论文部分内容阅读
产肠毒素大肠杆菌(ETEC)是一种导致新生犊牛和仔猪腹泻的主要病原体之一。ETEC的毒力因子主要有黏附素(CFs)、不耐热性肠毒素(LT)和耐热性肠毒素(ST)三种。在前期研究中,利用PCR和酶切连接技术成功构建了两种ETEC亚单位疫苗3STaM(G)-K99和3STaM(S)-K99,且在大肠杆菌中获得高效表达。本研究利用阴离子交换层析纯化融合蛋白3STaM(G)-K99 and3STaM(S)-K99,辅以弗氏佐剂免疫新西兰大白兔,通过Elisa分析其免疫学性质,并利用肠毒素中和实验在昆明系乳鼠中评价其激发抗STa中和抗体的能力。实验结果表明:亚单位疫苗3STaM(G)-K99 and 3STaM(S)-K99能够激发相对较高水平、可针对天然STa、ETEC和融合蛋白STa-K99的特异性抗体。其次,亚单位疫苗中STa突变体(STaM)组分的肠毒素活性显著降低,且其所激发的特异性抗体属于中和抗体,能有效抑制天然STa的肠毒素活性。亚单位疫苗3STaM(G)-K99 and3STaM(S)-K99为研制预防ETEC感染性腹泻的多价基因工程疫苗提供了基本素材和理论指导。
Enterotoxigenic Escherichia coli (ETEC) is one of the major pathogens causing diarrhea in newborn calves and piglets. ETEC virulence factors are mainly adhesin (CFs), heat-labile enterotoxin (LT) and heat-tolerant enterotoxin (ST) three. In previous studies, two ETEC subunit vaccines, 3STaM (G) -K99 and 3STaM (S) -K99, were successfully constructed using PCR and restriction enzyme ligation, and were highly expressed in E. coli. In the present study, New Zealand white rabbits were immunized with 3STaM (G) -K99 and3STaM (S) -K99 by anion exchange chromatography, and their immunological properties were analyzed by Elisa. The enterotoxin neutralization assay Kunming mice were evaluated for their ability to stimulate neutralizing antibodies against STa. The results showed that the subunit vaccines 3STaM (G) -K99 and 3STaM (S) -K99 could stimulate relatively high levels of specific antibodies against STa, ETEC and the fusion protein STa-K99. Second, the STa mutant (STaM) component of the subunit vaccine significantly reduced the enterotoxin activity, and the specific antibodies raised by it were neutralizing antibodies, which could effectively inhibit the enterotoxin activity of the natural STa. The subunit vaccine 3STaM (G) -K99 and 3STaM (S) -K99 provided the basic material and theoretical guidance for the development of multivalent genetic engineering vaccine against ETEC-infected diarrhea.