摘要
目的构建人源Fab噬菌体抗体库,筛选抗hPRLR抗体片段并进行初步鉴定。方法从乳腺癌患者外周血提取总RNA,通过RT-PCR扩增人抗体轻链和重链基因,构建抗hPRLR人源Fab抗体库。分别以His-hPRLR融合蛋白、BSA-hPRLR表位多肽融合蛋白和GST-hPRLR融合蛋白作为抗原包板,经过3轮循环的吸附-洗脱-扩增的筛选及1轮交叉筛选,挑单克隆用Phage-ELISA、DNA测序筛选阳性克隆,将筛选到的阳性克隆FabG2转化至Top10’受体菌,诱导表达可溶性Fab抗体,通过Western blot和ELISA进行特异性的鉴定。结果构建的人源Fab库容为1.0×109,4轮的筛选,获得6株能与hPRLR结合的人源抗体克隆。选取的FabG2能够进行可溶性Fab抗体蛋白的表达,ELISA初步鉴定,能够与hPRLR进行特异性地结合。结论成功构建了人源Fab噬菌体抗体库,筛选并鉴定了1株抗hPRLR Fab抗体的克隆。hPRLR特异性Fab抗体的获得将为高表达hPRLR乳腺癌的生物免疫治疗奠定了基础。
We aim to get specific Fab antibody against human prolactin receptor （hPRLR） from the human Fab antibody library constructed by phage display technology. Human lymphocytes were collected from peripheral blood of 24 breast cancer patients. And then the total RNA was extracted and reversely transcribed to eDNA. Genes of light and heavy chains of human antibody were amplified by RT-PCR to construct anti-hPRLR immunized human antibody library. After three rounds of panning and one round of crossed-panning with against his-hPRLR fusion protein, BSA- polypeptide （epitopes of hPRLR） and GST-hPRLR fusion protein, positive clones were chosen by Phage-ELISA and DNA sequencing. Then the positive clones were transformed into E. coli Topl0＇ and induced to express antibody protein. The results indicated that the human Fab phage-display library consisting of 1.0×l0^9 clones were successfully constructed, and six clones were selected after four rounds. One of them named FabG2 expressed protein correctly. ELISA and Western blot analysis showed that FabG2 could bind hPRLR specifically. We concluded that the hPRLR specific Fab antibody selected from large phage-display library could be used as candidates for therapy agent of breast cancer which over-expresses hPRLR.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2012年第6期489-492,496,共5页
Immunological Journal
基金
国家自然科学基金项目(81172142)
江苏省自然科学基金资助项目(BK2008442)
南京医科大学科技发展基金(08NMU2007)
中国博士后科学基金(20080431115
200902526)
江苏省博士后科研资助项目(0801018C)
