基于单个B淋巴细胞扩增技术筛选马传染性贫血病毒的囊膜蛋白单克隆抗体

Sorting of EIAV envelop protein monoclonal antibodies based on single B cell amplification technology

马传染性贫血病毒(EIAV)囊膜蛋白(Env)是参与病毒感染过程中的关键蛋白,由表面蛋白(Gp90)及跨膜蛋白(Gp45)组成,Env蛋白在介导病毒粘附、细胞间融合、疾病传播等方面发挥重要作用。为了通过单个B淋巴细胞扩增技术获得Env特异性单克隆抗体(MAb),本研究利用密码子优化后的Gp90基因构建了重组质粒pcDNA3.1-s-gp90-His,按常规方法将该重组质粒免疫小鼠6次后分离脾细胞,利用荧光激活细胞分选(FACS)技术分离免疫小鼠脾细胞中可识别Gp90的单个B淋巴细胞,结果显示,本研究从2×10^(8)个小鼠脾细胞中获得890个识别Gp90的单个B淋巴细胞。通过特异性引物以单个B淋巴细胞的基因组cDNA为模板扩增抗体重链(Ig VH)和轻链(Ig VL)的可变区基因,利用重叠延伸PCR方法分别将重链(Ig VH)和轻链(Ig VL)可变区基因克隆至pcDNA3.1中,测序鉴定结果显示分别正确构建了27对匹配的pcDNA3.1-VH和pcDNA3.1-VL表达质粒,将这27对匹配的表达质粒分别按照pcDNA3.1-VH:pcDNA3.1-VL=1:1的比例共转染至HEK293F细胞,收获细胞上清,经Protein A纯化获得Gp90 MAb。通过ELISA和western blot两种方法对纯化的Gp90 MAb进行鉴定,结果显示获得了27株可匹配的抗体基因对,经表达后有4株MAb可与Gp90蛋白结合,且可以识别Gp90蛋白的天然构象。本研究首次采用单个B淋巴细胞扩增技术对Gp90 MAb进行了筛选,获得的Gp90 MAb为Env蛋白检测方法的研究提供了物质基础,同时为深入研究Env蛋白的功能奠定了基础。

Equine infectious anemia virus(EIAV)envelope protein(Env)is a key protein involved in the infection process which consists of surface protein(Gp90)and transmembrane protein(Gp45).Env plays an important part in virus adhesion,cell fusion,disease transmission and other aspects.In order to obtain Env-specific monoclonal antibody(MAb),we constructed the codon-optimized Gp90 gene on pcDNA3.1,and built the recombinant plasmid pcDNA3.1-s-gp90-His.According to the conventional method,after immunizing mice with the recombinant plasmid pcDNA3.1-s-gp90-His 6 times,spleen cells were isolated.We used fluorescence activated cell sorting(FACS)technology to isolate the single B lymphocyte that can recognize Gp90 in the spleen cells of immunized mice.890 single B lymphocytes that recognize Gp90 were obtained from 2×10^(8)mouse spleen cells.Then,we amplified the variable region genes of the antibody heavy chain(Ig VH)and light chain(Ig VL)through specific primers using the genomic cDNA of single B lymphocyte as a template,and the heavy chain(Ig VH)and the light chain(Ig VL)variable region gene was ligated to pcDNA3.1 by overlapping PCR.The sequencing and identification results showed that 27 pairs of matching pcDNA3.1-VH and pcDNA3.1-VL expression plasmids were successfully constructed.The plasmids were co-transfected into HEK293F cells at the ratio of pcDNA3.1-VH:pcDNA3.1-VL=1:1,and the cell supernatant was harvested and purified by Protein A column to obtain Gp90 monoclonal antibody.The purified Gp90 monoclonal antibodies were identified by ELISA and western blot.Results showed that 4 monoclonal antibodies can bind with Gp90 protein and recognize the natural conformation of Gp90 protein from 27 matched antibody gene pairs were obtained.This study used single B lymphocyte amplification technology to screen Gp90 monoclonal antibodies for the first time.At the same time,the acquisition of Gp90 monoclonal antibodies provided a basis for the development of Env protein detection methods and laid a foundation for in-depth study of the function of Env protein.