Cloning And Prokaryotic Expression Of N-terminal Fragment Of Candida Albicans Hyphal Wall Protein1

Objective:With the extensive application of corticosteroids, immunosuppressants andbroad-spectrum antimicrobial agents, interventional therapy clinics, as well as theincrease in AIDS patients, the incidence of fungal infections is increasing quickly,mainly Candida albicans infection. Because of the difficulties in early diagnosis, theinvasive candidiasis often very poor prognosis. Candida albicans is a both with yeastform and the growth of fungi mycelium morphology to produce mycelium being anexpression of its virulence. Staab first discovered Candida albicans hyphal wall protein1(Hwp1), and Hwp1has Features of the mycelium specificity and antigenicity andspecies specificity, so that it may play an important role in the diagnosis of invasivecandidiasis. For the reason above, in this research we selected the gene Encoding Hwp1protein, constructed the recombinant prokaryotic expression plasmid of it and expressedthe recombinant proteins in E. coli. Follow-up to investigate the Hwp1antigen-antibodysystem laid the foundation for the role and status of invasive Candida albicans.Materials and Methods:1. Preparation of the target gene: Candida albicans total RNA was extracted by YeastDNAiso Kit then the target gene was obtained by PCR method.2. The cloning of target gene: The target gene was cloned into pMD18-T vector plasmidsuccessful cloning by blue-white screening method for sequencing after PCR anddigestion method identified target genes. 3. Expression of purpose protein: Recombinant prokaryotic expression plasmids weretransferred into E. coli BL21and induced by IPTG. Then BL-21cell transformed werecrumbled by ultrasonics and the results were analyzed by SDS-PAGE electrophoresis.Results:1. N-terminal fragment of Hwp1was obtained successfully by PCR. The sequenceanalysis shows that the target gene were homologous basically with the ones inGenBank.2. The recombinant expression plasmid pMD18-T-Hwp1and pET28a (+)-Hwp1hadbeen constructed successfully. PCR and restriction enzyme digestion results were in linewith expectations.3. Prokaryotic expression plasmid pET28a (+)-Hwp1was transformed to BL-21. Thetarget protein was expressed in Escherichia coli after induction with IPTG.Conclusion:1. Prokaryotic expression plasmid pET28a (+)-Hwp1was constructed successfully.2. Prokaryotic expression plasmid of Hwp1was induced and expressed fusioned proteinin E. coli.BL-21successfully, which provides a basis for further studies on candidiasis.