C型产气荚膜梭菌α、β_1、β_2毒素基因融合及免疫原性研究  

Fusion of alpha-toxin gene,beta1-toxin gene and beta2-toxin gene from Clostridium perfringens type C and its immunogenicity

作　　者：许崇波[1] 陈向东[2] 许崇利[3] 逄越[1] 高凤山[1] 

机构地区：[1]大连大学生物工程学院生物有机化学辽宁省重点实验室,辽宁大连116622 [2]武汉大学生命科学学院微生物学国家重点学科,湖北武汉430072 [3]吉林化工学院环境与生物工程学院,吉林吉林132022

出　　处：《浙江大学学报（农业与生命科学版）》2008年第4期379-384,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金资助项目(30360080);辽宁省高等学校科学研究项目(A类)攻关计划资助项目(05L018);教育部青年骨干教师国内访问学者资助项目(2007)

摘　　要：为了构建具有良好免疫原性的α-β1-β2融合蛋白,利用PCR技术,从含C型产气荚膜梭菌α毒素基因的克隆质粒pETXA1中扩增出0.95 kbα毒素基因,将其连接到经NcoⅠ单酶切并用碱性磷酸酶处理的含1.65 kbβ1-β2融合基因的重组质粒pETXB1B2上,构建含2.6 kbα-β1-β2融合基因的表达质粒重组菌株BL21(DE3)(pETXAB1B2).经酶切鉴定和序列测定证实,构建的重组质粒pETXAB1B2含有α-β1-β2融合基因,且基因序列和阅读框架正确.经ELISA检测,重组菌株表达的α-β1-β2融合蛋白能够被α、β1和β2毒素抗体识别.表达优化结果表明,以IPTG为诱导剂诱导α-β1-β2融合基因表达的优化条件是:培养基pH 7.5,培养温度37℃,IPTG浓度0.4 mmol?L-1,菌体生长密度OD600达到1.0时加入IPTG,诱导时间5 h,此时α-β1-β2融合蛋白表达量为31.2%.免疫试验结果表明,α-β1-β2融合蛋白免疫的小鼠可以抵抗1MLD(最小致死量,minimum lethal dose)C型产气荚膜梭菌标准株C59-44毒素攻击,表明构建的重组菌株可以作为预防仔猪红痢基因工程亚单位苗的候选菌株.In order to construct the good immunogenic α-β1-β2 fusion protein, a 0. 95 kb gene fragment from plasmid pETXA1 containing Clostridium perfringens type C alpha-toxin gene was amplified by PCR and it was inserted into recombinant plasmid pETXB1B2 containing 1.65 kb β1-β2 fusion gene, which was cleaved with NcoⅠ and processed with alkaline phosphatase. The recombinant plasmid pETXAB1B2 containing 2.6 kb α-β1-β2 fusion gene was constructed and then transformed into Escherichia coli BL21 （DE3） by DNA recombination technique. The recombinant expression plasmid pETXAB1B2 was further studied by restriction endonucleases analysis and nucleotide sequencing. The results show that the recombinant expression pETXAB1B2 possessed a positive α-β1-β2 fusion gene sequence and reading frame. BL21 （DE3） （pETXAB1B2） could produce α-β1-β2 fusion protein and the expressed products were recognized by alpha-toxin, betal-toxin and beta2-toxin antibodies with ELISA and Western blot analysis. The optimum IPTG induction conditions for the α-β1-β2 fusion gene expression were as follow： culture medium pH 7.5, 37 ℃, and the recombinant strain growth density OD600 at 1.0, and 0. 4 mmol·L^-1 IPTG for 5 h. The expression level of the α-β1-β2 fusion proteins was about 31.2% of total cellular protein with IPTG induction. More importantly, a mouse immunization with crude preparation containing the α-β1-β2 fusion protein inclusion bodies or inactivated recombinant strain could induce protection against at least 1MLD of the toxin challenging from Clostridium perfringens type C. It is indicated that the constructed recombinant strain BL21 （DE3） （pETXAB1B2） can be used as a candidate of vaccine strain.

关 键 词：C型产气荚膜梭菌 Α毒素基因 β1毒素基因 β2毒素基因 融合蛋白 免疫原性 

分 类 号：S852.6[农业科学—基础兽医学] Q785[农业科学—兽医学]