抗血管内皮生长因子鼠-人嵌合抗体真核表达载体的构建及在CHO DG44细胞中的表达

Construction of eukaryotic expression vector for mouse-human chimeric antibody against vascular endothelial growth factor in CHO DG44 cells

目的构建抗血管内皮生长因子(vascular endothelial growth factor,VEGF)鼠-人嵌合抗体真核表达载体,并在CHO DG44细胞中进行表达及初步鉴定。方法分别采用鸡胚尿囊膜试验和小鼠肿瘤抑制试验检测鼠抗人VEGF单克隆抗体2E5对VEGF促血管生成和肿瘤增生的抑制作用;采用RT-PCR法从鼠源性抗VEGF单克隆抗体的杂交瘤细胞2E5中扩增抗VEGF抗体重链可变区基因(VH)和轻链可变区基因(VL),利用IMGT数据库进行序列比对分析,筛选出功能性基因;采用重叠延伸PCR技术(gene splicing by overlap extension PCR,SOE PCR)将VH基因与人Ig G1重链恒定区基因连接,VL基因与人Ig G1轻链恒定区基因连接,分别构建嵌合抗体重链基因和轻链基因,将嵌合的重链基因连入p Opti VECTM-TOPO誖TA载体,轻链基因连入pc DNATM3.3-TOPO誖TA载体,构建重链表达质粒p Opti VEC-H和轻链表达质粒pc DNA 3.3-L;采用脂质体法将重链和轻链表达质粒共转染CHO DG44细胞,经缺陷性培养基筛选和抗性标记筛选、MTX加压后,利用有限稀释法挑选单细胞株,ELISA法检测嵌合抗体的表达量;取批次培养的嵌合抗体细胞株上清液,经Mab Select Sure亲和纯化和陶瓷羟基磷灰石纯化后,对纯化的嵌合抗体进行SDS-PAGE和高效液相色谱分析,并测定N-端序列、分子量和解离常数。结果鼠抗人VEGF单克隆抗体2E5能抑制VEGF的促血管生成和肿瘤生长。嵌合表达载体p Opti VEC-H和pc DNA 3.3-L经菌落PCR及测序证实构建正确。经缺陷性培养基筛选、抗性标记筛选、MTX加压及单克隆筛选后,嵌合抗体的表达量可达68.5μg/ml。纯化的嵌合抗体纯度达99%以上;N-端测序结果与预期重、轻链N-端氨基酸序列一致;质谱法测定分子量为149 290;解离常数为2×10-9 mol/L。结论成功构建了抗VEGF鼠-人嵌合抗体轻、重链表达载体,在CHO DG44细胞中表达了嵌合抗体,经层析纯化的嵌合抗体纯度较高,与VEGF抗原具有较强的亲和力,为后续进行抗VEGF单克隆抗体的人源化改造奠定了基础。

Objective To construct the eukaryotic expression vector for mouse-human chimeric antibody against vascular endothelial growth factor（VEGF）,express in CHO DG44 cells and preliminarily identify the expressed product. Methods The inhibitory effect of murine anti-human VEGF monoclonal antibody 2E5 on the activity of VEGF in promoting angiogenesis and tumor proliferation by chorioallantoic membrane assay and tumor suppression assay in mice. The VH and VL genes of VEGF antibody were amplified from hybridoma 2E5 cells secreting anti-VEGF monoclonal antibody from mouse origin by RT-PCR,of which the sequences were compared by using IMGT databank to screen the functional gene.The amplified VH gene was linked to the gene of constant region of heavy chain,while the VL gene to that of light chain of human Ig G1 by gene splicing by overlap extension PCR（SOE PCR）,to construct the heavy and light chain genes of chimeric antibody,respectively. The chimeric heavy chain gene was inserted to vector p Opti VECTM-TOPO誖TA,while the chimeric light chain gene to vector pc DNATM3. 3-TOPO 誖 TA,to construct recombinant plasmids p Opti VEC-H and pc DNA3. 3-L,respectively. The constructed recombinant plasmids were co-transfected to CHO DG44 cells,from which single cell strain was screened with defective medium,drug-resistant marker and MTX pressure and determined for the expression level of chimeric antibody by ELISA. The supernatant of batch culture of cell strain secreting chimeric antibody was collected,from which chimeric antibody was purified by Mab Select Sure affinity chromatography,and analyzed by SDS-PAGE,HPLC,as well as tests for N-terminal sequence,relative molecular mass and dissociation constant. Results Mouse anti-human VEGF monoclonal antibody 2E5 inhibited the promoting effect of VEGF on angiogenesis and tumor growth. Colony PCR and sequencing proved that recombinant plasmids p Opti VEC-H and pc DNA3. 3-L were constructed correctly. The expression level of chimeric antibody after screening with defective medium,drug-resistant marker,MTX pressure and screening of single clones reached 68. 5 μg / ml,while the purity reached more than 99% after purification.The N-terminal sequencing results were consistent with those of amino acid sequences of heavy and light chains expected.The molecular weight determined by mass spectrometry was 149 290,while the dissociation constant was 2 × 10-9 mol / L.Conclusion The expression vector for light and heavy chains of mouse-human chimeric antibody against VEGF were successfully constructed and expressed in CHO DG44 cells. The purified chimeric antibody by chromatography showed high purity and high affinity to VEGF antigen,which laid a foundation of further humanization of monoclonal antibody against VEGF.