艰难梭菌毒素AC端结构域在大肠杆菌中的可溶性表达与卵黄抗体的制备

Expression of C-terminal domain of Clostridium difficile toxin A and production of yolk antibody IgY against recombinant toxin A

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。

Objective To express the C-terminal domains of toxin A for the development of egg yolk immunoglobulin against Clostridium difficile-associated diarrhea.Methods The DNA sequence of C-terminal domain of toxin A（TcdA-C） was optimized and synthesized.The gene was cloned into pET32b（＋） vector before the recombinant vectors were transformed to E.coli BL21（DE3） competent cells.Fusion protein was purified by high affinity Ni＋resin.After thrombin digestion,TcdA-C without tag was recovered from flow through of another round of high affinity Ni＋resin purification.Biological activities of TcdA-C were assayed.High potency specific IgY antibodies against TcdA-C were prepared from immunization of egg laying hens,then the IgY antibodies were purified.Results Soluble recombinant TcdA-C protein was expressed in E.coli at a high level,and with good biological activity.The titer of prepared TcdA-C specific IgY antibodies was 1∶ 50 000.Purified IgY had neutralization activity in rabbit intestinal loop test.Conclusion Recombinant TcdA-C protein with native biological activities is successfully expressed and purified,which helps develop recombinant vaccines of C.difficile.IgY antibodies against the TcdA-C protein can be used for diagnosis and treatment of C.difficile-associated diarrhea.