对一个CDR区发生突变的改形抗CD28重链单域抗体的研究

The Study of a Reshaping Anti-CD28 Heavy-chain Variable Domain(V_H) Antibody with CDR Mutations

在构建改形抗CD2 8重链单域抗体时 ,得到一个具有较高免疫学活性的改形抗CD2 8重链单域抗体基因序列。其推导氨基酸序列与改形构建时所选的人源抗体的FRs同源性最高 ;其推导氨基酸序列与鼠源抗CD2 8单克隆抗体重链可变区氨基酸序列比较发现 ,该改形抗体在CDR2区 53位缺失Ala氨基酸残基 ;而在 65a位上插入一个Arg氨基酸残基。在CDR3区缺少Asp95,Lys96,Gly97,Tyr98氨基酸残基。在FR3区缺少Lys82a ,Ser82b ,Leu82c氨基酸残基。由于该序列发生的突变较大 ,作者进一步对其进行了研究。该改形抗CD2 8VH 单域抗体基因与人c myc及人IgG3’CL绞链区基因在大肠杆菌中融合表达。融合表达包涵体经复性 ,纯化处理后 ,仍具有较高与人CD2 8分子结合活性。

One gene with CDR mutations of the reshaping anti CD28 heavy chain variable domain(V H) antibodies, which had high antigen binding activity, was obtained from a phage library of the reshaping anti CD28 heavy chain variable domain(V H) antibodies. It's deduced amino acid sequence was most homologous as the acceptor human antibody FRs. To compare it's deduced amino acid sequence with the original mouse anti human CD28 V H amino acid sequence, We found that there were a deletion mutation of Ala 53 and a insertion mutation of Arg 65a in CDR2; deletion mutations of Asp 95 ,Lys 96 ,Gly 97 ,Tyr 98 in CDR3; and deletion mutations of Lys 82a ,Ser 82b ,Leu 82c in FR3. Because there were many mutations in this gene, a further study was carried out. It was co expressed with c myc tag and the hinge region of human IgG3'CL gene in E.coli. BL21, and the results of ELISA showed that antigen binding activity of the refolding fusion protein was still kept in a high level.