Bai Jia-Ning, Zhang Yan, Zhao Bao-Hua
College of Life Science, Hebei Normal University, Shijiazhuang 050016, Hebei Province, China.
World J Gastroenterol. 2006 Feb 28;12(8):1229-34. doi: 10.3748/wjg.v12.i8.1229.
To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression.
Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay.
The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta fusion gene was constructed and transformed into BL21(DE3). The recombinant strain BL21(DE3)(pZCPAB) was obtained. After the recombinant strain BL21(DE3)(pZCPAB) was induced by 42 degC, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.
The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.
研究产气荚膜梭菌α-β融合基因的克隆及其融合表达的免疫原性。
从产气荚膜梭菌NCTC64609株和C58-1株中PCR扩增α毒素和β毒素的保护性抗原基因后进行克隆。用质粒pZCPAB克隆该基因片段。该片段编码的基因具有α-β融合基因结合的稳定表达。为验证α-β融合基因的确切位置，构建了结构域质粒。将这两个基因融合到表达载体pBV221中。通过ELISA、SDS-PAGE、Western印迹和中和试验鉴定表达的α-β融合蛋白。
获得了保护性α毒素基因（cpa906）和β毒素基因（cpb930）。构建了携带α-β融合基因的重组质粒pZCPAB，并将其转化到BL21(DE3)中。获得了重组菌株BL21(DE3)(pZCPAB)。重组菌株BL21(DE3)(pZCPAB)在42℃诱导后，SDS-PAGE和薄层凝胶扫描分析显示其表达产物约占总细胞蛋白的22.14%。中和试验表明，用α-β融合蛋白免疫诱导的抗体可中和α毒素和β毒素的毒性。
获得的α毒素和β毒素基因正确。重组菌株BL21(DE3)(pZCPAB)可产生α-β融合蛋白。该蛋白可用于免疫且具有免疫原性。用α-β融合蛋白免疫诱导的抗体可中和α毒素和β毒素的毒性。
