重组抗c-Met嵌合抗体的构建及其利用慢病毒快速表达

发布时间：2018-10-16 14:24

【摘要】：文章采用兼并引物逆转录PCR(reverse transcription PCR,RT-PCR)从特异性分泌抗人c-Met阻断型单克隆抗体的杂交瘤细胞株3E1D7中扩增抗体重、轻链可变区基因(V_H和V_L)。通过重叠延伸PCR(splice overlap extension PCR,SOE-PCR)方法分别将鼠源V_H与人IgG1的重链恒定区基因Cγ1相连,鼠源V_L与人IgG1的轻链恒定区基因Cκ相连获得嵌合抗体重链基因(H)和轻链基因(L)。将嵌合抗体重、轻链基因连接到慢病毒表达载体中,经双酶切和测序鉴定成功构建了慢病毒表达质粒pRRL-CMV-ch3E-H和pRRL-CMVch3E-L。磷酸钙法转染293T细胞,表达的嵌合抗体经Protein A Sepharose 4B亲和层析柱纯化,通过SDSPAGE检测抗体的完整性,ELISA法检测嵌合抗体的特异性抗原结合活性及人源性。感染慢病毒的293T细胞上清纯化后,经SDS-PAGE分析,可见25kDa嵌合抗体轻链和50kDa的嵌合抗体重链蛋白。且纯化后的嵌合抗体能够与c-Met抗原特异性结合成功获得重组抗人c-Met嵌合抗体,该抗体减少了鼠源成分,降低了免疫原性,利用慢病毒可以快速获得大量重组抗体蛋白,为该抗体药物的体内外评估奠定基础。
[Abstract]:The anti-body weight and light chain variable region genes (VH and VL) were amplified by degenerate primer reverse transcriptase (PCR (reverse transcription PCR,RT-PCR) from hybridoma cell line 3E1D7 which specifically secreted monoclonal antibody against human c-Met blocking type (VH and VL). By overlapping extension PCR (splice overlap extension PCR,SOE-PCR), mouse VH was linked to human IgG1 heavy chain constant region gene C 纬 1, and mouse origin VL was linked to human IgG1 light chain constant region gene C 魏 to obtain chimeric antibody heavy chain gene (H) and light chain gene (L). The chimeric anti-body weight and light chain genes were ligated into the lentivirus expression vector. The lentivirus expression plasmids pRRL-CMV-ch3E-H and pRRL-CMVch3E-L. were successfully constructed by double enzyme digestion and sequencing. The chimeric antibody was purified by Protein A Sepharose 4B affinity chromatography column. The integrity of the antibody was detected by SDSPAGE. The specific antigen-binding activity and human origin of chimeric antibody were detected by ELISA method. After purification of 293T cells infected with lentivirus, 25kDa chimeric antibody light chain and 50kDa chimeric antibody heavy chain protein were found by SDS-PAGE analysis. The purified chimeric antibody could be successfully combined with c-Met antigen to obtain recombinant anti-human c-Met chimeric antibody. The antibody reduced the murine component and the immunogenicity, and a large number of recombinant antibody proteins could be obtained quickly by using lentivirus. To lay the foundation for the in vivo and in vitro evaluation of the antibody drug.
【作者单位】：同济大学生命科学与技术学院;烟台荣昌制药股份有限公司;
【基金】：国家自然科学基金青年科学基金资助项目(81402399) 中央高校基本科研业务费专项资金资助项目(2000219121) 江苏省自然科学基金资助项目(BK2012162)
【分类号】：R392

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