绵羊肺腺瘤病毒完整囊膜蛋白的多克隆抗体制备与特异性检测

Preparation and specific detection of polyclonal antibodies against complete envelope protein of sheep pulmonary adenoma virus

本研究旨在构建绵羊肺腺瘤病毒(JSRV)的囊膜基因(env)原核表达载体,制备多克隆抗体并对其特异性进行鉴定。以笔者所在实验室构建的pEGFP-C1/JSRV-env质粒为模板,PCR扩增env基因全长1848 bp,构建原核表达质粒并命名为pET-32a/JSRV-env,进行双酶切鉴定和测序分析后,转化到E.coli Transetta(DE3),经IPTG诱导及亲和层析法对其纯化。将纯化蛋白常规免疫成年新西兰大白兔制备多克隆抗体,通过Western-blot方法鉴定多克隆抗体的特异性,免疫组化方法评价其临床检测效果。双酶切鉴定及测序结果表明,已成功构建pET-32a/JSRV-env重组质粒且包含编码YXXM基序的基因序列,成功表达及纯化该蛋白,其大小约89 ku。以制备的多克隆抗体为一抗,经Western-blot检测自然感染绵羊肺腺瘤病(OPA)的病肺组织,在89 ku处出现单一特异性条带。免疫组化结果显示,OPA病肺肿瘤细胞可见棕黄色的阳性信号。结果表明,成功构建JSRV-env原核表达质粒,制备的多克隆抗体特异性较好,可用于生产实践中OPA的检测,且制备简便、成本低廉。同时也为进一步探讨JSRV Env的功能提供了实验基础。

The aim of this study was to construct the prokaryotic expression vector of envelope(env)of Jaagsiekte sheep retroviruse(JSRV)and prepare polyclonal antibody and identify its specificity.Using the pEGFP-C1/JSRV-env plasmid constructed in our laboratory as the template,the full-length 1848 bp of env gene was amplified by PCR,and the prokaryotic expression plasmid pET-32a/JSRV-env,was constructed and identified by double endonuclease digestion and sequencing,then transformed into E.coli Transetta(DE3).It was purified by IPTG induction and affinity chromatography.The purified protein was routinely immunized with adult New Zealand white rabbits to prepare polyclonal antibodies.The specificity of polyclonal antibodies was identified by Western-blot method,and the clinical detection effect was evaluated by immunohistochemical method.The results showed that the recombinant plasmid pET-32a/JSRV-env was successfully constructed and contained the gene sequence encoding YXXM motif.The protein was successfully expressed and purified,and its size was about 89 ku.Using the prepared polyclonal antibody as the first antibody,the lung tissues of naturally infected Ovine pulmonary adenomatosis(OPA)showed a single specific band at 89 ku by Western-blot.Immunohistochemical results showed that brown positive signals could be seen in lung tumor cells of sheep with pulmonary adenomatosis OPA.In summary,the prokaryotic expression plasmid of JSRV-env was successfully constructed and the polyclonal antibody prepared had good specificity,and could be used for the detection of OPA in production practice,and the preparation was simple and low cost.At the same time,it also provides an experimental basis for further discussion of the function of JSRV Env.