发形霞水母丝氨酸蛋白酶家族原核表达载体的构建及其表达

摘要

目的：获得发形霞水母丝氨酸蛋白酶，为深入研究发形霞水母丝氨酸蛋白酶的功能奠定基础。方法综合运用生物信息学分析发形霞水母丝氨酸蛋白酶分子的序列特征和活性功能。通过设计带有特异性酶切位点的引物，将编码发形霞水母丝氨酸蛋白酶的核苷酸序列（ CcSP1，CcSP2和CcSP3）从cDNA文库中扩增出来，利用pET－24a（＋）载体构建重组表达质粒（ pET24a－CcSP1、pET24a－CcSP2和pET24a－CcSP3），经过筛选和鉴定正确后转入表达菌Rosetta（DE3）．plysS。重组质粒转化表达菌在IPTG诱导下表达，利用SDS－PAGE电泳和Western－blot分析表达的重组蛋白。结果 SDS－PAGE电泳结果可见重组质粒转化的表达菌分别在34、42和30kDa处有蛋白条带，Western－blot检测显示，这些蛋白可特异性与抗His抗体反应，分别与预测重组蛋白分子量一致，鉴定为正确表达的目的重组蛋白。结论本研究成功构建了发形霞水母丝氨酸蛋白酶家族原核表达载体，获得了目标蛋白，为进一步研究水母丝氨酸蛋白酶的活性和功能奠定基础。%Objective To obtain a single toxin component from the jellyfish Cyanea capillata and provide a foundation for the further study on bioactivity and function of the serine proteases from C.capillata.Methods Primers designed with restriction enzyme were used to amplify the coding region of cDNAs (CcSP1, CcSP2 and CcSP3).PCR fragments were ligated with the pET-24a( +) vector to construct the recombinant plasmids (pET24a-CcSP1, pET24a-CcSP2 and pET24a-CcSP3).After screening and identification,the recombinant plasmids were transformed into the Rosetta (DE3).plysS for protein expression.After induction with IPTG, SDS-PAGE and Western-blot were used to detect the expression of the recombinant proteins.Results SDS-PAGE showed that the proteins of rCcSP1, rCcSP2 and rCcSP3 were expressed in a single band at about 34 kDa, 42 kDa and 42kDa, respectively.Western-blot detection with anti-His antibody further confirmed that these recombinant proteins were His-tagged CcSP1, CcSP2 and CcSP3 fusion protein were obtained.Conclusion Prokaryotic recombinant plasmids of C.capillata serine proteases are contructed and recombinant proteins are obtained, which establishes the foundation for future study on the function of serine proteases from jellyfish.