抗HEV嵌合抗体的构建及在CHO细胞中的表达

通过RT-PCR方法从分泌戊型肝炎(戊肝)病毒中和性鼠源单克隆抗体(单抗)8C11的杂交瘤细胞中克隆出抗体基因的重链可变区(VH)、轻链可变区(VK)序列,并分别克隆到含有人gamma 1重链和kappa轻链恒定区序列的pcDNA3.1/Hygro和pcDNA3.1( )质粒中,共转染中华仓鼠卵巢癌细胞(CHO)细胞.RT-PCR结果表明,转染的CHO细胞转录了嵌合重链及轻链基因,间接ELISA及Western blot结果表明:翻译出的两种多肽在细胞内正确组装成嵌合抗体分子,并可分泌至细胞外,表达的嵌合抗体保留了原鼠单抗的抗原结合特异性及对8H3结合抗原的增强作用.8C11嵌合抗体的成功表达可降低鼠源性,为探讨戊肝抗体治疗的可能性奠定了基础.

Construction and Expression of a Mouse-human Chimeric Antibody against Hepatitis E Virus

A neutralizing mouse anti-HEV monoclonal antibody(McAb),designated as 8C11,has previously been produced and characterized by our laboratory.We reported here the construction and the expression of mouse-human chimeric antibody derived from the McAb.cDNAs encoding variable regions of heavy and light chains were isolated from 8C11 cells by RT-PCR,and introduced into mammalian expression vectors pcDNA3.1/Hygro and pcDNA3.1(+)containing cDNA of human gamma 1 and kappa constant regions,respectively.The vectors were cotransfected into CHO cells.The result of RT-PCR showed that the introduced genes for chimeric heavy and light chains were transcribed.Indirect ELISA and Western blot indicated that the peptides were assembled correctly to form native IgG molecule, which could be secreted outside the cells.The expressed chimeric antibody held the binding specificity of its original McAb to HEV and enhanced the binding activity of 8H3 to antigen.The chimeric antibody described here is expected to be less immunogenic and thus more suitable for possible antibody therapy for hepatitis E.