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目的:利用大肠杆菌表达牛FMDV受体整合素β6亚基的配体结合域(LBD)片段,纯化重组目的蛋白后制备兔多克隆抗体并对其进行初步应用。方法:以含有牛整合素β6亚基全长cDNA的质粒为模板PCR扩增得到β6LBD基因片段,与原核表达载体pGEX4T-1相连,构建原核表达质粒pGEX/β6LBD,测序确认开放阅读框正确后,转化感受态细胞BL21(DE3),以IPTG诱导表达重组牛β6LBD融合蛋白。SDS-PAGE和Westernblot鉴定后提取包涵体,电泳分离纯化重组融合蛋白,免疫新西兰白兔制备多克隆抗体,以ELISA、琼脂扩散实验、免疫组化鉴定该抗体的效价和特异性。结果:重组牛β6LBD融合蛋白在大肠杆菌中获得了高效表达,重组蛋白主要以包涵体形式表达,相对分子质量(Mr)约为45000。制备的兔多克隆抗体效价达1∶12800以上,该抗体可与牛舌上皮细胞中的抗原分子结合,具有很好的特异性。结论:实现了牛FMDV受体整合素β6亚基LBD的原核表达和抗体制备,为研究受体β6亚基在牛体内的分布及其在FMDV感染过程中的作用机制提供了工具。
OBJECTIVE: To clone polyclonal antibody of rabbit FMDV integrin β6 subunit of integrin β6 by E. coli and purify the recombinant protein. Methods: The β6LBD gene fragment was amplified by PCR from the full length cDNA of bovine integrin β6 subunit. The prokaryotic expression vector pGEX4T-1 was ligated with the prokaryotic expression vector pGEX / β6LBD. The correct open reading frame was confirmed by sequencing. Transforming competent cells BL21 (DE3) to induce the expression of recombinant bovine β6LBD fusion protein by IPTG. Inclusion bodies were identified by SDS-PAGE and Western blot. The recombinant fusion proteins were isolated by electrophoresis and immunized New Zealand white rabbits to prepare polyclonal antibodies. The titer and specificity of the antibodies were determined by ELISA, agar diffusion and immunohistochemistry. RESULTS: The recombinant β6LBD fusion protein was highly expressed in Escherichia coli. The recombinant protein was mainly expressed in inclusion bodies. The relative molecular mass (Mr) was about 45,000. The prepared rabbit polyclonal antibody titer of more than 1: 12800, the antibody can be combined with antigen molecules in the tongue epithelial cells, has very good specificity. CONCLUSION: Prokaryotic expression and antibody preparation of integrin β6 subunit LBD of FMDV receptor were achieved, which provided a tool for studying the distribution of receptor β6 subunit in cattle and its mechanism of action in FMDV infection.