携带非典型cpb2基因牛源A型产气荚膜梭菌重组α毒素的免疫保护性研究  

Protective effect of recombinant α toxin of bovine type A Clostridium perfringens carrying the atypical cpb2 gene

作　　者：德艳艳[1,2] 姜志刚[2] 牛思博[2] 孙刚[1,3] 于力[2] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/大动物病研究室,黑龙江哈尔滨150001 [3]黑龙江省动物卫生监督所,黑龙江哈尔滨150090

出　　处：《中国预防兽医学报》2012年第7期563-567,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划(2006BAD04A09)

摘　　要：为验证重组α毒素对携带非典型cpb2基因的A型产气荚膜梭菌的免疫保护性,本研究应用PCR技术,从某牛场牛源A型产气荚膜梭菌G1分离株中扩增出1 194 bp的α毒素编码基因(cpa)和795 bp的β2毒素编码基因(cpb2)。经BLAST分析显示,G1分离株携带的cpb2基因与14个菌株的非典型cpb2基因的氨基酸序列同源性为95.1%～98.9%,与典型cpb2基因(L77695)的氨基酸序列同源性为61.7%。这表明,G1的cpb2基因为非典型cpb2基因。同时分别将cpa和cpb2基因扩增产物克隆于原核表达载体中,构建重组表达质粒pET-a和pET-b2,重组菌经IPTG诱导表达重组蛋白,将其纯化后单独及联合免疫小鼠进行免疫保护试验。结果显示,单独免疫重组α毒素蛋白组以及联合免疫重组β2毒素蛋白组的小鼠,均可以抵抗至少6倍最小致死量(MLD)的G1外毒素(包含α毒素和非典型β2毒素)的攻击,也可以完全抵抗至少6 MLD的G2外毒素(不包含β2毒素)的攻击。表明,重组α毒素蛋白对含有及不含有非典型β2毒素的A型产气荚膜梭菌均具有良好的免疫保护作用。Clostn＇dium perfingens is a pathogen to human and animals. In this study, the cpa gene encodingαtoxin and cpb2 gene encoding β2 toxin were amplified from C. perfringens type A isolate G1 by PCR, respectively. The PCR product of the cpb2 gene was sequenced and alignment to reference strains of cpb2 gene in GenBank by BLAST, the result showed that the deduced amino acid sequence of the cpb2 gene with atypical cpb2 genes （n=14） was 95.1% to 98.9% identity, but only 61.7% homology with typical cpb2 gene （Acce.No.L77695）, which indicated that the cpb2 gene was atypical cpb2 gene, and it was expressed in C. perfn＇ngens G1 isolate as show in western blot analysis. The cpa and cpb2 gene were cloned into pET-30a and pET-32a for expression in E. coil, respectively. Mice were immunized with each or both of the purified recombinant proteins, respectively, and the results showed that 100% of the immunized mice with recombinant α toxin or both α toxin and β2 toxin were protected against challenging with either 6 MLD toxin of C. perfringens G1 （including （x and 132 toxin） or 6 MLD toxin of C. perfringens G2 （without β2 toxin）. The results demonstrated that the recombinant ct toxin was able to protect the mice from challenge of C. perfnngens type A whether it expresses atypical β2 toxin or not.

关 键 词：非典型cpb2基因 A型产气荚膜梭菌 重组α毒素 重组非典型β2毒素 

分 类 号：S852.4[农业科学—基础兽医学]