CAJ | 학술논문

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79000，经凝血酶酶切后的相对分子质量约65000；目的蛋白免疫产蛋鸡后获得效价为1∶20000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。
목적：재대장간균중가용성표체간난사균독소B최기단（TcdB-c），면역산단계，획득침대TcdB-c적란황항체（IgY）。방법：인공합성TcdB-c적기인，장기극륭지pET32b（+）재체중，전화대장간균BL21（DE3），유도표체산물경금속오합층석순화，응혈매매절후득도목적단백TcdB-c；이용토홍세포응집화토장번실험검측목적단백활성，용TcdB-c면역산단계제비계란황항체，분리순화란황항체병경ELISA측정항체효개，용토장번실험검측항체적중화활성。결과：구건료TcdB-c적중조표체재체，유도표체적융합단백상대분자질량약위79000，경응혈매매절후적상대분자질량약65000；목적단백면역산단계후획득효개위1∶20000적항TcdB-c란황항체，차해항체가이중화TcdB-c대토소장적독성작용。결론：획득료구유생물학활성적TcdB-c，병제비료침대TcdB-c적계란황항체，위이용기인공정방법방치간난사균감염타하료기출。
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.