HBsAg And Mycobacterium Tuberculosis HSP70 Fusion Of Basic Research Of Hepatitis B DNA Vaccine

Objective Nucleic acid vaccines represent a new approach to the control of infectious agents. These novel vaccines are both easy to construct and produce. However, there is still a need to increase the potency of DNA vaccines. Heat shock proteins (LISPs) are major targets of the immune responses to bacterial and parasitic pathogens. Mycobacteriuni tuberculosis heat shock protein 70 (Mt.HSP7O) is an especially powerful antigen containing multiple B and T cell epitopes, and possesses adjuvant properties when used as a carrier in immunization .ln order to enhance hepatitis B DNA vaccines antivirus effect, we investigated whether M. tuberculosis HSP7O can be used as an adjuvant-free carrier to stimulate the humoral and cellular immune responses to an accompanying HBsAg in the form of chi.meric DNA vaccine. Methods 1. Construction of an enkaryotic expression plasmid pcDNA3. 1 -HSP7O The full length of HSP7O gene from the genome of M. tuberculosis H37Rv was amplified and cloned into pGEM-T vector with A-T cloning technique. Then the recombinant plasmid was digested with BainH I and EcoR I and subcloned into pcDNA3.1(+).Restriction enzyme digestion and DNA sequecing confirmed that the recombinant eukaryotic expression plasmid containing M. tuberculosis HSP7O gene (pcDNA3.1 -HSP 70) had been constructed correctly. 2. Construction of expression vectors used for nucleic acid immunization The HSP7O-encoding fragment of M. tuberculosis was obtained from the plasmid pcDNA3.1 -HSP7O.The HBsAg-encoding fragment of HBV,subtype adw ,obtained from the plasmid pEcob6. (1) pCI-S The HBsAg-encoding fragment was amplified by PCR using primers designed to generate HindlII and BamH I restriction sites at the 5'and 3'ends of the amplified fragments, respectively. Then the amplified HBsAg DNA was cloned into unique HindIII and BamH I cloning sites of the plasmid pcDNA3.1(+) (pcDNA3. 1-5). Finally,the recombinant plasmid pcDNA3. 1 -S was digested with EcoR I and Nhe I ,and subcloned into plasmid pCI-neo(pCI-S).In this construct the HBsAg-encoding gene is expressed under HCMV immediate early promoter control. (2) pCl-S-HSP7O HBsAg DNA fragment(without stop codon) was amplified by PCR using primers designed to generate HindlII and BamH I restriction sites at the 5 and 3 ends of the amplified fragments, respectively. Then the amplified HBsAg DNA was cloned into unique Hind III and BamH I cloning sites of the plasmid pcDNA3. 1 -HSP7O (pcDNA3. 1 -S-HSP7O). Thus, the C terminus of HBsAg gene was fused to the N terminus of HSP7O gene. Finally, the recombinant plasmid pcDNA3. 1-S-HSP7O was digested with EcoR I and Nhe I ,and subcloned into plasmid pCI-neo(pCI-S-HSP7O).In this construct the S-HSP7O chimera-encoding gene is expressed under HCMV immediate early promoter control. (3) pCI.-S-HSP7OC The amplified HBsAg DNA fragment(without stop codon) was cloned into the plasmid pcDNA3.l(+) (pcDNA3.1-S).The C terminus of I-ISP7O fragment (780bp) was amplified by PCR using primers designed to generate BamH I and EcoR I restriction sites at the 5'and 3'ends of the amplified fragments, respectively. Then the amplified fragment was cloned into unique BamH I and EcoR I cloning sites of the plasmid pcDNA3.l-S(pcDNA3.l-S-HSP7OC).Thus,the N...