犬细小病毒VP2蛋白的表达及间接ELISA方法的建立

Expression of VP2 gene of canine parvovirus virus and development of a recombinant protein-based indirect ELISA for antibodies detection

以犬细小病毒(Canine parvovirus virus,CPV)VP2基因的原核表达产物为抗原建立检测CPV抗体的间接ELISA方法。在分析CPV VP2基因稀有密码子(大肠杆菌系统)的基础上,克隆去除5′和3′端的稀有密码子的VP2基因,构建了VP2蛋白原核表达载体pET28a-VP2和pET32a-VP2,利用HIS标签对融合表达产物进行纯化。经SDS-PAGE和Western-blot检测证实该基因获得表达,并存在低相对分子质量产物,其中纯化后的pET28a-VP2具有良好的免疫学活性。以pET28a-VP2蛋白为基础,建立检测VP2抗体的i VP2-ELISA方法:抗原包被质量浓度为17.8 mg/L,血清最佳稀释度为1∶20,阳性判定标准初步定为:待测样品D值>0.367,且待测样品D值/阴性样品D值>2.0。采用i VP2-ELISA对20份背景清晰的犬血清样品进行检测,结果显示,i VP2-ELISA与HI试验的阴阳性符合率一致,但不呈现正比关系。本试验为犬场CPV抗体水平检测和进行CPV流行病学调查提供了一种简便的血清学诊断方法。

We developed an indirect ELISA method for detecting canine parvovirus virus（CPV） antibodies based on the recombinant pertactin protein expressed in Escherichia coli（DE3） strain.Base on analysis of CPV VP2 gene,we design a pair of primers to amplify the gene fragment except 5＇ and 3＇ rare codons in bacteria system,and constuct prokaryotic expression vector pET28a-VP2 and pET32a-VP2.The recombinant plasmids were transformed into E.coli BL21,and expressd after induced with IPTG.The expressed products were purified by His Tag of pETs vector,and identified by SDS-PAGE assay and Western-blot.The recombinant protein fragment pET28a-VP2 showed a great activity of immunity.The pET28a-VP2-based indirect ELISA was developed for detecting antibodies against CPV named iVP2-ELISA.The result showed that the opimal concentration of coated antigen was 17.8 mg/L and the best dilution of of serum was 1∶20.The positive criterion of this ELISA assay was D the tested serum 0.367 and D the tested serum/D the negtive serum 2.0.The method result was consistent with HA/HI assay and iVP2-ELISA,but not suggest a direct proportion relation.The assay showed excellent specificity,sensitivity and reduplication,and can be useful for epidemiological survey and clinical diagnosis of canine parvorivus.