蛇足石杉HsCAO2基因的克隆、生物信息学分析及载体构建

Cloning,bioinformatics analysis and expression vector construction of HsCAO2 from Huperzia serrata

目的为筛选高活性的蛇足石杉铜胺氧化酶(copper-containing amine oxidase,CAO),通过生物工程方法实现石杉碱甲(huperzine A,HupA)的合成、为加快新药创制进度提供理论依据。方法基于蛇足石杉转录组测序得到的CAO序列片段,利用c DNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)技术,扩增、拼接并验证获得该基因的全长c DNA序列,并将其命名为HsCAO2。结果通过RACE技术得到Hs CAO2的c DNA全长为2696 bp,CDS为2199 bp,编码732个氨基酸。氨基酸序列同源性比对发现,HsCAO2具有保守的氨基酸位点和Cu2+结合位点;进化树分析结果表明,HsCAO2与蛇足石杉中的另一个CAO(HsCAO1)同源性最高,达91.67%;蛋白质结构分析结果表明,HsCAO2也可能在生物体里以同源二聚体的形式存在。在此基础上,进一步分别构建了超表达载体pOX-Hs CAO2和体外蛋白表达载体pET-28a(+)-HsCAO2。结论HsCAO2很可能是蛇足石杉中的一个新的功能性CAO,且超表达载体和体外蛋白表达载体的成功构建为进一步研究其功能奠定了基础。

Objective To screen copper-containing amine oxidase(CAO)with high activity from Huperzia serrata,reinforce the synthesis of huperzine A(HupA)by bioengineering method,so as to provide the theoretical basis for the progress of new drug development.Methods Based on RNA-seq data and rapid amplification of cDNA ends(RACE)technology,a CAO-like gene named Hs CAO2 was identified from Huperzia serrata.Results The full length of Hs CAO2 c DNA and coding sequence(CDS)was 2696 and 2199 bp,respectively,encoding a 732-amino acids deduced protein.Sequence alignment results indicated HsCAO2 contained both conserved domains and Cu2+binding sites.Phylogenetic analysis showed that HsCAO2 shared the highest similarity(91.67%)with HsCAO1,another CAO verified in H.serrata.The secondary and 3-D structures of HsCAO2 simulated by online softwares indicated that HsCAO2 might exist as homodimers in H.serrata.Furthermore,the overexpression vector p OX-Hs CAO2 and in vitro protein expression vector p ET-28 a(+)-Hs CAO2 were constructed.Conclusion Hs CAO2 might be a new functional CAO which has not been reported before in H.serrata.The successful construction of the overexpression vector and in vitro protein expression vector lays a foundation for further study of its biological function.