自身免疫性疾病噬菌体抗体库的构建和初步鉴定  

Construction and preliminary identification of phage display antibody library against autoimmune diseases

作　　者：吴炜霖[1,2] 夏维[3] 陆慧琦[1,2] 熊怡淞[1,2] 杨再兴[1,2] 周琳[1,2] 韩焕兴[1,2] 仲人前[1,2] 

机构地区：[1]第二军医大学长征医院实验诊断科 [2]全军医学免疫诊断中心,200003 [3]上海市第一人民医院检验科,200080

出　　处：《国际检验医学杂志》2009年第7期625-626,629,共3页International Journal of Laboratory Medicine

基　　金：科技部高科技863基金(2006AA02Z496);国家自然科学基金(30471616);上海市科委研究计划项目(04DZ19106)

摘　　要：目的构建系统性红斑狼疮（SLE）、类风湿性关节炎（RA）、强直性脊柱炎、干燥综合症（SS）、原发性胆汁性肝硬化（PBC）等自身免疫性疾病的噬菌体抗体库。方法从多例确诊的高滴度自身免疫病患者外周血淋巴细胞中抽提总RNA，用RT—PCR方法分别扩增免疫球蛋白分子轻链κ、λ基因及重链Fd基因。经SacI和XbaI限制性双酶切后，在T4连接酶的作用下，将轻链基因片段克隆入同样双酶切的pComb3Hss载体中以构建轻链文库，随后将重链Fd段PCR产物经XhoI和SpeI双酶切后载入同样处理的κ/λpComb3Hss载体，然后将重组质粒κ/λ—Fd-pComb3Hss转化大肠杆菌XL1-Blue。用辅助噬菌体M13K07感染XL1-Blue，则可在噬菌体表面表达出随机的重组抗体库。结果提取的淋巴细胞总RNA及合成的cDNA质量好，PCR扩增了长度为660bp的轻链κ、λ基因及重链Fd基因，成功构建了库容为4×10^4的轻链抗体库和库容为6×10^4的Fab片段噬菌体抗体库，酶切及PCR鉴定均正确无误。结论成功构建了自身免疫性疾病的噬菌体抗体库，为进-步筛选出高亲和力抗体打下了基础。Objective To construct the phage antibody library against autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, Sjogren syndrome and primary billary cirrhosis. Methods The total RNA was extracted from peripheral blood lympho- cytes of some patients who suffered from autoimmune diseases. The heavy chain Fd and light chain ~, k gene were amplified by RT-PCR. The amplification products were digested by ScaI and XbaI, then ligated into the phagemid vector pComb3Hss to construct light chain library via T4 ligase. The ampli- fication products of Fd and the light chain library were digeseted by XhoI and Spe I. The ligated sam- ple of Fd and light chain library was then transformed into competent E. eoli XL1-Blue. The trans- formed cells were infected with M13KO7 helper phage to yield recombinant phage antibody. Results The quantity of total RNA and cDNA were qualified. Human immunoglobulin light chain and heavy chain Fd genes （660 bp） were amplified successfully. The light chain library with the capacity size of 4 ×10^4 and the human recombinant Fab antibody library with the capacity size of 6×10^4 were also suc- cessfully obtained, while both the digestion by enzymes and PCR identification showed the right re- sults. Conclusion A phage antibody library against autoimmune diseases is successfully constructed, which lays a foundation for the following panning of high affinity antibody.

关 键 词：自身免疫疾病 细菌噬菌体 抗体 

分 类 号：R392[医药卫生—免疫学]