艰难梭菌毒素B羧基端的表达与卵黄抗体制备

Expression of Receptor Binding Region of Clostridium difficile Toxins B and Production of IgY Antibody

目的:在大肠杆菌中可溶性表达艰难梭菌毒素B羧基端(TcdB-c),免疫产蛋鸡,获得针对TcdB-c的卵黄抗体(IgY)。方法:人工合成TcdB-c的基因,将其克隆至pET32b(+)载体中,转化大肠杆菌BL21(DE3),诱导表达产物经金属螯合层析纯化,凝血酶酶切后得到目的蛋白TcdB-c;利用兔红细胞凝集和兔肠袢实验检测目的蛋白活性,用TcdB-c免疫产蛋鸡制备鸡卵黄抗体,分离纯化卵黄抗体并经ELISA测定抗体效价,用兔肠袢实验检测抗体的中和活性。结果:构建了TcdB-c的重组表达载体,诱导表达的融合蛋白相对分子质量约为79 000,经凝血酶酶切后的相对分子质量约65 000;目的蛋白免疫产蛋鸡后获得效价为1∶20 000的抗TcdB-c卵黄抗体,且该抗体可以中和TcdB-c对兔小肠的毒性作用。结论:获得了具有生物学活性的TcdB-c,并制备了针对TcdB-c的鸡卵黄抗体,为利用基因工程方法防治艰难梭菌感染打下了基础。

Objective： To express carboxyl terminal of Clostridium difficile toxin B（TcdB-c） in E.coli and gener ate chicken yolk antibodies（IgY） against TcdB-c. Methods： The gene sequence of TcdB-c was optimized and syn thesized, and then it was cloned into pET32b（＋） vector, followed by transformed into E.coli BL21（DE3） compe tent cells. After induction, recombinant fusion protein was expressed and purified, and it was digested by thrombin to release TcdB-c. Biological activities of TcdB-c were assayed by hemagglutination and rabbit intestinal loop test. IgY antibodies against TcdB-c were generated from immunized egg laying hens, and specificity and activity of which were detected by ELASA and rabbit intestinal loop test respectively. Results： Recombinant protein with rela tive molecular weight of 79 000 was solubly expressed in E.coli, and the released TcdB-c of 65 000 showed tox ic effect on small intestine of rabbit. IgY antibodies specifically against TcdB-c had titer of 1：20 000 and had neutralization activity. Conclusion： IgY antibodies against TcdB-c protein were prepared, laying foundation for the diagnosis and treatment of C.difficile associated diarrhea by gene engineering strategy.