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.
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