Construction Of Eucaryote Expression Vector And Procaryotic Expression Of Secreted Aspartyl Proteinase Gene Sap2 Of Candida Albicans

Background:Candida albicans,is the most common opportunistic pathogen fungi, causing a wide variety of infections ranging from mucosal infections in generally healthy persons to life-threatening systemic infections in individuals with impaired immunity.Because few classes of drugs are effective against these fungal infections,and all of them have limitations with regard to efficacy and side-effects,the prospects for the successful development of a vaccine against candidiasis are greater now than at any time in the past.Secreted aspartic proteinases have been described as virulence factors implicated in the mechanisms of host colonization by the yeast Candida albicans in different types of candidiasis.In this study,SAP2,the target gene fragment which encodes Secreted aspartic proteinases 2,was recombined into eukaryotic expression vector and procaryotic expression vector,and then the recombined vector pMAL-c2x/SAP2 was transferred into engineered strain BL21(DE3) to express soluble protein.The study will provide foundation for further investigation in the virulence and pathogenic mechanism of Secreted aspartic proteinases and developing C.albicans DNA vaccine.Objective:To construct eukaryotic expression plasmid pcDNA3.1_SAP2 used as gene vaccine in Further investigation;construct procaryotic expression plasmid and induce and purify solvable protein to be antigen for checking the created antibody in immunized mouse. Material and Methods:The target gene fragment SAP2(169₁194bp) was obtained by standard PCR amplification while genome DNA of C.albicans SC5314 strain was used as template.Then the SAP2 and plasmids pcDNA3.1(+)myc-HisC were cleaved with two restriction endonucleases EcoRâ… ,Xhoâ… ,and the digested products were separated and purified in low melting temperature agarose gels.The purified SAP2 and plasmid pcDNA3.1(+)myc-HisC were recombined by T4DNA ligase,ligation products were transformed into competent cell,Escherichia coli TOP10.Transformed colonies were screened by Ampr LB plate,then recombined plasmids were isolated and identified by restricted endonuclease cutting and DNA sequencing.Procaryotic expression plasmid pET32a/SAP2 and pMAL-c2x/SAP2 were constructed by the same method.After pMAL-c2x/SAP2 was transformed into E.coli strain BL21(DE3),it was induced by IPTG.The induced MBP-Sap2 was purified by amylose resin affinity chromatography,and then was cleaved by Xa factor.Result:1.Identified by agarose gel electrophoresis,the target gene SAP2 obtained PCR amplification had the same molecular size as predicted. It was indicated that recombined plasmids contained inserted SAP2 gene fragment by restricted endonuclease cut analysis,the sequencing data also indicated that inserted SAP2 gene had correct DNA sequence and orientation according to DNA sequence of C.albicans SC5314.2.At 16℃,After transferred into engineered strain BL21(DE3),both Procaryotic expression plasmid pET32a/SAP2 and pMAL-c2x/SAP2 can express soluble fusion protein when induced by IPTG.The target protein Sap2 was obtained after fusion protein MBP-Sap2 purifying and cleaving. Conclusion:1.We successfully construct Eukaryotic Expression vector pcDNA3.1/SAP2.Using Endo-free Plasmid Maxi Kit,we got enough recombined plasmid for gene vaccine production.2.We successfully construct Procaryotic Expression vector pET 32a/SAP2,DMAL-c2x/SAP2,which can express soluble fusion protein when induced by IPTG under low temperature.Totally,we got 4mg target protein to be antigen for antibody checking in immunized animals through affinity chromatography and proteinase cleavage.