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目的:根据人抑制素α、βA、βB3个亚基特点,选择各亚基特定的抗原表位进行融合表达,并制备相应的多克隆抗体。方法:根据各亚基抗原表位氨基酸序列,合成其基因序列,并选用大肠杆菌偏爱密码子,插入pGEX-4T-2中作融合表达,纯化表达产物,免疫绵羊,制备抗体,测定了抗体水平。结果:经酶切鉴定,分别获得抑制素α、βA、βB与pGEX-4T-2连接的阳性重组子pGEX-INHA、pGEX-INHBA、pGEX-INHBB。其表达产物GST-INHA、GST-INHBA、GST-INHBB及纯化后融合蛋白,经考马斯亮蓝染色鉴定,大小正确。用常规免疫法免疫绵羊抗体,经ELISA检测抗体效价,结果显示,直接提取融合蛋白GST-INHA、GST-INHBA、GST-INHBB免疫绵羊抗体效价分别为:1∶64、1∶128、1∶32,纯化后的融合蛋白GST-INHA、GST-INHBA、GST-INHBB免疫绵羊抗体效价分别为:1∶32、1∶32、1∶64。结论:可在本研究所制备的抗体的基础上建立测定血清抑制素水平的免疫诊断方法。
OBJECTIVE: According to the characteristics of human subunit α, βA and βB subunits, the specific epitope of each subunit was selected for fusion expression and the corresponding polyclonal antibody was prepared. Methods: According to the amino acid sequence of each subunit epitope, the gene sequence was synthesized. The preferred codons of E. coli were selected and inserted into pGEX-4T-2 for fusion expression. The expressed product was purified and immunized sheep to prepare antibody. The antibody level . Results: The recombinant plasmids pGEX-INHA, pGEX-INHBA and pGEX-INHBB with inhibin α, βA, βB and pGEX-4T-2 were identified by restriction enzyme digestion. The expression products GST-INHA, GST-INHBA, GST-INHBB and purified fusion protein, identified by Coomassie brilliant blue, the correct size. The antibody of sheep was immunized by routine immunization and the antibody titers were detected by ELISA. The results showed that the antibody titers of GST-INHA, GST-INHBA and GST-INHBB were respectively 1:64, 1:12, : 32, the purity of the purified fusion protein GST-INHA, GST-INHBA, GST-INHBB immune sheep antibody titer: 1:32,1:32,1:64. CONCLUSIONS: Immunodiagnostic methods for the determination of serum inhibin levels can be established on the basis of the antibodies prepared in this study.