草鱼潜在过敏原α-烯醇化酶原核表达条件的优化

Optimization of prokaryotic expression conditions for potential allergen α-Enolase in Ctenopharyngodon idella

中文关键词:  草鱼  烯醇化酶  原核表达  包涵体  重组蛋白

英文关键词:Ctenopharyngodon idella  Enolase  prokaryocyte expression  inclusion body  recombinant protein

中文摘要:

      目的 通过对草鱼α-烯醇化酶(α-Enolase)原核表达的条件进行优化, 分别从表达的上清液与包涵体中纯化获得重组α-Enolase。方法 本研究基于前期克隆的草鱼α-Enolase基因序列设计表达引物, 将草鱼α-Enolase基因克隆至pET-30a质粒中, 得到pET-30a-α-Enolase重组质粒。然后, 将重组质粒转入大肠杆菌Origami 2 (DE3)感受态细胞中, 并用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)进行体外诱导表达。采用十二烷基硫酸钠-聚丙烯酰胺电泳方法分析重组蛋白的可溶性, 并对表达条件进行优化。利用亲合层析法纯化目的蛋白。结果 在20℃诱导15 h得到的重组蛋白以两种形式存在: 一部分为可溶性蛋白存在于上清液中, 一部分以包涵体形式存在于沉淀中; 在37℃诱导4 h得到的重组蛋白则只有包涵体形式。包涵体蛋白表达量最大的优化条件为: 诱导温度为37℃, IPTG终浓度1.0 mmol/L, 诱导时间4 h。采用亲合层析法分别纯化上清液(20℃诱导15 h)和包涵体中(37℃诱导4 h)的重组蛋白, 得率分别为3.5 mg/100 mL和4.9 mg/100 mL菌液。结论 利用原核表达从上清液与包涵体中均可纯化得到重组α-Enolase。但这两种α-Enolase在生物活性与免疫原性是否存在差异性, 以及通过重组α-Enolase是否可以代替天然蛋白用于鱼类过敏原的研究, 有待进一步研究。

英文摘要:

      Objective To obtain the purified recombinant α-Enolase from the supernatant and inclusion bodies, optimize the prokaryotic expression conditions for α-Enolase in Ctenopharyngodon idella. Methods The expression primers were designed based on the α-Enolase gene sequence of Ctenopharyngodon idella cloned previously in our laboratory. The recombinant plasmid pET-30a-α-Enolase was constructed, afterward, the recombinant plasmid was transferred into Escherichia coli Origami 2 (DE3) competent cells, and the expression of recombinant protein was induced at different temperature by isopropyl-β-D-thiogalactopyranoside (IPTG). The solubility of the recombinant protein was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the expression conditions were optimized. Finally, the target protein was purified using affinity chromatography. Results The recombinant proteins under the induction condition of 20℃ for 15 hours showed two forms, in which the soluble protein was in the supernatant while the inclusion bodies was in the sediment. However, the recombinant proteins under the induction condition of 37℃ for 4 hours were only inclusion bodies. The optimal conditions for the maximum expression of inclusion body protein were as follows: Induction temperature was 37℃, final IPTG concentration was 1.0 mmol/L, and induction time was 4 h. The target protein was purified from the supernatant (induced at 20℃ for 15 h) and inclusion bodies (induced at 37℃ for 4 h) using affinity chromatography. The 3.5 mg of recombinant protein was obtained from the supernatant of every 100 mL of bacterial suspension, and 4.9 mg of recombinant protein was obtained from the sediment of every 100 mL of bacterial suspension. Conclusion Recombinant α-Enolase can be purified from the supernatant and inclusion bodies respectively. However, whether there are differences in the structure, biological activity, and immunogenicity between the 2 kinds of forms of recombinant α-Enolase, and whether recombinant α-Enolase can substitute for the natural one in the study of fish allergen. These problems are still unclear, which need further research.