Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator bacteria.

Objective To express and purify the pneumococcal surface adhesin A(PsaA) protein,discuss its application as a protein carrier in conjugates vaccine. Methods The gene encoding for the PsaA protein was amplified from the genomic DNA of Streptococcus pneumoniae using PCR. The PCR product was then cloned into the prokaryotic expression vector pET-28a and the recombinant was transformed into host cell E. coli BL21 (DE3). The expression of the recombinant protein(rPsaA) was induced by IPTG and purifled by using DEAE anion-exchange chromatography. The rPsaA was successfully conjugated with group A meningococcal polysaccharide(GAMP). The mice were immunized subcutaneously with the conjugate and the immune responses against GAMP and PsaA were detected by ELISA. Results The recombinant PsaA was expressed as a 37 × 103 soluble protein without His-Tag. The rPsaA was successfully conjugated with GAMP. In addition to the immune response against PsaA, The antibody response against GAMP was significant improved in the mice immunized with conjugate vaccine in comparison with those immunized with GAMP alone. Conclusion The recombinant protein PsaA without His-Tag was obtained and conjugated with GAMP. The strong antibody responses against PsaA and CAMP were obtained in the immunized mice at the same time which may provide the protection against pneumonia and meningitis simultaneously.