凡纳滨对虾造血激素基因全长序列分析及其克隆与表达

虾造血激素(astakine,AST)是节肢动物中具有促进造血组织细胞分化和增殖的一种细胞因子。在对白斑综合征病毒(WSSV)感染分子机制的研究中发现,WSSV的一个可能受体蛋白与AST存在特异性结合。为进一步了解WSSV感染与对虾细胞分化之间的关系,本研究应用反转录聚合酶链式反应(RT-PCR)结合cDNA末端快速扩增(RACE)技术,成功克隆了凡纳滨对虾Litopenaeus vannamei造血激素基因(lvast)全长cDNA(GenBank登录号:HM594944),并对该基因进行了生物信息学分析。lvast全长共1846bp,含有1个372bp的开放阅读框,编码124个氨基酸,估算分子量为13.3kD。其编码的氨基酸序列包含一个前动力蛋白(Prokineticin)结构域。基因序列和氨基酸序列同源性比较表明,凡纳滨对虾造血激素(LvAST)与斑节对虾和软尾太平蝲蛄AST同源性较高,与脊椎动物的同源性较低。通过构建包含lvast基因ORF重组表达载体pBAD/gIIIA-lvast,本研究成功表达出与预期相符合的目的蛋白,为进一步研究LvAST的结构与功能提供了理论依据和实验基础。

Cloning, full-length sequence analysis and prokaryotic expression of astakine gene of Litopenaeus vannamei

Astakine (AST) is a cytokine that promotes cell differentiation and proliferation of hemocytes in the hematopoietic tissue, which plays important roles in crustacean immune responses. Evidence from our previous studies showed that WSSV may compete with AST for a common receptor. By rapid amplification of cDNA ends (RACE) PCR, the full-length gene of Litopenaeus vannamei astakine gene (lvast) was obtained, which consisted of 1,846 bp with a 372 bp ORF, encoding 124 amino acids with the predicted molecular mass of 13.3kDa. The deduced protein contained a prokineticin (PK) domain. Homological comparison among LvAST and other species showed that the homogeneities were higher in comparison with those of Penaeus monodon and Pacifastacus leniusculus, and lower in comparison with those of vertebrates.The recombinant protein of LvAST was successfully expressed with the constructed recombined vector pBAD/g IIIA-lvast containing the lvast ORF. The results laid a theoretical basis for further functional studies of astakine.