猪瘟病毒E~（rns）蛋白的高效表达及其ELISA诊断试剂盒的建立研究
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摘要
本研究拟通过基因工程手段对猪瘟病毒E~(rns)蛋白进行高效表达,建立一种大规模筛查猪瘟特异性抗体的血清学诊断试验,为E2蛋白标记疫苗的推广应用打下基础。
应用RT-PCR技术对中国猪瘟兔化弱毒株(C株)E~(rns)基因进行扩增,得到约768bp的基因片段。将这个基因克隆到原核表达载体pET32a(+)中,经酶切、PCR扩增和测序分析确证其正确插入到表达载体中,且阅读框正确,从而构建成重组表达载体。重组质粒转化至宿主菌BL21(DE3)中,用异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导表达,用SDS-PAGE电泳,Western-blotting分析表达的蛋白。结果表明,E~(rns)基因可以在大肠杆菌中表达,表达产物的分子量约为48ku(目的基因的蛋白分子质量为26ku,载体HIS分子质量为22ku),与理论推测的蛋白分子量一致;蛋白的表达占菌体蛋白的35%左右,经Western-blotting证明,表达的E~(rns)蛋白可被猪瘟标准阳性血清所识别。
E~(rns)蛋白在pET-32a(+)表达载体中表达产量较高,其中80%的蛋白以可溶性形式存在,具有生物学活性。收集诱导表达的菌液,超声波破碎后离心取上清,应用Ni-NTA HisBind Resins蛋白纯化试剂盒对表达蛋白进行纯化,获取纯化蛋白。SDS-PAGE分析电泳结果表明,其纯度可达90%。Western-blotting试验结果显示,纯化蛋白与CSFV阳性血清的特异性很高,纯化蛋白可用于基因工程诊断抗原。
用纯化的猪瘟病毒重组E~(rns)蛋白作为抗原包被酶标板,以辣根过氧化物酶(HRP)标记的兔抗猪IgG为二抗,建立了检测猪瘟病毒抗体的间接ELISA方法,并确定了ELISA最佳工作条件:抗原包被浓度为6.25ug/mL,4℃包被过夜,猪瘟阳性血清(1:50)在37℃作用40min,二抗(1:10000)37℃作用30min,底物溶液37℃显色10min,2M硫酸终止反应。通过重复性试验,特异性试验和符合性试验等试验结果表明该方法重复性好,特异性强,灵敏度高;与用猪瘟抗体检测的ELISA(IDEXX公司)试剂盒比较,特异性为89%,敏感性为88.7%,两者的符合率为89.2%。用已建立的方法检测临床血清样本148份,总阳性率为90%。本研究结果为建立野毒感染与疫苗免疫相区别诊断方法的标准化提供了实验基础,为猪瘟的诊断与监测提供一种良好的技术手段,也为E2蛋白标记疫苗的推广应用打下基础。
The amplified E~(rns) fragments of the C strains were 768bp in length. And they were ligated with plasmid pGM-T vector.The recombinant plasmids and expression vector pET-32a(+)were digested by the same restriction endonucleases.These genes were ligated and transformed into Escherichia coli.The insert position,the size and the reading frame all were corrected by PCR and the sequence analysis.The result showed that the prokaryotic expression vectors were constructed successfully.Then the recombinants were transformed into BL21 (DE3) for E~(rns) expression with IPTG inducing.The expressed proteins were measured by SDS-PAGE and western-blotting.The results showed that the E~(rns) genes can expressed successfully in E.coli.The western-blotting results indicated that the expressed protein can be recognized by the CSFV positive serum.The rate of the expressed proteins in the induced bacteria protein was about 35%.
The protein was expressed massly.Soluble test showed that 80%of the protein exsits in suspension.The fusion protein was purified by Ni-NTA HisBind Resins Kits.Purified protein were analysised by SDS-PAGE and western-blotting.The results showed that the purity of fusion protein were 90%,purified protein specified with the CSFV positive serum.
Using the purified recombinant proteins,an indirect ELISA for detection of anti-CSFV antibodies was developed and its optimal reactions were determined: coating antigen for 4℃overnight at a concentration of 6.25ug/ mL,serum sample (1:50) and HRP labeled anti-porcine IgG (1:10000) being incubated at 37℃for 40 min.The ELISA assay was confirmed to have a good repeatability,specificity and sensitivity by repeated test,crossing test and coincidence test. And this ELISA method was also compared with the established routine ELISA test kit.The specificity and sensitivity of the ELISA is 89% and 88.7% respectively. In addition,148 serum samples collected from swine farms were detected by the developed assay,it was showed that the positive rate was 90% for antibody against CSFV. The E~(rns)-ELISA can be used as differential diagnostic assays to differentiate animals infected with wild-type viruses from those vaccinated with E2 based marker vaccines.
应用RT-PCR技术对中国猪瘟兔化弱毒株(C株)E~(rns)基因进行扩增,得到约768bp的基因片段。将这个基因克隆到原核表达载体pET32a(+)中,经酶切、PCR扩增和测序分析确证其正确插入到表达载体中,且阅读框正确,从而构建成重组表达载体。重组质粒转化至宿主菌BL21(DE3)中,用异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导表达,用SDS-PAGE电泳,Western-blotting分析表达的蛋白。结果表明,E~(rns)基因可以在大肠杆菌中表达,表达产物的分子量约为48ku(目的基因的蛋白分子质量为26ku,载体HIS分子质量为22ku),与理论推测的蛋白分子量一致;蛋白的表达占菌体蛋白的35%左右,经Western-blotting证明,表达的E~(rns)蛋白可被猪瘟标准阳性血清所识别。
E~(rns)蛋白在pET-32a(+)表达载体中表达产量较高,其中80%的蛋白以可溶性形式存在,具有生物学活性。收集诱导表达的菌液,超声波破碎后离心取上清,应用Ni-NTA HisBind Resins蛋白纯化试剂盒对表达蛋白进行纯化,获取纯化蛋白。SDS-PAGE分析电泳结果表明,其纯度可达90%。Western-blotting试验结果显示,纯化蛋白与CSFV阳性血清的特异性很高,纯化蛋白可用于基因工程诊断抗原。
用纯化的猪瘟病毒重组E~(rns)蛋白作为抗原包被酶标板,以辣根过氧化物酶(HRP)标记的兔抗猪IgG为二抗,建立了检测猪瘟病毒抗体的间接ELISA方法,并确定了ELISA最佳工作条件:抗原包被浓度为6.25ug/mL,4℃包被过夜,猪瘟阳性血清(1:50)在37℃作用40min,二抗(1:10000)37℃作用30min,底物溶液37℃显色10min,2M硫酸终止反应。通过重复性试验,特异性试验和符合性试验等试验结果表明该方法重复性好,特异性强,灵敏度高;与用猪瘟抗体检测的ELISA(IDEXX公司)试剂盒比较,特异性为89%,敏感性为88.7%,两者的符合率为89.2%。用已建立的方法检测临床血清样本148份,总阳性率为90%。本研究结果为建立野毒感染与疫苗免疫相区别诊断方法的标准化提供了实验基础,为猪瘟的诊断与监测提供一种良好的技术手段,也为E2蛋白标记疫苗的推广应用打下基础。
The amplified E~(rns) fragments of the C strains were 768bp in length. And they were ligated with plasmid pGM-T vector.The recombinant plasmids and expression vector pET-32a(+)were digested by the same restriction endonucleases.These genes were ligated and transformed into Escherichia coli.The insert position,the size and the reading frame all were corrected by PCR and the sequence analysis.The result showed that the prokaryotic expression vectors were constructed successfully.Then the recombinants were transformed into BL21 (DE3) for E~(rns) expression with IPTG inducing.The expressed proteins were measured by SDS-PAGE and western-blotting.The results showed that the E~(rns) genes can expressed successfully in E.coli.The western-blotting results indicated that the expressed protein can be recognized by the CSFV positive serum.The rate of the expressed proteins in the induced bacteria protein was about 35%.
The protein was expressed massly.Soluble test showed that 80%of the protein exsits in suspension.The fusion protein was purified by Ni-NTA HisBind Resins Kits.Purified protein were analysised by SDS-PAGE and western-blotting.The results showed that the purity of fusion protein were 90%,purified protein specified with the CSFV positive serum.
Using the purified recombinant proteins,an indirect ELISA for detection of anti-CSFV antibodies was developed and its optimal reactions were determined: coating antigen for 4℃overnight at a concentration of 6.25ug/ mL,serum sample (1:50) and HRP labeled anti-porcine IgG (1:10000) being incubated at 37℃for 40 min.The ELISA assay was confirmed to have a good repeatability,specificity and sensitivity by repeated test,crossing test and coincidence test. And this ELISA method was also compared with the established routine ELISA test kit.The specificity and sensitivity of the ELISA is 89% and 88.7% respectively. In addition,148 serum samples collected from swine farms were detected by the developed assay,it was showed that the positive rate was 90% for antibody against CSFV. The E~(rns)-ELISA can be used as differential diagnostic assays to differentiate animals infected with wild-type viruses from those vaccinated with E2 based marker vaccines.
引文
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