论文部分内容阅读
为获得纯化的山羊地方性鼻内肿瘤病毒(enzootic nasal tumor virus of goats,ENTV)表面蛋白(surface protein,SU protein)和抗SU蛋白的多克隆抗体,根据Gen Bank已登录的ENTV Shaanxi株SU基因序列,设计合成1对特异性引物,PCR扩增SU基因并将其连接于原核表达载体p ET-28a(+)中,构建重组质粒p ET-28a-SU,经鉴定正确后转化大肠杆菌Rosetta(DE3)株进行诱导表达,并进行SDS-PAGE分析。重组菌经IPTG诱导后成功表达出分子质量为43 ku的重组蛋白。将诱导表达的蛋白经镍柱亲和层析纯化、复性后免疫小鼠制备多克隆抗体。Western-blot分析结果表明,原核表达纯化的SU蛋白能够与制得的小鼠抗SU蛋白多克隆抗体特异性结合。上述结果表明,本试验成功获得了纯化的ENTV SU蛋白和小鼠抗ENTV SU蛋白的多克隆抗体,为进一步研究SU蛋白在ENTV感染过程中的作用和建立诊断方法提供了材料。
In order to obtain purified polyclonal antibodies to enzootic nasal tumor virus of goats (ENTV) surface protein (SU protein) and anti-SU protein, based on the SU gene Sequence, a pair of specific primers were designed and synthesized. The SU gene was amplified by PCR and ligated into the prokaryotic expression vector p ET-28a (+). The recombinant plasmid p ET-28a-SU was constructed and transformed into Escherichia coli Rosetta (DE3) strain was induced and analyzed by SDS-PAGE. Recombinant bacteria induced by IPTG successfully expressed a molecular weight of 43 ku recombinant protein. The induced protein was purified by nickel column affinity chromatography, and the mice were immunized to prepare polyclonal antibodies. The results of Western-blot analysis showed that the purified SU protein prokaryotic expressed specifically with the mouse anti-SU protein polyclonal antibody. The above results show that the purified ENTV SU protein and mouse anti-ENTV SU protein polyclonal antibody were successfully obtained in this study, which provided a material for further study on the role of SU protein in ENTV infection and the establishment of diagnostic methods.