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根据Nectin-4基因序列设计特异性引物,进行PCR扩增,将其克隆至原核表达载体pGEX-4T-1中,经PCR、酶切和测序鉴定,成功构建了重组表达质粒pGEX-4T-Nectin-4。将重组质粒转化大肠埃希氏菌Rosetta感受态细胞,经IPTG诱导表达目的蛋白。表达产物经SDS-PAGE和Western blot鉴定正确后,纯化后免疫BALB/c小鼠,制备多克隆抗体血清,并利用琼脂扩散试验检测其效价。SDS-PAGE结果表明该重组蛋白获得高效表达,表达的蛋白以包涵体形式存在。Western blot结果表明表达的蛋白能与GST单抗发生反应,具有较好的反应原性。琼脂扩散试验结果表明制备的抗Nectin-4蛋白多克隆抗体效价达1∶8。由于Nectin-4是小反刍兽疫病毒新近发现的受体,这一研究为后续Nectin-4与小反刍兽疫病毒的相互作用研究奠定了一定基础。
According to the sequence of Nectin-4 gene, specific primers were designed and amplified by PCR. Cloned into the prokaryotic expression vector pGEX-4T-1, the recombinant plasmid pGEX-4T-Nectin was successfully constructed by PCR, restriction enzyme digestion and sequencing. -4. The recombinant plasmid was transformed into Escherichia coli Rosetta competent cells, induced by IPTG expression of the target protein. The expressed product was identified by SDS-PAGE and Western blot. After purification, the BALB / c mice were immunized and polyclonal antibody was prepared. The titer of polyclonal antibody was determined by agar diffusion assay. The results of SDS-PAGE showed that the recombinant protein was highly expressed, and the expressed protein was in the form of inclusion body. Western blot results showed that the expressed protein reacted with GST McAb and had good reactionogenicity. Agar diffusion test results show that the anti-Nectin-4 protein polyclonal antibody titer reached 1: As Nectin-4 is a newly discovered receptor for PCV, this study lays the foundation for the subsequent study of the interaction between Nectin-4 and PCV.