猪伪狂犬病鉴别诊断研究与猪繁殖与呼吸综合征病毒基因组序列的测定
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摘要
伪狂犬病(Pseudorabies，PR)是由α疱疹病毒科的伪狂犬病病毒(Pseudorabies Virus，PrV)引起的包括多种家畜和野生动物共患的一种烈性传染病。猪是该病毒的天然宿主和贮存者。该病给我国养猪业造成了巨大的经济损失。预防、控制和最终根除该病是我国当前面临的一项艰巨任务。在欧美等发达国家，伪狂犬病的根除计划多采用gE或gG基因标记疫苗和相应的gE或gG鉴别诊断方法来进行。gG糖蛋白是PrV的一种分泌蛋白，gE糖蛋白在决定病毒的毒力及神经嗜性等方面起着重要作用，但两者都不是PrV增殖所必需的蛋白，因此可作为缺失的候选基因。目前我国已研制成功gE和gG基因标记疫苗。为了建立适合我国国情的gE和gG鉴别诊断方法，开展了以下研究。
1．PrV gG基因的克隆及其在大肠杆菌中的表达
依据伪狂犬病病毒(Pseudorabies Virus PRV)Rice株序列，合成针对gG基因的特异性引物，从PRV鄂A株中扩增出gG基因，并将其克隆到pBluescriptⅡ SK+质粒中并测序，用SacⅠ和HindⅢ酶切，将gG基因融合到pET—28a质粒中T_7启动子下游，构建成原核表达质粒pET—28a-gG_1，结果并未获得表达。再用NotⅠ切除gG基因后小半部分片段，构建成原核表达质粒pET—28a-gG_2，使其在E.coli BL21(DE3)中以IPTG诱导表达，经SDS—PAGE分析，表达特异带为47kD，斑点杂交证实表达产物具有抗原性。表达产物主要以可溶性蛋白的形式存在于细菌裂解液的上清之中，上清经饱和硫酸铵溶液沉淀，透析，PEG20000浓缩，ELISA分析表明，经初步纯化的表达产物可与抗PRV猪血清发生特异性免疫反应。
2．gG-LAT和gG-ELISA的建立
以PRV gG基因表达提纯产物为抗原分别致敏乳胶和包被酶标板，摸索最佳的致敏和包被条件，建立了gG-LAT和gG-ELISA。方阵滴定确定gG-LAT最佳致敏温度为37℃，最佳致敏时间为30分钟，抗原最佳致敏浓度为1.3mg／ml。以gG-LAT检测各类血清340份，结果表明gG-LAT的特异性及敏感性分别为100％(43／43)和91.4％(96／105)，检测192份部分接种TK-／gG-／Laz+弱毒苗的猪血清，gG-LAT检出阳性率为36.5％(70／192)。
方阵滴定确定gG-ELISA的抗原最佳包被量为1.1μg／孔，血清1：40稀释时P／N值最大，为10.5(1.16／1.1)。故确定1.1μg／孔为抗原最佳包被量，1：40为最佳血清稀释浓度。以gG-ELISA分别检测两PRV清洁猪场gG基因缺失疫苗免疫的猪血清50份与PRV全病毒灭活苗免疫的猪血清50份，结果在gG缺失疫苗免疫猪血清中检出阳性2份，其它为阴性；在PRV全病毒灭活苗免疫猪血清中检出阳性49份，阴性1份。此结果显示，gG-ELISA诊断敏感性为98％(49／50)，诊断特异性为96％(48／50)。以5块不同批次的包被抗原的酶标板检测5份血清，结果显示阳性血清
猪伪狂犬病鉴别诊断研究与猪繁殖与呼吸综合征病毒基因组序列的测定
的变异系数均小于10%，表明该方法重复性好。以gG一EuSA检测69份样本并与
PRv病理诊断及PRV-PcR诊断结果比较。结果显示gG一EuSA与PRV病理诊断符
合率为84.0%(58/69)。gG一ELISA与pRv-pCR诊断符合率为94.2%(65/69)，以
gG一EuSA检测842份血清，检出阳性242份，阳性率为28.74%。以上结果表明本
研究建立的gG一LAT和gG一ELISA特异性强、敏感性高，可用于区分gG基因缺失疫
苗活苗免疫猪与自然感染野毒的血清学阳性猪。
3.PrVgE基因的主要抗原表位区在大肠杆菌中的表达
将伪狂犬病病毒(PRv)Ea株gE基因主要抗原表位区段融合到原核表达载体
pET28b的”启动子下游，构建成原核表达质粒，经IpTG诱导，SDs一pAGE和western
blot印迹分析，证实gE基因主要抗原表位区在大肠杆菌中获得了表达，表达产物具
有抗原性，分子量为38kD，位于包涵体中。
4.gE.1‘AT和gE一EUsA的建立
乒基因主要抗原表位区表达的包涵体蛋白经变性、复性处理后，复性产物致敏
乳胶，并对抗原致敏浓度、致敏时间、致敏温度进行优化，建立了gE乳胶凝集试验
(薛.LAI，)。该方法不仅能显著区分gE基因缺失疫苗免疫猪血清和野毒感染猪血清，
而且具有简便、快速等优点。检测360份猪血清，阳性率为57.77%(208/360)，比国
外阻断gE一ELISA的58.05%(209/360)略低，阳性符合率为94.86%(203/214)，总符
合率为%.94%(349/360)，两种检测方法结果差异不显著(P>0.05)。
将经纯化、变性、复性等处理后的gE蛋白作为抗原建立伪狂犬病的gE一EL工SA
鉴别诊断方法。方阵滴定确定7.5 pg/孔为抗原最佳包被量，1:40为最佳血清稀释
浓度。以该方法检测115份己知背景猪血清，结果表明，该方法诊断特异性为94.5%，
诊断敏感性为%.7%。以5块不同批次包被抗原的酶标板检测5份血清，结果显示
阳性血清的变异系数均小于10%，表明该方法重复性好。对比国外阻断gE一ELISA
同时检测356份猪血清，两者阳性符合率为87.44%(195/223)，总符合率为92.13%
(328/356)。以上结果表明该gE一LAT和gE一ELISA特异、敏感且重复性好，可用于
猪伪狂犬病的鉴别诊断。
5.猪繁殖与呼吸综合征基因组序列的测定
猪繁殖与呼吸综合征是另一种危害我国养猪业的传染病，它主要以母猪发热、
厌食、流产、早产、死胎、弱仔等繁殖障碍及各年龄猪的呼吸道症状为主要特征，
Pseudorabies virus (PrV), a member of Alpha Herpesvirus, is the causative agent of Pseudorabies (Aujeszky's disease), one of the most serious infectious diseases of several domestic and wild animals. Swine was the natural host and reservoir of PrV. Pseudorabies was an economically important disease of swine industry in China. Currently it is an important and difficult task to prevent, control and eradicate this disease in China. Use of an effective gE or gG gene-deleted PrV vaccine combined with a differential diagnostic kit for PrV glycoprotein gE or gG has proven successful in several Pseudorabies-eradication. gE and gG are unessential for viral replication in cell culture, however gE played an important role in determining virulence and neurotropism. In order to develope a PrV-gE(or gG) differential diagnostic method for the pseudorabies eradication program in China, the following researches were explored.
1. The cloning of PrV gG gene and it's expression in E.coli.
The complete glycoprotein gG gene of pseudoiabies virus Ea strain was amplified by PCR technique and cloned into pBluescriptIISK+. The gG gene was sequenced by sanger's sequencing technique. Then, the gG gene was inserted into downstream of the T7 promoter of an expression vector, pET-28a, to yield the recombinant plasmid pET-28a-gGi, but the expression failed. Then the plasmid-pET-28a-gGi was digested by Not I to delete the short fragment of downstream of gG gene and linked to yield the recombinant pET-28a-gG2. After induced by IPTG, a high expression of fusion protein was obtained. SDS-PAGE analysis showed that the fusion protein was 47kD and the product of expression was specific to antisera against PRV by protein dot blot analysis. The fusion protein existed mainly in form of soluble protein. After be purified by saturated (NH4)2SO4 solution, the product was specific to swine antisera against PRV in indirect ELISA.
2. The development of gG-LAT and gG-ELISA
The gG latex agglutination test (LAT) and the gG Enzyme linked Immunosorbent Assay (ELISA) based on recombinant glycoprotein gG for differentiation between infected and vaccinated pigs was developed. gG-LAT can significantly differentiate the infected from vaccinated (gG deleted caccine) pigs. Statistical result of testing 340 serum samples indicated that the diagnosis method has high specificity and sensitivity. The specificity and sensitivity of the developed gG-ELISA were evaluated by testing 100 serum samples. The result of clinical application indicated that there is high coincidence rate between gG-ELISA and other clinical diagnosis methods. 842 serum samples of pigs that were vaccinated by gG- marker vaccines have been tested by gG-ELISA for
epidemiologic investigation, and 242 sera of them were examined to be positive. The positive rate was 28.74%. The above results indicated that the gG-LAT and gG-ELISAcan be used for the differentiation between the pseudorabies virus (PrV) infected and vaccinated (gG' vaccine) pigs.
3. The expression of the major epitope domain of glycoprotein gE of pseudorabies virus in E.coli.
A 0.8kb DNA fragment encoding the major epitope domain of glycoprotein gE of pseudorabies virus Ea strain was inserted into the downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induced by IPTG, a high level expression of fusion protein was obtained. SDS-PAGE and Western blotting analysis showed that the fusion protein was 38kD and the protein was specific to antisera against PRV Ea strain.
4. The development of gE-LAT and gE-ELISA
The gE protein existed mainly in form of inclusion body. After being denatured and renatured, the protein was used to sensitize latex to prepare the latex antigen. Then, the concentration of antigen, times and temperature for sensitization were optimized. The latex agglutination test (LAT) based on recombinant glycoprotein gE for differentiation between infected and vaccinated (gE deleted vaccine) pigs was developed. The specificity and sensitivity of the developed gE-LAT were also
1．PrV gG基因的克隆及其在大肠杆菌中的表达
依据伪狂犬病病毒(Pseudorabies Virus PRV)Rice株序列，合成针对gG基因的特异性引物，从PRV鄂A株中扩增出gG基因，并将其克隆到pBluescriptⅡ SK+质粒中并测序，用SacⅠ和HindⅢ酶切，将gG基因融合到pET—28a质粒中T_7启动子下游，构建成原核表达质粒pET—28a-gG_1，结果并未获得表达。再用NotⅠ切除gG基因后小半部分片段，构建成原核表达质粒pET—28a-gG_2，使其在E.coli BL21(DE3)中以IPTG诱导表达，经SDS—PAGE分析，表达特异带为47kD，斑点杂交证实表达产物具有抗原性。表达产物主要以可溶性蛋白的形式存在于细菌裂解液的上清之中，上清经饱和硫酸铵溶液沉淀，透析，PEG20000浓缩，ELISA分析表明，经初步纯化的表达产物可与抗PRV猪血清发生特异性免疫反应。
2．gG-LAT和gG-ELISA的建立
以PRV gG基因表达提纯产物为抗原分别致敏乳胶和包被酶标板，摸索最佳的致敏和包被条件，建立了gG-LAT和gG-ELISA。方阵滴定确定gG-LAT最佳致敏温度为37℃，最佳致敏时间为30分钟，抗原最佳致敏浓度为1.3mg／ml。以gG-LAT检测各类血清340份，结果表明gG-LAT的特异性及敏感性分别为100％(43／43)和91.4％(96／105)，检测192份部分接种TK-／gG-／Laz+弱毒苗的猪血清，gG-LAT检出阳性率为36.5％(70／192)。
方阵滴定确定gG-ELISA的抗原最佳包被量为1.1μg／孔，血清1：40稀释时P／N值最大，为10.5(1.16／1.1)。故确定1.1μg／孔为抗原最佳包被量，1：40为最佳血清稀释浓度。以gG-ELISA分别检测两PRV清洁猪场gG基因缺失疫苗免疫的猪血清50份与PRV全病毒灭活苗免疫的猪血清50份，结果在gG缺失疫苗免疫猪血清中检出阳性2份，其它为阴性；在PRV全病毒灭活苗免疫猪血清中检出阳性49份，阴性1份。此结果显示，gG-ELISA诊断敏感性为98％(49／50)，诊断特异性为96％(48／50)。以5块不同批次的包被抗原的酶标板检测5份血清，结果显示阳性血清
猪伪狂犬病鉴别诊断研究与猪繁殖与呼吸综合征病毒基因组序列的测定
的变异系数均小于10%，表明该方法重复性好。以gG一EuSA检测69份样本并与
PRv病理诊断及PRV-PcR诊断结果比较。结果显示gG一EuSA与PRV病理诊断符
合率为84.0%(58/69)。gG一ELISA与pRv-pCR诊断符合率为94.2%(65/69)，以
gG一EuSA检测842份血清，检出阳性242份，阳性率为28.74%。以上结果表明本
研究建立的gG一LAT和gG一ELISA特异性强、敏感性高，可用于区分gG基因缺失疫
苗活苗免疫猪与自然感染野毒的血清学阳性猪。
3.PrVgE基因的主要抗原表位区在大肠杆菌中的表达
将伪狂犬病病毒(PRv)Ea株gE基因主要抗原表位区段融合到原核表达载体
pET28b的”启动子下游，构建成原核表达质粒，经IpTG诱导，SDs一pAGE和western
blot印迹分析，证实gE基因主要抗原表位区在大肠杆菌中获得了表达，表达产物具
有抗原性，分子量为38kD，位于包涵体中。
4.gE.1‘AT和gE一EUsA的建立
乒基因主要抗原表位区表达的包涵体蛋白经变性、复性处理后，复性产物致敏
乳胶，并对抗原致敏浓度、致敏时间、致敏温度进行优化，建立了gE乳胶凝集试验
(薛.LAI，)。该方法不仅能显著区分gE基因缺失疫苗免疫猪血清和野毒感染猪血清，
而且具有简便、快速等优点。检测360份猪血清，阳性率为57.77%(208/360)，比国
外阻断gE一ELISA的58.05%(209/360)略低，阳性符合率为94.86%(203/214)，总符
合率为%.94%(349/360)，两种检测方法结果差异不显著(P>0.05)。
将经纯化、变性、复性等处理后的gE蛋白作为抗原建立伪狂犬病的gE一EL工SA
鉴别诊断方法。方阵滴定确定7.5 pg/孔为抗原最佳包被量，1:40为最佳血清稀释
浓度。以该方法检测115份己知背景猪血清，结果表明，该方法诊断特异性为94.5%，
诊断敏感性为%.7%。以5块不同批次包被抗原的酶标板检测5份血清，结果显示
阳性血清的变异系数均小于10%，表明该方法重复性好。对比国外阻断gE一ELISA
同时检测356份猪血清，两者阳性符合率为87.44%(195/223)，总符合率为92.13%
(328/356)。以上结果表明该gE一LAT和gE一ELISA特异、敏感且重复性好，可用于
猪伪狂犬病的鉴别诊断。
5.猪繁殖与呼吸综合征基因组序列的测定
猪繁殖与呼吸综合征是另一种危害我国养猪业的传染病，它主要以母猪发热、
厌食、流产、早产、死胎、弱仔等繁殖障碍及各年龄猪的呼吸道症状为主要特征，
Pseudorabies virus (PrV), a member of Alpha Herpesvirus, is the causative agent of Pseudorabies (Aujeszky's disease), one of the most serious infectious diseases of several domestic and wild animals. Swine was the natural host and reservoir of PrV. Pseudorabies was an economically important disease of swine industry in China. Currently it is an important and difficult task to prevent, control and eradicate this disease in China. Use of an effective gE or gG gene-deleted PrV vaccine combined with a differential diagnostic kit for PrV glycoprotein gE or gG has proven successful in several Pseudorabies-eradication. gE and gG are unessential for viral replication in cell culture, however gE played an important role in determining virulence and neurotropism. In order to develope a PrV-gE(or gG) differential diagnostic method for the pseudorabies eradication program in China, the following researches were explored.
1. The cloning of PrV gG gene and it's expression in E.coli.
The complete glycoprotein gG gene of pseudoiabies virus Ea strain was amplified by PCR technique and cloned into pBluescriptIISK+. The gG gene was sequenced by sanger's sequencing technique. Then, the gG gene was inserted into downstream of the T7 promoter of an expression vector, pET-28a, to yield the recombinant plasmid pET-28a-gGi, but the expression failed. Then the plasmid-pET-28a-gGi was digested by Not I to delete the short fragment of downstream of gG gene and linked to yield the recombinant pET-28a-gG2. After induced by IPTG, a high expression of fusion protein was obtained. SDS-PAGE analysis showed that the fusion protein was 47kD and the product of expression was specific to antisera against PRV by protein dot blot analysis. The fusion protein existed mainly in form of soluble protein. After be purified by saturated (NH4)2SO4 solution, the product was specific to swine antisera against PRV in indirect ELISA.
2. The development of gG-LAT and gG-ELISA
The gG latex agglutination test (LAT) and the gG Enzyme linked Immunosorbent Assay (ELISA) based on recombinant glycoprotein gG for differentiation between infected and vaccinated pigs was developed. gG-LAT can significantly differentiate the infected from vaccinated (gG deleted caccine) pigs. Statistical result of testing 340 serum samples indicated that the diagnosis method has high specificity and sensitivity. The specificity and sensitivity of the developed gG-ELISA were evaluated by testing 100 serum samples. The result of clinical application indicated that there is high coincidence rate between gG-ELISA and other clinical diagnosis methods. 842 serum samples of pigs that were vaccinated by gG- marker vaccines have been tested by gG-ELISA for
epidemiologic investigation, and 242 sera of them were examined to be positive. The positive rate was 28.74%. The above results indicated that the gG-LAT and gG-ELISAcan be used for the differentiation between the pseudorabies virus (PrV) infected and vaccinated (gG' vaccine) pigs.
3. The expression of the major epitope domain of glycoprotein gE of pseudorabies virus in E.coli.
A 0.8kb DNA fragment encoding the major epitope domain of glycoprotein gE of pseudorabies virus Ea strain was inserted into the downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induced by IPTG, a high level expression of fusion protein was obtained. SDS-PAGE and Western blotting analysis showed that the fusion protein was 38kD and the protein was specific to antisera against PRV Ea strain.
4. The development of gE-LAT and gE-ELISA
The gE protein existed mainly in form of inclusion body. After being denatured and renatured, the protein was used to sensitize latex to prepare the latex antigen. Then, the concentration of antigen, times and temperature for sensitization were optimized. The latex agglutination test (LAT) based on recombinant glycoprotein gE for differentiation between infected and vaccinated (gE deleted vaccine) pigs was developed. The specificity and sensitivity of the developed gE-LAT were also
引文
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