利用苏云金芽胞杆菌细胞表面展示系统表达禽流感病毒NP蛋白

Display of Avian influenza virus nucleoprotein on Bacillus thuringiensis cell surface

首次利用苏云金芽胞杆菌(Bacillus thuringiensis,Bt)S-层蛋白CTC表面展示系统研究在Bt细胞表面展示禽流感病毒NP蛋白的可行性和最佳方案,为研制能常温长期保藏和运输的禽用口服疫苗奠定基础。用全长np基因或部分np基因(npp)代替S-层蛋白ctc基因的3′-端或中部,构建了4个重组质粒pSNP(含ctc-np)、pCSA-SNP(csa-ctc-np)、pCTC-NPP(ctc-npp)和pCSNPP(csa-ctc-npp)。将重组质粒分别电转化入Bt受体菌株BMB171中,获得了5个重组菌株BN、BCN、C-S、BCCN和CN。用5个重组菌株的营养细胞做玻片凝集试验,结果显示5个重组菌株均成功地在细胞表面展示了NP蛋白。用5个重组菌株的营养细胞免疫小鼠,ELISA测定血清抗体效价,结果显示5个重组菌株均具有免疫原性,其中重组菌株CN的免疫原性最高,其含融合基因csa-ctc-npp,证明该种融合基因的构建方式最佳。这为利用S-层蛋白CTC表面展示系统构建展示其它禽类病原体抗原的重组菌株以研制禽用热稳定性口服疫苗奠定了基础。

S-layer protein CTC surface display system of Bacillus thuringiensis （Bt） was used to test the possibility of displaying avian influenza virus nucleoprotein （NP） on Bt cell surface. Four recombinant plasmids were constructed by replacing 3＇- terminal or central part below the surface anchor sequence slh of S-layer protein gene ctc with full length of np gene or part np gene （ npp ）. The four resulting plasmids were pSNP （ harboring fusion gene ctc-np ）, pCSA-SNP （harboring fusion gene csa-ctc-np, csa represents csaAB operon which is very important to the anchoring of S-layer protein on the bacterial cell surface）, pCTC-NPP （harboring fusion gene ctc-npp） and pCSNPP （harboring fusion gene csa-ctc-npp）. Five recombinant Bt strains were constructed by electro-transferring recombinant plasmids to Bt plasmid- free derivative strain BMB171. The resulting strains were BN （harboring pSNP）, BCN （harboring pSNP as well as the plasmid pMIL-CSA which carried csaAB operon）, C-S （harboring pCSA-SNP）, BCCN （harboring pCTC-NPP and pMIL- CSA） and CN （harboring pCSNPP）. The vegetative cells of five recombinant strains were used as agglutinogens of slide agglutination assay. Slide agglutination assay showed recombinant NP proteins were successfully displayed on the surface of five recombinant strains, respectively. After immunizing mice with vegetative cells of five recombinant straics respectively,five recombinant strains all elicited humoral respones to NP and exhibited immunogenicity as assayed by enzyme-linked immunosorbent assay （ELISA）. Meanwhile these assays showed recombinant strain CN （harboring fusion gene csa-ctc-npp ） exhibited the highest immunogenicity among five recombinant strains. That means the best way of constructing S-layer fusion gene is csa-ctc-^＊ （＊ denotes heterologous antigen gene） which means the central part of Slayer protein gene ctc replaced by the heterologous antigen gene and csaAB operon located on the upstream of fusion gene. The strategy developed in this study gives a possibility to generate heat stable, oral, veterinary vaccine with Bt S- layer protein CTC surface display system.