人分泌型磷脂酶A2(hsPLA2-IIA)基因的克隆、表达及活性鉴定

Gene Coloning,Expression and Enzymatic Assay of Human sPLA2-IIA

目的为了构建人分泌型磷脂酶A2(secretaryphospholipaseA2,sPLA2-IIA)的有效表达系统,从胎脾中提取总RNA。方法采用RT-PCR方法扩增出编码sPLA2-IIA的基因定向地克隆于硫氧环蛋白基因融合表达载体pET32a的TrxA基因3′末端,构建符合读码框的融合表达载体pET32a-sPLA2-IIA。37℃下经IPTG诱导,hsPLA2-IIA融合蛋白在大肠杆菌BL21(DE3)中获得高效表达,表达产物以包涵体的形式存在。包涵体经8mol/L尿素溶解、复性后检测结果显示具有较高的催化活性并呈现剂量依赖关系。结论以大肠杆菌为宿主,成功表达了hsPLA2-IIA蛋白,为进一步进行hsPLA2-IIA的大量生产和功能研究奠定了基础。

Objective： To clone the cDNA of human sPLA2-ⅡA, construct the engineered Escherischia coli expressing human sPLA2-ⅡA and identify the expressed human sPLA2-ⅡA. Methods： Total RNAs were purified from human fetal spleen. The cDNA of human sPLA2-ⅡA was cloned by RT-PCR and inserted into plasmid pET32a（ ＋ ） between NcoⅠ and EcoRⅠ sites for expressing the recombinant human sPLA2-ⅡA in Escherischia coli BL21 （DE3）. The recombinants were screened by SDS-PAGE. The engineered Escherischia coli expressing trxAhuman sPLA2-ⅡA fusion protein was established. The expressed human sPLA2-ⅡA exists in the form of inclusion body and accounts for about 25% of the total proteins of Escherischia coli BL21 （DE3）. Conclusion： the engineered E. coli methods are suitable for preparing plenty of human sPLA2-ⅡA which has laid base for the large-scale expression, purification and basic studies of human sPLA2-ⅡA.