Abstract 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 IPTG (isopropy-β-D-thiogalactopyranoside)(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 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 significant difference among the 4, 8 and 24 h with injection of PBS (P＞0.05). Our results indicated exogenous MIH recombination protein could shortly promoted the expression of endogenous MIH protein, and then partly restrain the molting in crab. This result provided insight into the molting mechanism of crab and made a foundation for antibody preparation in future.
CHEN Jiao,CHEN Xiao-Wen,YU Jun, et al. Prokaryotic Expression of MIH Gene and Effect of Its Exogenous Recombinant Protein on Endogenous Gene Expression in Eriocheir sinensis[J]. , 2016, 24(4): 576-583.
[1]曹佳培, 2013.中华绒螯蟹蜕皮抑制激素(MIH)基因的原核表达及其分泌特征的研究[D]. 硕士学位论文, 河北大学, 导师: 康现江. pp. 32-35. [2]蔡生力, 杨丛海.2000. 体外注射激素对中国对虾卵巢发育的影响[J]. 中山大学学报: 自然科学版(z1): 91-95. [3]崔剑, 2012.TAT-Apoptin原核表达载体的构建, 可溶性表达及体外活性检测[D]. 硕士学位论文, 辽宁大学, 导师: 贲松彬. pp. 34-48. [4]郭豫杰, 2004.中华绒螯蟹蜕皮抑制激素1(Ers—MIH1)-GST融合蛋白在大肠杆菌中的表达[D]. 硕士学位论文, 南京师范大学, 导师: 周开亚, pp. 38-47. [5]郭松, 傅明骏, 赵超, 等.2015. 斑节对虾cyclinH基因的原核表达和蛋白纯化[J]. 广东农业科学, 42(3): 125-130. [6]李世东, 2012.重组质粒pET-OmpU的构建, 表达及其对锦鲤的免疫保护作用[D]. 硕士学位论文, 安徽农业大学, 导师: 李槿年. pp. 14-29. [7]刘泳, 2010.肌肉生长抑素原核表达条件优化与表达产物鉴定[D]. 硕士学位论文, 华中农业大学, 导师: 杨利国. pp. 1-7. [8]林静云, 白志毅, 李家乐, 等.2013. 三角帆蚌(Hyriopsis cumingii)Perlucin 蛋白原核表达条件的优化及表达产物的鉴定[J]. 生物技术通报, 7: 021. [9]苗田田, 赵金良, 苌建菊, 等.2012. 外源生长激素对尼罗罗非鱼生长, 骨骼肌纤维增生及肥大的影响[J]. 农业生物技术学报. 20(11): 1315-1320. [10]邱高峰, 张爱萍, 楼允东.2003. 锯缘青蟹蜕皮抑制激素 cDNA 的分子克隆及其表达分析[J].水产学报, 27(3): 207-212. [11]宋霞, 周开亚, 马长艳.2003. 中华绒螯蟹蜕皮抑制激素1(MIH1)基因的 cDNA 片段克隆和 Northern 印迹分析[J]. 中国水产科学, 10(5): 353-358. [12]孙妍, 张亦陈, 刘逸尘, 等.2011. 中华绒螯蟹蜕皮抑制激素基因全长cDNA克隆和重组表达[J]. 水生生物学报, 35(2): 210-217. [13]孙赛红, 2014.红鳍东方鲀4个免疫基因的原核表达及群体EST-SSRs分析[D]. 硕士学位论文, 大连海洋学院, 导师: 王秀利. pp. 21-44. [14]王在照, 相建海, 崔朝霞.2002. 编码中华绒螯蟹蜕皮抑制激素基因的cDNA片段克隆和序列分析[J]. 海洋与湖沼, 33(4). [15]吴秀梅, 徐黎明, 赵景壮, 等.2015. 哲罗鱼胰岛素样生长因子-II的原核表达与活性分析[J]. 中国水产科学, 22(2): 243-249. [16]姚燕, 周开亚, 宋大祥.2006. 中华绒螯蟹蜕皮抑制激素基因的表达及抗体制备[J].动物学报. 52(1): 209-214. [17]赵杰, 2014.草鱼IL-8基因在炎症反应中的功能研究[D]. 硕士学位论文, 苏州大学, 导师: 宋学宏, pp. 52-58. [18]臧坤, 徐永江, 柳学周, 等.2015. 星突江鲽胰岛素样生长因子II的原核表达与生物活性分析[J]. 中国水产科学, 22(2): 214-223. [19]张喆, 李健, 王芸, 等.2011. 中国对虾细胞色素P450基因CYP4原核表达条件优化[J]. 海洋科学, 35(9): 49-55. [20]Chang E S, Mykles D L.2011. Regulation of crustacean molting: a review and our perspectives. General and comparative endocrinology[J], 172(3): 323-330. [21]Chung J, Webster S.2005. Dynamics of in vivo release of molt-inhibiting hormone and crustacean hyperglycemic hormone in the shore crab, Carcinus maenas. Endocrinology, 146(12): 5545-5551. [22]Hopkins P M.2009. Crustacean ecdysteroids and their receptors[J]. Ecdysone: structures and functions, 9: 73-97. [23]Lee K J, Elton T S, Bej a k.et al. 1995. Molecular cloning of a cDNA encoding putative molt-inhibiting hormone from the blue crab, Callinectes sapidus[J]. Biochemical and biophysical research communications, 209(3): 1126-1131. [24]Livak K J, Schmittgen T D, 2001.Analysis of relative gene expression data using real-time quantitative PCR and the 2? ΔΔCT method[J]. methods, 25(4): 402-408. [25]Lee S G, Bader B D, Mykles D L, et al.2007. Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases inthe tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signalingin the molting gland[J]. Experimental Biology, 210: 3245-3254. [26]Nakatsuji T, Sonobe H, Watson R D, et al.2006. Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish (Procambarus clarkii): involvement of cyclic GMP and cyclic nucleotide phosphodiesterase[J]. Molecular and cellular endocrinology, 253(1): 76-82. [27]Nakatsuji T, Lee C Y, Watson R D.2009. Crustacean molt-inhibiting hormone: structure, function, and cellular mode of action[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 152(2): 139-148. [28]Stewart M J, Stewart P, Sroyraya M, et al.2013. Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus[J]. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 164(2): 276-290. [29]Techa S, Chung J S.2015. Ecdysteroids Regulate the Levels of Molt-Inhibiting Hormone (MIH) Expression in the Blue Crab, Callinectes sapidus[J]. Plos one, 4(7). [30]Sonobe H, Kamba M, Ohta k, et al.1991. In vitro secretion of ecdysteroids by Y-organs of the crayfish, Procambarus clarkii[J]. Experientia, 47(9): 948-952.
