Gene Coloning,Expression and Enzymatic Assay of Human sPLA2-IIA / 中国生物工程杂志

ABSTRACT

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 trxA-human 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.