VPAHPND毒力蛋白PirAB的原核表达与纯化

Prokaryotic Expression and Purification of VPAHPND Virulence Protein PirAB

急性肝胰腺坏死病(Acute Hepatopancreatic Necrosis Disease, AHPND)是一种新型对虾病害,其病原为一类携带编码二元毒素蛋白PirAB的弧菌(VPAHPND),可感染对虾且致死率高。本研究利用GenBank公布的pirAB全基因序列设计特异性引物,通过PCR获得目的基因pirA、pirB、pirAB并克隆至表达载体pET 21b(+)中,构建BL21-pET21b-pirA、BL21-pET21b-pirB、BL21-pET21b-pirAB原核表达体系;通过对诱导剂浓度、诱导温度和诱导时间进行优化,分析重组蛋白的最佳表达条件;采用Ni磁珠对重组蛋白进行纯化,并以Western-blot方法分析其抗原性。结果表明,PirA、PirB、PirAB的重组蛋白分子质量分别约为17 kDa、50 kDa和(50+17) kDa,与预期大小一致。重组蛋白的最佳诱导条件:IPTG浓度0.01 mmol/L,PirA和PirAB 30℃诱导24 h、PirB 30℃诱导4 h,蛋白均呈现可溶性表达;纯化后的重组蛋白具有较好的反应原性,可为下一步的抗体制备和AHPND致病机理研究提供充足的实验材料。

Acute Hepatopancreatic Necrosis Disease(AHPND) is a new disease of shrimp.The pathogen of AHPND is a kind of Vibrio(VPAHPND) carrying a binary toxin PirAB,which can infect shrimp with high mortality.In this study, the whole gene sequence of pirAB published by GenBank was used to design specific primers, and the target genes of pirA,pirB,and pirAB were obtained by PCR and cloned into the expression vector pET 21b(+) to construct prokaryotic expression systems of BL21-pET21b-pirA,BL21-pET21b-pirB,and BL21-pET21b-pirAB.The optimal expression conditions of recombinant protein were analyzed by optimizing the concentration of inducer, induction temperature, and induction time.The Ni magnetic beads were used to purify the recombinant protein, and its anti-genicity was analyzed by Western blot method.The results showed that the molecular weights of the recombinant proteins of PirA,PirB and PirAB were about 17 kDa, 50 kDa and(50+17) kDa, respectively, which were consistent with the expected size.The optimum induction conditions of recombinant protein were as follows: IPTG concentration was 0.01 mmol/L,PirA and PirAB were induced at 30℃ for 24 h, and PirB was induced at 30℃ for 4 h.The protein was expressed in a soluble form.The purified recombinant protein has a good reactivity, which can provide sufficient experimental materials for the next antibody preparation and AHPND pathogenesis research.