红笛鲷p38β MAPK基因的克隆及原核表达

Cloning and Prokaryotic Expression of p38β MAPK from Lutjanus sanguineus

鱼类p38 MAPK基因在宿主免疫调控及抵御病原侵染的过程中具有重要作用。利用RACE技术克隆了红笛鲷p38βMAPK基因(Ls-p38β,Gen Bank登录号为KJ502277)。序列分析结果显示,Ls-p38βc DNA全长1 628 bp,开放阅读框1 086 bp,可编码361个氨基酸,预测其编码蛋白的分子量为41.6 k D,理论等电点为5.74。该基因推导的氨基酸序列含有p38 MAPK家族保守的双磷酸化激活环(TGY),与尼罗罗非鱼的p38β有97%的相似性。将此基因定向插入原核表达载体构建重组表达质粒p ET32-p38β后导入BL21(DE3)菌株,利用IPTG进行诱导表达。SDS-PAGE与Western blot分析显示约在60 k D出现特异蛋白条带,与预期分子量大小相符,说明Ls-p38β融合蛋白表达成功。

Humphead snapper （ Lutjanus sanguineus ） p38β MAPK （ Ls-p38β ） was cloned using RACE method. The GenBank accession number is KJ502277. The full-length cDNA of p38β MAPK was 1 628 bp containing an open reading frame of 1 086 bp, encoding 361 amino acids with an estimated molecular weight of 41.6 kD and a theoretical pI of 5.74. Amino acid alignment indicated that Ls-p38β possessed a Thr-Gly-Tyr （ TGY ） dual phosphorylation motif of p38 MAPK family and shared 97% similarity with Oreochromis niloticus p38β. The prokaryotic expression vector pET-p38β was constructed and then transformed into BL21 （ DE3 ） . SDS-PAGE and Western blotting analysis indicated that the recombinant protein of Ls-p38β was successfully expressed and molecular weight of expressed fusion protein was 60 kD.