【摘要】 目的　构建抗腮腺炎病毒抗体Fab段基因重组表达载体。方法　从腮腺炎患者和腮腺炎抗体IgG阳性正常人群的淋巴细胞中提取总RNA ,逆转录成cDNA。用相应的引物进行PCR ,扩增出轻链和重链Fd段基因 ,经酶切后分别和噬粒载体pComb3连接 ,再经电穿孔转化大肠杆菌XL1 blue菌株 ,将轻链和重链Fd基因先后克隆入pComb3中。结果　从分离出的淋巴细胞中共提取高质量RNA约 1 1 0 μg ,经逆转录、PCR ,分别扩增出约 70 0bp大小的κ、λ和Fd基因。PCR产物和载体经纯化、双酶切后进行连接 ,在 2 5kv,2 0 0Ω ,2 5 μF的条件下电转化 ,最终转化率为 :2 4 8× 1 0 7。随机挑取电转化后的 1 0个菌落 ,经PCR鉴定 ,共有 5个克隆同时含有轻链和重链Fd基因 ,抗体Fab的重组率为 5 0 %。结论　获得的抗体Fab重组表达载体是高效、实用的 ,为下一步构建抗腮腺炎病毒噬菌体抗体库打下基础更多还原

【Abstract】 Objective To construct the recombinant expression vector of genes encoding antibody Fab fragments against Mumps Viruses.Methods The total RNA extracted from PBLs of patients or volunteers infected with Mumps Viruses was reverse transcribed, and light chain and heavy chain Fd fragments of the Immunoglobulin genes were amplified by PCR using primers flanking κ,λ and Fd. After digestion with SacI+XbaI and SpeI+XhoI, the amplified fragments were cloned into phagemid pComb3 and electrotransinfected into competent E coli XL1 blue. Results 110μg total RNA was extracted from PBLs with high quality. About 700bp κ,λ and Fd gene fragments were amplified by RT PCR. The PCR products were ligated with pComb3 after purification and double digestion, and then electrotransinfection was conducted with final 2 48×10 7 transforming ratio under the condition of 2 5kv, 200Ω, 25μF. PCR identification was conducted on 10 random clones. There were 5 clones containing light chain and heavy Fd gene fragments. Conclusion The recombinant expression plasmid harboring antibody Fab gene fragments against Mumps Viruses was constructed. Further construction of phage Fab library against Mumps Viruses was in progress.更多还原