人源抗汉坦病毒核衣壳蛋白T7噬菌体抗体库的构建  

Construction of T7 Phage Display Library against Nucleocapsid Protein of Hantaan Virus

作　　者：刘兵[1] 王美莲[1] 王斯[1] 史俊岩[1] 郑兰艳[1] 牟玲[2] 罗恩杰[1] 

机构地区：[1]中国医科大学基础医学院病原生物教研室,辽宁沈阳110001 [2]沈阳市传染病院肾综合征出血热研究所,辽宁沈阳110003

出　　处：《微生物学杂志》2007年第2期16-20,共5页Journal of Microbiology

基　　金：辽宁省自然科学基金资助(20062101)

摘　　要：构建人源T7噬菌体单链抗体(scFv)库筛选抗汉坦病毒核衣壳蛋白(NP)抗体。从肾综合征出血热恢复期患者外周血淋巴细胞中提取总RNA,反转录合成cDNA第一条链,PCR分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL),经重叠延伸拼接(SOE)PCR组成scFv基因,并将其与T7噬菌体载体的2个臂相连接。体外包装后,在宿主菌BLT5403中,扩增重组噬菌体抗体库。以基因工程表达NP进行4轮“吸附-洗脱-扩增”的筛选,酶免疫实验检测抗体活性。所建抗体库库容为1.35×107,扩增后初级库滴度为2.12×1010pfu/mL。以NP抗原筛选后抗体出现特异性富集,经酶免疫实验鉴定,得到2株与NP抗原特异结合的噬菌体抗体。结果表明,研究成功构建了人源抗NP蛋白T7噬菌体抗体库。A T7 phage display library of human single-chain fragment of variable region （scFv） was constructed to screen antibodies against nueleoeapsid protein （NP） of Hantaan virus. Total RNA was extracted from peripheral lymphocyte of hemorrhagic fever with renal syndrome （HFRS） patients during the recovery stage. The first strand of eDNA was synthesized by reverse transcription assay. After amplification of variable region of splicing overlap extension （SOE）PCR, and then ligated into 2 arms of T7 phage vector. And after packing in vitro, the reeombinants of T7 phage were propagated in BLT 5403 host, Phages from the library were screened by 4 rounds of ＂absorption-elution- propagation＂ with gene-engineering expressed NP and individual clones were assayed by enzyme immunoassay. Phage display library with size of 1.35 × 107 was obtained. The titer of the phage library was 2.12× 10^10 pfu/mL after propagation, Specific phage was enriched after the screening with NP and 2 clones with ability of specific binding to NP were identified by enzyme immunoassay. The results demonstrated that the T7 display library of human antibodies against NP was constructed successfully.

关 键 词：汉坦病毒 SCFV t7噬菌体抗体库 

分 类 号：R373.3[医药卫生—病原生物学]