鸡传染性支气管炎M41毒株S1基因的原核表达及间接ELISA抗体检测方法初步建立

Prokaryotic expression of S1 gene of the M41 strain of avian infectious bronchitis virus (IBV)and the initial establishment of an indirect ELISA assay for detecting IBV antibodies

为获得鸡传染性支气管炎病毒(IBV)M41株的全长S1蛋白,并以S1蛋白为包被抗原建立间接ELISA抗体检测方法,试验采用RT-PCR方法扩增出S1基因,将其克隆到pBluescriptⅡKS(+)载体中,酶切、测序鉴定后将S1基因导入融合表达载体pMAL-c2X中,将重组质粒转化大肠杆菌TB1,IPTG诱导后,应用SDS-PAGE及Western-blot检测蛋白表达情况。结果表明:融合蛋白得到了表达;以纯化的重组蛋白作为包被抗原,在包被量为3.2μg/孔、血清160倍稀释条件下P/N值最大,初步建立了IBV抗体间接ELISA检测方法。

To obtain the S1 whole-length protein from the strain M41 of infectious bronchitis virus（IBV）,and to establish an indirect enzyme-linked immunosorbent assay（ELISA） for measuring antibodies,taking the S1 protein as coating antigen.The S1 gene was amplified by RT-PCR and cloned into the pBluescript Ⅱ KS（＋） vector.After confirmed by the restriction endonuclease analysis,the positive clone of S1 gene was sequenced and then transferred into the expression vector of fused protein pMAL-c2X.The recombinant plasmid was transformed into E.coli TB1.After inducing with IPTG,the protein expression was analyzed and determined using SDS-PAGE and Western-blot analysis.The results showed that the fusion protein was expressed.Using the purified recombinant protein as a coating antigen,the P/N reached the maximum value when the coating antigen was used at 3.2 μg per well with 160-fold diluted immune serum.It indicates that an indirect ELISA assay for detecting IBV antibodies is initially established.