人源尖吻蝮蛇毒蛋白抗体单链可变区片段噬菌体文库的初建  

Primary construction of single variable region fragment phage library against Agkistrodon acutus echidnotoxin from human origin

作　　者：刘明华[1] 曹宇亮[1] 张雷[1] 陈翔宇[1] 向强[1] 田君[1] 

机构地区：[1]第三军医大学西南医院急救部,重庆400038

出　　处：《中国生物制品学杂志》2014年第1期19-22,27,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81071537);重庆市科技攻关项目(CSTC\2010AC5026);第三军医大学临床创新基金(2009XLC15)资助

摘　　要：目的构建人源尖吻蝮蛇毒蛋白抗体单链可变区片段(scFv)噬菌体文库。方法采用Trizol试剂提取尖吻蝮蛇咬伤后恢复期患者的外周血淋巴细胞的总RNA,用Oligotex誖mRNA Mini Kits纯化获得总mRNA,逆转录合成总cDNA,针对人抗体轻链和重链可变区基因设计一系列特异性引物,以合成的总cDNA为模板,扩增得到抗体轻链和重链可变区基因库,再通过重叠延伸PCR(splicing by overlap extension PCR,SOE-PCR)将轻链和重链拼接得到scFv文库;将scFv基因双酶切后,插入T7噬菌体骨架进行包装,以尖吻蝮蛇蛇毒蛋白抗原进行4轮淘选,建立蛇毒蛋白抗体scFv噬菌体文库,采用噬斑法检测scFv噬菌体文库滴度并进行PCR及测序鉴定。结果成功地将轻链和重链可变区基因库混合拼接得到scFv文库;经4轮淘选,蛇毒蛋白抗体scFv噬菌体文库滴度为6.0×1014PFU/ml,抗体基因在噬菌体中表达的阳性率达50%以上,表达的scFv片段均为轻链和重链的杂合体,可较好的重现可变区的多样性。结论已成功构建了人源尖吻蝮蛇毒蛋白抗体scFv噬菌体文库,为下一步噬菌体文库的克隆构建和筛选奠定了基础。Objective To construct a single variable region fragment phage library againsl Agkistrodon aciaus echidnotox- in from hnnlan origin. Methods Total RNA was extracted f＇ronl peripheral blood lymphncyles of viciilns bitten [iv Agk- istrndon aeuins, at convalescence period, by using Trizol kit, from which total mRNA was purified by using Oligolex mRNA Mini Kits and reversely transcribed to total el）NA. A series of specific primers for lhe light chain variable region （V l,） and heavy chain variable region （VH） of human antibody were designed, based on which a geoe library was con- slrut＇ted by amplification using the synthetic eDNA as a template. The VI, and VH amt）licons were combined randomly by splicing by overlap extension PCR （SOE-PCR） to construct a scFv library. The scFv geue was digesled with EcoR [ and Hind HI , and inserted into T7 phage backbone for packaging. After four cycles of scanning wilh echidnoloxin antigen, a human s＇Fv phage libra,＂ specific for eehidnotoxin was constructed, determined for tiler by plaque Jssay, anti identified hy PCR and sequencing. Results A scFv library was suceessfillly constructed by mixing the VI, and Vtt libraries. Alter four cycles of scanning, the tiler of seFv phage library specific for echidnotoxin was 6. 0 × 1014 PFU/ml, while the pnsi- live expression rate of antibody gene in phage reached more than 50%. All the expressed seFv fragments were heterozy- gote of light and heavy chains, and the diversity of variable regions was reproduced effectively. Conclusion A seF phage library against eehidnotoxin from human origin was suecesslhlly constructed, which laid a foundation of further cloning, construction and screening.

关 键 词：尖吻蝮蛇 蛇毒蛋白 噬菌体 抗体文库 

分 类 号：R595.8[医药卫生—内科学] Q789[医药卫生—临床医学]