应用哺乳动物细胞表面抗体展示技术构建登革病毒特异性全长人源抗体库

Construction of dengue virus- specific full- length fully human antibody libraries by mammalian display technology

目的应用哺乳动物细胞表面抗体展示技术构建登革病毒特异性全长人源抗体库。方法采集登革确诊患者恢复期外周血,分离外周血淋巴细胞并提取其总RNA,用RT-PCR扩增全长Kappa轻链(LCκ)和重链可变区(VH)基因,构建抗体轻链基因库和抗体重链基因库;将抗体基因库转染CHO细胞、用流式细胞仪分析抗体在CHO细胞表面的表达。结果以1.2μg总RNA为模板,用6套引物扩增全长Kappa轻链和重链可变区基因,插入表达载体,轻链基因库容量为1.45×104,重链基因库容量为1.8×105;随机挑选的10个轻链克隆和10个重链克隆,经过测序鉴定,轻链有8个含有正确的阅读框架,编码8个不同的氨基酸序列,重链有7个含有正确的阅读框架,编码7个不同的氨基酸序列。流式细胞仪分析含有正确阅读框架的单克隆和抗体基因库,均检测到全长抗体在CHO细胞表面的表达,抗体库中可表达的抗体多样性达到109。结论以1.2μg总RNA为模板,通过RT-PCR扩增,成功构建可表达库容量达到109的登革病毒特异性全长人抗体基因库。

Objective To construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique. Methods Total RNA was extracted from peripheral blood mononuclear cells （PBMCs） from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions （LC~ and VH） of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry. Resluts Using 1.2 pg of the total RNA isolated from the PBMCs as the template, the LCK and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45~104 and the heavy chain gene library had a size of 1.8~10L Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46 ~ 109 [（1.45 ~ 104x 80%）x （1.8 x 105~ 70%）]. Conclusion Using 1.2 gg of total RNA as template, the LOc and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 109.