重组抗c-Met嵌合抗体的构建及其利用慢病毒快速表达

Construction of chimeric anti-c-Met antibody and its rapid expression using lentiviral vector

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。

The variable region of the heavy chain（V H） and light chain（V L） genes were amplified directly from the cDNA of 3EID7 hybridoma cells which secreted mouse anti-human c-Mesenchymal epithelial transition factor（c-Met） antibody using the reverse transcription PCR（RT-PCR）. Full length chimeric IgG was assem- bled by fusing the VH and VL with the constant region of human heavy and light chains（IgG1 C71 and Cx） respectively using the splice overlap extension PCR（SOE-PCR）. The constructed chimeric antibody heavy chain （H） and light chain（L） were inserted into the lentiviral transfer plasmid pRRL-CMV respectively, which were identified by the restriction enzyme digestion and sequencing. The lentiviral expression vectors were transfected into 293T cells with lentiviral packaging plasmid and the transfected cell pool was used to produce the recombinant antibody. The expressed chimeric antibody was purified by Protein A Sepharose 4B affinity chromatography, then identified by reduced SDS-PAGE and its biological affinity was determined by ELISA. Restriction enzyme digestion analysis proved that recombinant plasmid pRRL-CMV-ch3E-H and pRRL-CMV-ch3E-L were constructed correctly, Light and heavy chains of the chimeric antibody, with molecular masses of about 25 and 50 kDa, were ob^rved on reduced 10~~ SDS-PAGE. ELISA results showed the purified chime- ric antibody could bind to c-Met specifically. Chimeric anti-c-Met antibody was successfully prepared, which was expected to be less immunogenic due to the decrease of components from mouse origin. It laid a founda- tion for further study of anti-c-Met therapeutic antibody.