H3N2亚型犬流感病毒NP蛋白的表达纯化及多克隆抗体制备

EXPRESSION AND CHARACTERIZATION OF RECOMBINANT NUCLEOPROTEIN OF H3N2 SUBTYPE CANINE INFLUENZA VIRUS

将分离的H3N2亚型犬流感病毒(Canine influenza virus,CIV)A/Canine/Nanjing/11/2012(H3N2)株的核蛋白(NP)基因克隆至原核表达载体p ET28a(+)中,构建重组表达质粒p ET-NP,然后转化大肠杆菌E.coli Rosetta(DE)感受态细胞,经IPTG诱导后,采用SDS-PAGE和Western blot进行分析。结果显示,大肠杆菌表达的重组NP蛋白的分子量约为61 k Da,与预期相符,并能与犬CIV阳性血清发生特异性反应。将表达的重组NP蛋白进行纯化后,免疫大白兔制备抗NP蛋白多克隆抗体血清,Western blot检测该血清可与CIV的NP蛋白发生特异性反应,间接ELISA检测该多克隆抗体血清的效价达1:30 000,显示了较高的抗体效价。本研究为CIV快速检测方法的建立和流行病学调查奠定了科学依据。

The nucleoprotein(NP)gene of H3N2subtype Canine infl uenza virus(CIV)A/Canine/Nanjing/11/2012(H3N2)isolated from Nanjing,China,was cloned into pET-28a(+)for generation of a recombinant plasmid pET-NP.The resulting recombinant pET-NP was then transformed into E.coli Rosetta(DE3)competent cells following by induction with IPTG.The expression of the NP protein was detected in SDS-PAGE and Western blot.The results showed that the recombinant NP was61kDa protein corresponding to the expected molecular mass and reacted with the antiserum against H3N2subtype CIV.The recombinant NP of CIV was purifi ed via Ni-NTA affi nity chromatography and the polyclonal antibodies were produced by immunizing rabbits with purifi ed recombinant NP.Western blot indicated that the polyclonal antibodies specifically recognized the recombinant NP.The polyclonal antibodies were titrated in indirect ELISA using the purifi ed recombinant NP as the coating antigen and the titer was as high as1:30000.The availability of the recombinant NP and polyclonal antibodies laid a foundation for development of rapid detection and epidemiological methods for CIV.