H5N1型禽流感病毒血凝素HA1蛋白在苏云金芽胞杆菌细胞表面的展示及其免疫原性

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H5N1型禽流感病毒HA1蛋白的可行性及其免疫原性，为研制既安全有效又能常温长期保藏和运输的禽流感口服基因工程疫苗奠定基础。用部分ha1基因（ha1p）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，构建了融合基因ctc-ha1p和 csa-ctc-ha1p；利用电脉冲转化法将含融合基因的重组质粒转入苏云金芽胞杆菌无质粒突变株BMB171中，获得了重组菌株BCCH（含ctc-ha1p，并导入一个载有协助细胞表面展示的csaAB操纵子的质粒）和CH（含csa-ctc-ha1p）。通过血凝和血凝抑制试验以及小鼠免疫学实验证实，2个重组菌株均成功地在细胞表面展示了重组HA1蛋白并具有一定的特异性和免疫原性，其中，CH的效果强于BCCH，说明csa-ctc-ha1p这种融合基因的构建方式更胜一筹。研究结果表明，苏云金芽胞杆菌S-层蛋白CTC表面展示系统可用来研制禽流感口服疫苗。

Display of H5N1 Avian influenza virus Haemagglutinin HA1 on Bacillus thuringiensis Cell Surface and Its Immunogenicity for Mice

S-layer protein CTC surface display system of Bacillus thuringiensis (Bt) was used to test the possibility of displaying H5N1 Avian influenza virus haemagglutinin HA1 on B. thuringiensis cell surface. Two recombinant plasmids were constructed by replacing the central part below the surface anchor sequence slh of S-layer protein gene ctc with part ha1 gene (halp ). The two resulting plasmids were pCTC-HA1P (harboring fusion gene ctc-halp ) and pCSHA1P (harboring fusion gene csa-ctc-halp, csa represents csaAB operon which was very important to the anchoring of S-layer protein on the bacterial cell surface). Two recombinant B. thuringiensis strains were constructed by electro-transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The resulting strains were CH (harboring pCSHA1P) and BCCH (harboring pCTC-HA1P as well as the plasmid pMIL-CSA which carried csaAB operon). The vegetative cells of CH and BCCH were used as antigens of haemagglutination (HA) assay and haemagglutination inhibition (HI) assay. HA assay showed recombinant HA1 proteins were successfully displayed on the cell surface of CH and BCCH, respectively. HI assay showed recombinant HA1 proteins displayed on the cell surface were specific to standard positive HI (haemagglutination inhibition test) serum of subtype H5 AIV. After immunizing mice with vegetative cells of CH and BCCH respectively, CH and BCCH all elicited a humoral response to HA1 and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay (ELISA). ELISA also showed CH exhibited a higher immunogenicity than BCCH. The strategy developed in this study gives a possibility to generate heat-stable, oral, veterinary vaccine against AIV with B. thuringiensis S-layer protein CTC surface display system.