胞壁形式表达融合蛋白Der p2-PEB1重组BCG的构建和鉴定

利用重组BCG技术,构建能以胞壁形式表达屋尘螨Der p2和PEB1抗原的重组BCG口服哮喘疫苗pCW-Der p2-PEB1-rBCG,以pCW-Der p2质粒和pUC-PEB1质粒DNA为模板扩增基因,构建出重组质粒pCW-Der p2-PEB1.将重组质粒电穿导入BCG感受态细胞,潮霉素抗性筛选rBCG阳性克隆.阳性rBCG经PCR特异性扩增目的基因片段Der p2及PEB1,证实其中含有目的基因.用抗Der p2单克隆抗体和制备的小鼠PEB1抗血清对rBCG进行间接免疫荧光鉴定.结果成功扩增了Der p2及PEB1基因,构建出重组质粒pCW-Der p2-PEB1,并将质粒电穿入BCG中,免疫荧光法鉴定其成功.说明成功地构建了胞壁表达Der p2-PEB1融合蛋白重组BCG pCW-Der p2-PEB1-rBCG.为基因工程疫苗的研制奠定了基础.

Construction and expression of the recombinant BCG expressing Der p2-PEB1 fusion protein in form of lipoprotein

Objective To construct the recombinant BCG,on which was able to express house dust mite antigen Der p2 and C.jejuniPEB1 in form of lipoprotein for oral asthma vaccine, the pCW-Der p2-PEB1-rBCG.Methods:The pCW-Der p2 and pUC-PEB1 plasmid DNA were amplified and recombinant plasmid pCW-Der p2-PEB1 was synthesized. The recombinant plasmid was transferred into competent BCG, after that rBCG positive bacterium strains were selected by hygromycin resistance screening. Target gene was detected in positive rBCG by specific amplification using PCR. rBCG was checked by indirect immunofluorescence with anti-Der p2 monoclonal antibody and antiserum against mouse PEB1. Results:We successfully amplified Der p2 and peb1 gene, constructed recombinant plasmid pCW-Der p2-PEB1 and transferred the plasmid into BCG,which was confirmed by indirect immunofluorescence.Conclusion:Recombinant BCG pCW-Der p2-PEB1-rBCG Expressing Der p2-PEB1 Fusion Protein on its Surface was successfully constructed and this product may provide basis for the further research of genetic engineering vaccine.