兔出血症病毒衣壳蛋白VP60基因的克隆、表达及ELISA检测方法的建立与初步应用

【作者】 付瑞；

【导师】 贺争鸣；

【作者基本信息】 中国药品生物制品检定所 ， 免疫学， 2004， 硕士

【摘要】 兔出血症病毒(Rabbit Haemorrhagic Disease Virus，RHDV)是实验动物必须排除的病源微生物之一。由于至今还没有适于RHDV增殖的敏感细胞，使得RHDV的检测成为实验动物质量控制中的一个难点。 为建立兔出血症病毒(Rabbit haemorrhagic disease Virus，RHDV)抗体ELISA检测方法，我们克隆了RHDV的VP60全长基因，构建了表达质粒。对表达产物进行了纯化，将其作为抗原，在国内首次建立了用于RHDV抗体检测的RHDV-VP60-ELISA方法，并对国内7省市的338份普通级兔血清和49份SPF兔血清进行RHDV抗体检测。 根据几株已知的RHDV VP60序列设计引物，从中国农业大学动物医学院提供的发病兔肝中提取了RHDV RNA，用Oligo(dT)作为引物反转录合成cDNA，用PCR扩增出VP60基因片段。将纯化的基因产物连接到pGEM-T-easy载体，测序得到克隆基因的核苷酸序列，经BLAST程序搜索证实为RHDV VP60全长基因，将重组质粒命名为pGEM-T-VP60。同时，利用分子生物学软件DNASTAR将得到的目的基因序列同其它已知RHDV VP60序列进行比较，并进行进化树分析。 我们构建了原核表达质粒并在大肠杆菌中高效表达了包含全长VP60序列的pET-VP60重组蛋白，该重组蛋白为包涵体表达。通过亲和层析方法对该蛋白进行了纯化，得到了纯度为98％的目的蛋白。 用表达的该目的蛋白作为包被抗原，以HRP标记的山羊抗兔IgG作为二抗，按间接酶联免疫试验原理，建立了RHDV抗体的ELISA方法，在对方法的敏感性、特异性、精密性、重复性和稳定性测定、比较分析的基础上，利用ELISA方法对来自国内7省市的338只普通级兔和49只SPF兔进行RHDV抗体检测，以此对实验用兔RHDV免疫效果做出客观评价。 根据实验数据显示，实验中得到的重组蛋白具有良好的抗原性，RHDV-VP60-ELISA方法的敏感性、特异性、重复性、精密性与稳定性好。RHDV-VP60的克隆表达以及RHDV-VP60-ELISA检测方法的建立，解决了RHDV病毒制备和提纯需要一定实验条件和复杂的问题，也克服了以往实验用兔RHDV抗体检测方法敏感性不高、操作繁琐、标准化程度低并难以推广应用的实更多还原

【Abstract】 Rabbit haemorrhagic disease virus (RHDV) is the pathogeny microbe which must be ridded from laboratory animals. Because there isn’t appropriate susceptive cell, the test of RHDV is the difficulty in control of the quality for laboratory animals.To analyze the antigenic activity of the expression products from VP60 gene of RHDV, the full-length VP60 gene was cloned and a recombinant expression plasmid was constructed. The expression product was purified and used as antigen to detect the anti-RHDV serum. And, 338 ordinary rabbit serum and 49 SPF rabbit serum samples from 7 provinces and cities have been examined.PCR primers were designed according to the sequences of several known VP60 gene. Viral RNA was extracted from the liver of a rabbit with RHDV . The cDNA was reverse-transcripted using Oligo(dT) as 3’ out primer and PCR was carried out to achieve the full length of VP60 gene. The PCR products were purified and ligated to pGEM-T-easy vector and sequenced. The sequences were search searched against database using BLAST program and conformed that they were the RHDV VP60 sequences. We designated this recombinant plasmid as pGEM-T-VP60. As the same time, multiple sequence alignment and phylogenetic tree analysis was done by DNASTAR software.The prokaryotic expressing plasmid was constructed, and it was designated as pET-VP60. The pET-VP60 recombinant protein was expressed as inclusion bodies with high efficiency in E.coli. The protein has been purified with the affinity chromatography, and the purity of the target protein was 98%.The data show the purified recombinant protein pET-VP60 possesses good antigenicity. The pET-VP60 was coated as antigen, the HRP-conjugated goat anti-rabbit IgG(H+L) as the secondary antibody, the poly anti-RHDV diagnostic kit更多还原