羊口疮病毒蛋白ORFV035的表达、纯化和多克隆抗体的制备

Expression and Purification of orf Virus Protein ORFV035 and Preparation of Polyclonal Antibody

克隆表达羊口疮病毒蛋白ORFV035,并制备其多克隆抗体,为后续对病毒复制、装配、形态发生和成熟过程的研究奠定基础。PCR扩增羊口疮病毒ORFV035基因,将其与质粒pET-30a(+)经Bam HⅠ和HindⅢ双酶切后连接,构建重组质粒pET30a-035。重组质粒经双酶切和测序鉴定,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,纯化后目的蛋白免疫小鼠,制备多克隆抗体并对其进行鉴定。成功构建了重组质粒pET30a-035,在大肠杆菌BL21中以包涵体形式高效表达。包涵体洗涤、溶解后进行Ni柱纯化,得到纯度较高的ORFV035-his融合蛋白。以纯化蛋白免疫小鼠获得多克隆抗体。Western blot检测显示该多抗可以识别天然ORFV035蛋白。成功诱导表达、纯化ORFV035蛋白并制备ORFV035多克隆抗体。

This work aims to express and purify recombinant orf virus encoded protein 035（ORFV035）in Escherichia coli and preparea polyclonal antibody（p Ab）against ORFV035 for immunoassays,as well as lay foundation for the further researches on the replication,assembly,morphogenesis,and mature process. The gene of ORFV035 was amplified by PCR from orf viral genome,and sub-cloned intothe expression vector p ET-30a（＋）,and recombinant plasmid p ET-30a-035 was constructed by enzymatic digestion and ligation of Bam HI and Hind Ⅲ. Then the plasmid p ET-30a-035 after double enzyme digestion and sequencing was transformed into Escherichia coli BL21,the transformants were induced by IPTG,and SDS-PAGE was used to identify the expression of the protein. The His-tagged fusion proteinORFV035 was ultrasonic-treated and then purified through Ni-chelating affinity chromatography. The purified ORFV035 was used to immuneinto BALB/c mouse for producing anti-ORFV035 p Ab. The results showed that the recombinant plasmids p ET30a-035 was successfullyconstructed,and the protein ORFV035 in E. coli BL21 was in inclusion body. The ORFV035-his fusion protein was obtained after washing,dissolving,and purifying the inclusion body,and the p Ab anti-ORFV035 was prepared. The Western blot analysis showed that this p Abrecognized natural ORFV035.