抗冻蛋白AFPⅢ的原核表达、纯化及多克隆抗体的制备

Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Antifreeze Protein AFPⅢ

采用基因重组技术将AFPⅢ基因与原核表达载体pET32a(+)连接,转化大肠杆菌BL21(DE3),通过PCR、单双酶切及测序鉴定构建结果。用IPTG诱导蛋白表达,将融合蛋白纯化后,免疫6-7周龄的小白鼠,制备AFPⅢ多克隆抗体,采用Western印迹法检验抗体特异性。通过酶联免疫吸附试验(ELISA)测定多克隆抗体滴度。成功地构建了AFPⅢ的原核表达载体,经在大肠杆菌中诱导表达、镍柱亲和层析纯化,得到较纯的相对分子质量约26 000 Da的融合蛋白,免疫小白鼠后得到多抗血清,Western印迹结果显示此多克隆抗体与AFPⅢ蛋白特异性结合。该试验为进一步研究AFPⅢ在转基因鱼中的定点表达以及作用机制奠定了基础。

Gene recombination technology was used to link AFPⅢ gene and prokaryotic expression vector pET32a（＋） and then the reeombinant plasmid was transformed into Escherichia coli（BL21）and the positive clones were identified by restriction enzyme digestion, PCR and sequencing. Expression AFPⅢ was induced with IPTG and the expression product was purified, which the purified AFPⅢ was used to immunize mouse to obtain the antiserum. The specificity of the antibodies was examined by Western blotting. The purified antigen was found to have antigenicity by ELISA and the prokaryotie expression vector pET32a-AFPⅢ to be successfully constructed. A fusion protein with a molecular weight of about 26 kD was obtained after induction with IPTG and affinity chromatography. The anti-AFPⅢ antibody was obtained from the immunized mouse. The results of Western blotting indicated that the polyclonal antibody had high specificity to AFPⅢ, providing a foundation for further research on the biological function of AFPⅢ and expression of AFPⅢ in transgenic fish tissues.