人抑制素βB亚基片段的合成及其单克隆抗体的制备

SYNTHESIS OF HUMAN INHIBIN βB FRAGMENTS, PREPARATION AND GENERATION OF MONOCLONAL ANTIBODIES AGAINST HUMAN INHIBIN βB SUBUNITS

目的 合成人抑制素 βB亚基两个片段 βB(1 2 8) (GLECDGRTNLC CRQQFFIDFRLIGWNDW ) ,βB(85 115 ) (LSTMSMLYFDDEYNIVKRDVPNMIVEECGCA)并制备其单克隆抗体。方法 应用固相多肽合成法人工合成了 2个 βB亚基片段 ,以化学合成的片段作为半抗原 ,经抗原的制备、单抗的制备获得 4株杂交瘤细胞株。单抗的鉴定及特异性分析实验表明这些单克隆抗体具有特异性 ,敏感性和实用性。对抑制素α,βA ,βB亚基的单抗进行了进一步的纯化。用纯化后的单抗进行了乳腺癌的免疫组化研究。结果 相关肿瘤组织对各亚基单抗的免疫组化反应无明显差异 ;乳腺癌对各亚基单抗的免疫组化反应阳性率无明显差异。

AIM To synthesize fragments of inhibin subunits βB(1 28) (GLECDGRTNLC CRQQFFIDFRLIGWNDW), βB(85 115) (LSTMSMLYFDDEYNIVKRDVPNMIVEECGCA) and to obtain their monoclonal antibodies. METHODS Two segments of inhibin βB subunit were synthesized as semiantigens by solid phase peptide synthesis with Fmoc chemistry. These peptide products were conjugated with TG as antigens to prepare McAbs against inhibin βB subunit while EDC was coupling reagent. After immunization, fusion, selection and cloning procedures, 4 hybridoma cell lines were obtained. Ascites titer, specificity and relative affinity were identified. Four hybridoma cell lines were obtained through preparations of immunogens and monoclonal antibodies. The analysis of these McAbs indicated that they were highly special, sensitive and practical. The McAbs of inhibin α, βA and βB subunits were further purified. The immunohistochemistry in breast cancer was carried out with these three subunits′ McAbs. RESULTS The McAbs of inhibin βB subunit were highly sensitive, specific and useful. There was no notable divergence on the reactions of relative tumor tissues to the McAbs of inhibin α, βA and βB subunits in immunohistochemistry study. There was also almost the same positive ratio of the reaction of breast cancer tissues to these McAbs. CONCLUSION The method of detecting serum inhibin level will be established on the basis of present research.