摘要
为探究水貂细小病毒(MEV)VP2蛋白的结构和功能,对MEV VP2蛋白进行原核表达并纯化,用ELISA初步评价重组蛋白在血清学诊断中的价值。通过大肠埃希菌表达外源基因的方法构建MEV VP2原核表达体系,经诱导表达得到以包涵体形式存在的目的蛋白,对蛋白进行纯化、透析复性并鉴定;加入分子伴侣pTf16质粒构建共表达系统,优化表达条件以提升可溶性目的蛋白的表达量,对表达产物进行纯化及鉴定。将纯化后的可溶性蛋白、复性后的包涵体蛋白及纯化后的全病毒蛋白作为抗原包被酶标板,用间接ELISA方法对MEV标准阴、阳性血清进行检测,初步对比评价3种抗原的血清学诊断价值。结果表明,经过双酶切和测序鉴定,成功构建重组蛋白原核表达载体;重组VP2蛋白的分子质量约为67 ku;优化诱导温度和诱导试剂浓度未能解决包涵体表达问题;构建了共表达系统,优化表达条件后可溶性目的蛋白的表达量得到明显提高;SDS-PAGE和Western blot鉴定结果表明,两种重组蛋白皆具有良好的反应原性;间接ELISA结果表明可溶性表达蛋白更适合作为MEV的候选诊断抗原。通过分子伴侣共表达和包涵体蛋白复性的方法获得了大量有活性的目的蛋白,为建立水貂细小病毒血清学诊断方法和制备MEV病毒样颗粒(VLPs)及VP2蛋白多克隆抗体奠定了基础。
In order to explore the structure and function of the VP2 protein of Mink enteritis virus(MEV),the enzyme-linked immunosorbent assay(ELISA)was used to evaluate the prokaryotic expression and purification of MEV VP2 protein in the value of serological diagnosis in this experiment.The prokaryotic expression system of MEV VP2 was constructed by E.coli expression of foreign genes,and the target protein in the form of inclusion body was obtained by induction expression.The inclusion body protein was purified,renatured and identified.The molecular chaperone pTf16 plasmid was added to construct the co-expression system,and the expression conditions were optimized to increase the expression level of the soluble target protein,and the expression product was purified and identified.Three antigens including the purified soluble protein,the renatured inclusion body protein and the purified whole virus protein were used as coating antigens,and indirect ELISA method was used to evaluate the detection values of MEV serum antibodies by three antigens.The results showed that the recombinant plasmid was successfully constructed by double enzyme digestion and sequencing.The molecular weight of recombinant VP2 protein is about 67 ku.Optimization of induction temperature and concentration of induction reagent failed to solve the problem of inclusion body expression.The co-expression system was successfully constructed,and the expression level of soluble target protein was significantly increased after the optimizing conditions.The results of SDS-PAGE and Western blotting showed that the two recombinant proteins have good reactogenicity.The results of indirect ELISA showed that soluble expressed proteins were more suitable for candidate diagnostic antigens for MEV.Conclusion:This experiment obtained a large number of active target proteins by co-expression of foreign genes and molecular chaperone into E.coli and renaturation of inclusion bodies,which provided a theoretical basis for the establishment of serological diagnostic methods for MEV.At the same time,it laid a foundation for the preparation of MEV virus-like particles(VLPs)and VP2 protein polyclonal antibodies in the future.
作者
朱翔宇
蔡熙姮
史宁
王洋
由海波
鲁荣光
闫喜军
侯金利
李滋睿
胡博
徐超
ZHU Xiang-yu;CAI Xi-heng;SHI Ning;WANG Yang;YOU Hai-bo;LU Rong-guang;YAN Xi-jun;HOU Jin-li;LI Zi-rui;HU Bo;XU Chao(Institue of Special Wild Economic Animal and Plant Science,Chinese Academy of Agricultural Science,Changchun,Jilin,130112,China;Key Laboratory of Special Animal Epidemic Disease,Changchun,Jilin,130112,China;College of Animal Science and Technology,Jilin Agricultural University,Changchun,Jilin,130118,China;Xinzhuang Veterinary Station,Yantai,Shandong,265401,China)
出处
《动物医学进展》
北大核心
2020年第6期1-6,共6页
Progress In Veterinary Medicine
基金
国家重点研发计划项目(2017YFD0501600)。
关键词
水貂细小病毒
VP2基因原核表达
可溶性蛋白
包涵体复性
血清学评价
Mink enteritis virus
prokaryotic expression of VP2 gene
soluble protein
inclusion body renaturation
serologic evaluation
