人源化乙肝表面抗体重链克隆、表达及免疫学特性初步鉴定

Cloning,Expression and Immunological Traits of Humanized HBsAb Heavy Chain

目的建立一种利用单细胞分选技术克隆人源化乙肝表面抗体重链的方法。方法从乙肝疫苗接种志愿者外周血中分选得到单个抗体分泌细胞,单细胞RT-PCR筛选20个单菌落,挑选其中有IgG保守区域的克隆全长,并将IgG(H)基因全长克隆到pEGFP-N1载体,转染COS-7细胞,取上清液进行Western blotting验证,并采用Batty饱和法测定亲和力常数。结果筛选得到IgG(H)能与HBsAg结合,且Ka值达到2.39×109L/mol。结论利用单细胞分选克隆技术获得的抗体重链亲和力较高。

Objective To establish a method for cloning humanized HBsAb heavy chain by single - cell sorting and single - cell RT - PCR techniques. Methods Single antibody - secreting cell was isolated by single - cell sorting from peripheral blood of HBV - vacci- nated subjects. 20 colonies were screened by single - cell RT - PCR. The full - length coding HBsAb heavy chain cunserw^d sequence was cloned into pEGFP - N1 vector. Then the vector was transfeeted into COS -7 cells. The binding affinity of 1G（H） heavy chain in cell - free supernatant expressed by COS -7 cells was deteeed by western hlot. Results The IG（H） heavy chain can specific bind to HbsAg and the value of Ka was 2.39 ×10^9L/tool. Conclusion The results denmnstrated that humaried antibody heavy chain has a higtl affinity to HBsAg