中华绒螯蟹MIH基因的原核表达及其外源重组蛋白对内源性基因表达的影响  

Prokaryotic Expression of MIH Gene and Effect of Its Exogenous Recombinant Protein on Endogenous Gene Expression in Eriocheir sinensis

作　　者：陈娇[1] 陈晓雯[1] 王军[1] 黄姝[1] 岳武成 王成辉[1] 

机构地区：[1]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306

出　　处：《农业生物技术学报》2016年第4期576-583,共8页Journal of Agricultural Biotechnology

基　　金：上海市中华绒螯蟹产业技术体系项目(沪农科(产)字2015-4号);上海市科委崇明科技专项项目(No.13391912102);上海市工程技术中心建设项目(No.03DZ2251800)

摘　　要：蜕皮抑制激素(molt inhibiting hormone,MIH)对甲壳动物的蜕壳生长起着至关重要的调控作用。本研究通过优化MIH基因的原核表达条件,获得其外源重组蛋白,研究了外源重组蛋白注射到中华绒螯蟹(Eriocheir sienesis)体内后,对眼柄中内源性MIH基因表达的影响。结果发现,构建的重组表达质粒(p ET-28a-MIH)在27℃条件下用1.0 mmol/L的异丙基-β-D-硫代半乳糖苷(isopropy-β-Dthiogalactopyranoside,IPTG)诱导6 h,目的蛋白表达量最高,此为该基因的原核表达优化条件。进而将MIH重组蛋白注射到活体中华绒螯蟹,发现注射4 h后在眼柄中的内源MIH表达水平显著高于8、24 h和空白对照组,表明外源性MIH重组蛋白只能在短时期内对内源性MIH表达有促进作用,从而会在一定程度上抑制中华绒螯蟹的蜕壳。研究结果为中华绒螯蟹的蜕壳机制研究和后续的抗体制备提供了基础资料。Molt inhibiting hormone （MIH） is an essential hormone in regulating molting and growth of crustacean, which is produced in eyestalk of crustacean. However, it is very difficult to obtain MIH content in single individual because separation and purification of this protein require large numbers of samples, which limits the physiology and biochemical study of MIH gene. Thus, an alternative way is to use prokaryotic expression for conducting large amount of recombinant proteins. By now, the influence of the exogenous recombinant MIH protein on the endogenous MIH gene expression of Chinese mitten crab （Eriocheir sienesis） has been remained unclear. In this study, a series of experiments were conducted to identify the optimal conditions for prokaryotic expression of MIH gene. Its exogenous recombinant protein was firstly used via injecting into the eyestalk of Chinese mitten crab for observing the expression profiles of endogenous MIH gene. The experiments conditions included different concentration of isopropy-β-D-thiogalactopyranoside （IPTG） （0.5, 1.0, 2 and 4 mmoL/L）, different induction time （0, 2, 4 and 6 h） , and different induction temperature （27 and 37 ℃ ）. The results indicated that the highest amount of content of recombinant MIH protein were obtained under the conditions of IPTG with concentration of 1.0 mmol/L for 6 h at 27 ℃. The MIH recombinant protein concentration was 2.3 mg/mL under this condition. Then the recombinant protein from the induced bacterial expression （pET-28a-MIH） were injected into living crabs. It was found that the expression level of MIH gene in eyestalk was significantly higher after 4 h injection than those of phosphate buffered saline （PBS） control group （P〈0.05）, while no significant difference was found among 8 h, 24 h injection and PBS control groups （P〉0.05）. Furthermore, the expression level of endogenous MIH gene in eyestalk was significant higher after 4 h than that after 8 and 24 h （P〈0.05）, while there were no signif

关 键 词：中华绒螯蟹 蜕皮抑制激素(MIH) 原核表达 条件优化 基因表达 

分 类 号：S917[农业科学—水产科学]