amplification of the 5 the specimens collected from ducks not infected with DPV.These results demonstrated that the established UL24 PCR detection method is reliable,sensitivity and could be used in 1 the clinical diagnosis and surveillance of duck plague infection.
Comparisons and analysis of the established indirect immunohistochemistry, immunofluorescent,AC-ELISA and PCR detection methods indicates that positive results could be obtained by the four methods for examination of the DPV positive specimens, specimens not infected with DPV,whereas negative results could be obtained by the four methods for examination of blank control,goat serum and other duck pathogens.These examinations indicated that these four methods are well specific.The examinations of the specimens from the artificially infected ducks indicated that positive results could be obtained in the specimens of spleen,thymus,liver and duodenum collected 4 hours postinfection.Immunohistochemistry examination indicated that UL24 gene encoding protein could be detected in almost all the tissues 24 hours postinfection,whereas UL24 gene encoding protein could be detected by immunofluorescent and PCR detection methods 12 hours postinfection.UL24 gene encoding protein could be detected in the 11 specimens by AC-ELISA methods 24 hours postinfection.The examination of the clinically infected DPV infected specimens indicated that 88.4%positive results could be obtained by immunohistochemistry method,96.1%positive results could be obtained by immunofluorescent method,85.2%positive results could be obtained by AC-ELISA methods,100%positive results could be obtained by PCR methods.
引文
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