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目的重组表达艰难梭菌毒素A的C端结构域,制备针对艰难梭菌毒素A C端结构域的卵黄抗体。方法将优化合成的艰难梭菌毒素A的C端结构域DNA片段克隆到表达载体pET32b(+)上,重组质粒转入大肠杆菌中诱导表达,经高亲和镍离子树脂纯化得到融合蛋白,凝血酶酶切后用高亲和镍离子树脂吸附多余多肽获得目的蛋白。检测目的蛋白的生物学活性,用目的蛋白免疫产蛋鸡制备针对艰难梭菌毒素A受体结合区的鸡卵黄抗体,分离纯化鸡卵黄抗体并用ELISA测定卵黄抗体的效价。结果获得了优化的艰难梭菌毒素A受体结合区的基因片段,继而制备了具有生物学活性的毒素A C端结构域重组蛋白。免疫产蛋鸡后获得了效价1∶50 000的抗艰难梭菌毒素A的卵黄抗体。结论获得了具有生物学活性的艰难梭菌毒素A C端结构域重组蛋白,为建立艰难梭菌基因工程疫苗打下了基础,并制备了针对艰难梭菌毒素蛋白A的卵黄抗体,可用于艰难梭菌相关性腹泻的诊断和治疗。
Purpose Recombinant expression of the C-terminal domain of C. difficile toxin A yolk antibodies against the C terminus of C. difficile toxins were prepared. Methods The C - terminal domain DNA fragment of Clostridium difficile toxin A was cloned into the expression vector pET32b (+). The recombinant plasmid was transformed into E. coli and induced to express. The fusion protein was purified by high affinity nickel ion resin. Enzyme digestion with high affinity nickel ion resin adsorption of excess polypeptide to obtain the desired protein. Detecting the biological activity of the target protein, immunizing laying hens with the target protein to prepare hen egg yolk antibody against the binding region of C. difficile toxin A receptor, isolating and purifying the egg yolk antibody and measuring the yolk antibody titer by ELISA. As a result, the gene fragment of the optimized C. difficile toxin A receptor binding region was obtained, and the biologically active toxin A C-terminal domain recombinant protein was prepared. Egg yolk antibody against Clostridium difficile toxin A with a titer of 1:50 000 was obtained after immunization of laying hens. CONCLUSION: The biologically active recombinant Clostridium difficile toxin AC-terminal domain recombinant protein has laid the foundation for the establishment of C. difficile genetic engineering vaccine and yolk antibody against toxin A of C. difficile has been prepared for use in C. difficile Related diarrhea diagnosis and treatment.