利用 S-层蛋白CTC在苏云金芽胞杆菌细胞表面展示鸡毒霉形体粘附素蛋白

利用苏云金芽胞杆菌S-层蛋白CTC表面展示系统研究在细胞表面展示鸡毒霉形体粘附素蛋白PMGA1.2的可行性及其免疫原性,为研制能常温长期保藏和运输的禽用口服疫苗奠定基础.用部分pmga1.2基因(pmga1.2p)代替S-层蛋白ctc基因中部且位于表面锚定序列slh下游的片段,构建了2个融合基因ctc-pmga1.2p和csa-ctc-pmga1.2p(csa表csaAB操纵元,其与S-层蛋白牢固地锚定到细胞表面密切相关);将含融合基因的重组质粒电转化入苏云金芽胞杆菌无质粒突变株BMB171中,获得了2个重组菌株BCCG(含ctc-pmga1.2p和携带csaAB操纵元的质粒)和CG(含csa-ctc-pmga1.2p).血凝和血凝抑制试验结果显示,2个重组菌株均成功地在细胞表面展示了重组蛋白PMGA1.2P;小鼠免疫学实验证实,2个重组菌株所展示的重组蛋白均具有免疫原性,其中,重组菌株CG的免疫效果优于BCCG.结果表明,苏云金芽胞杆菌S-层蛋白CTC表面展示系统可被用来研制热稳定性禽用口服疫苗.

Display of Mycoplasma gallisepticum Agglutinin on Cell Surface of Bacillus thuringiensis using S-layer Protein CTC

S-layer protein display system of Bacillus thuringiensis was used to test the possibility of displaying the protein of Mycoplasma gallisepticum agglutinin on cell surface. Two fusion genes ctc-pmga1.2p and csa-ctc-pmga1.2p were constructed by replacing the central part below the surface anchoring sequence slh of S-layer protein gene ctc with part of gene pmga1.2 (i.e., pmga1.2p). The csa represented csaAB operon which was very important to the anchoring of S-layer protein on the bacterial cell surface. Two recombinants, B. thuringiensis strains BCCG and CG were obtained by electro-transferring recombinant plasmids harboring fusion genes to B. thuringiensis plasmid-free derivative strain BMB171. BCCG harbored ctc-pmga1.2p and a plasmid carrying csaAB operon. CG harbored csa-ctc-pmga1.2p. Haemagglutination assay showed that recombinant protein PMGA1.2P was successfully displayed on the cell surface of BCCG and CG, respectively. Haemagglutination inhibition assay showed that recombinant protein PMGA1.2P was specific to positive serum of M. gallisepticum. After immunizing mice with BCCG and CG respectively, both BCCG and CG elicited a humoral respone to PMGA1.2P and exhibited immunogenicity when assayed by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that recombinant strain CG exhibited a higher immunogenicity than BCCG, which demonstrated that the construction way of fusion gene csa-ctc-pmga1.2p was more appropriate to S-layer protein displaying heterologous antigen on cell surface. The strategy indicated the possibility of generating heat-stable oral veterinary vaccine with B. thuringiensis S-layer protein display system.