关键词: 白念珠菌, 分泌型天冬氨酸蛋白酶, SAP2基因, 重组质粒

Abstract: Objective To construct the SAP2 prokaryotic expression vector, to express and purify solvable protein, so as to lay a foundation for antibody preparation and Sap2 antigen detection.Methods After the target gene fragment SAP2 was obtained by standard PCR amplification, the SAP2 and plasmids pMAL-c2x (+) were cleaved with two restriction endonucleases, and the digestion products were connected. Ligation products were transformed into competent cell,E.coli TOP10. Then positive clones of recombined plasmid were screened and identified by DNA sequencing. After recombinant plasmid pMAL-c2x/SAP2 was transformed into E.coli strain BL21 (DE3) which was induced by IPTG subsequently. The fusion protein was purified by Amylose Resin affinity chromatography,and then was cleaved by Xa factor.Results The target gene SAP2 amplified by PCR had the same molecular size as predicted. It was inserted directionally into vector pMAL-c2x (+). Procaryotic expression plasmid pMAL-c2x/SAP2 can express soluble fusion protein MBP-Sap2 by IPTG induction 14 h later. Total 32 mg target protein Sap2 was obtained after purifying and cleaving label.Conclusion The recombined plasmid pMAL-c2x/SAP2 is successfully and highly expressed in BL21 (DE3) with soluble form. Target protein Sap2 was successfully obtained through affinity chromatography and proteinase cleavage.

Key words: Candida albicans, secreted aspartic proteinases (Sap), SAP2 gene, recombined plasmid