Mycobacterium Tuberculosis Hsp70 Fusion Protein Expression In Pichia Pastoris And Immunological Studies, And Hbsag

Objective It has been shown that hsp-peptide isolated from cancer and hsp fusion proteins can elicit protective antitumor or antiviral cellular immune response without any additional adjuvant. In this study, we explore the novel protein vaccines to cure HBV chronic infection. Hsp70 and hsp70 fused with HBsAg yeast expression vectors were constructed. The exogenous genes can be integrated into Pichia pastoris chromosome by integrative expression plasmid generating genetic state secretion expression strain. Expression proteins were characterized as a basis of further studying their biological functions.MethodsConstruction and identification of hsp70 and hsp70 fused with HBsAg yeast expression vectors: Applied PCR and molecular clone technology, the yeast shuttle plasmids including pPIC9K/hsp70,pPIC9K/s,1. pPIC9K/hsp(1-370)-s,pPIC9K/hsp(161-370)-s,pPIC9K/s-hsp(1-370),pPIC9K/s-hsp(161-370) were constructed. Restriction enzyme digestion and DNA sequencing analysis confirmed whether the recombinant plasmids were correct.2. Expression of hsp70 and hsp70 fusion proteins in Pichia pastoris system: First,the plasmids were linearized with SacI; Second,Pichia pastoris strain GS115 was transformed by electroportion; Third,the transformants were selected by PCR; Last,the recombinant Pichia strains were induced by 0.5% methanol.The supernatants from cultures were identified by SDS-PAGE,ELISA and western-blot analysis.Results1. Restriction digestion and DNA sequence analysis confirmed the six vector correct.Expression of recombinant Pichia strains:Hsp70 was secreted into the supernatant,western-blot analysis showed that the positive band with about 70KD molecular weight was recongnized by anti-Mt.hsp70 antibody.Yeild of expression was up to 0.15mg/ml and the expression amount was more than 30% of the supernatant total proteins.However,the other five recombinant Pichia strains didn't secret the expected fusion proteins into the supernatant.The cell lysates were positive by measure with HBsAg ELISA.According to these,they2. maybe accumulated expression protein in cell.Conclusion1. We succeeded in constructing six yeast shuttle expression vectors.2. We utilized the Pichia expression system to secret hsp70 at first time and got a high expression recombinant Pichia strain,which would provide a opportunity to further study the application and biologic function of hsp70.