藤壶cp19k基因mRNA二级结构对其在大肠杆菌中表达的影响

Effect of secondary structure of barnacle cp19k gene mRNA on its expression in Escherichia coli

目的探究藤壶cp19k基因5′端mRNA二级结构对其在大肠杆菌中表达的影响。方法通过对3个藤壶物种的cp19k基因(balcp19k、mrcp19k、bicp19k)序列进行分析,并利用大肠杆菌密码子偏好性对其进行优化,将优化后的3个基因序列利用BioSun软件分析其5′端和3′端的mRNA二级结构和自由能。利用大肠杆菌原核表达系统对3个基因进行表达,并通过SDS-PAGE观察蛋白表达情况。结果3个基因的3′端mRNA二级结构和自由能差异较小;5′端mRNA二级结构相比,balcp19k结构相对简单,自由能较高,mrcp19k和bicp19k二级结构较稳定,自由能较低。SDS-PAGE及Western印迹结果显示,Balcp19k蛋白有表达,而Mrcp19k蛋白和Bicp19k蛋白无表达。结论cp19k基因5′端mRNA二级结构稳定且自由能低不利于蛋白表达。此结果为藤壶cp19k的进一步研究奠定了基础,也为其他外源基因在原核系统中表达提供了借鉴。

Objective To explore the effect of the secondary structure of the 5′-end mRNA of barnacle cp19k gene on its expression in Escherichia coli.Methods The cp19k gene(balcp19k,mrcp19k and bicp19k)sequences of three barnacle species were analyzed and optimized by E.coli codon preference.The optimized three gene sequences were analyzed by BioSun software for their 5′ends,the mRNA secondary structure and free energy at the 3′-end.The three genes were expressed by E.coli prokaryotic expression system,and protein expression was observed by SDS-PAGE.Results The secondary structure and free energy of the 3′-end mRNA of the three genes were not significantly different.Compared with the secondary structure of the 5′end mRNA,the structure of balcp19k was relatively simple and free energy was high.The secondary structure of mrcp19k and bicp19k was relatively stable,but free energy lower.The results of SDS-PAGE and Western blotting showed that Balcp19k protein was expressed,but Mrcp19k and Bicp19k proteins were not.Conclusion The low free energy and stable secondary structure of cp19k gene 5′-end mRNA are not conducive to protein expression,which can contribute to further study of cp19k in barna-barna-barna-barna-cp19k and provide references for the realization of expressions of other exogenous genes in the prokaryotic system.