摘要
应用噬菌体表面呈现技术,从腮腺炎病毒抗体阳性者淋巴细胞中提取总RNA,经RT-PCR扩增出抗体重链Fd和轻链基因,经XhoI/SpeI、SacI/XbaI双酶切,先后克隆入噬粒载体pComb3中,再电转化大肠杆菌XL1-blue,以辅助噬菌体VCSM13进行超感染。从分离出的淋巴细胞中共提取高质量RNA约110μg,经逆转录、PCR,分别扩增出约700 bp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2.5 kV、200 Ω、25μF的条件下电转化,转化率为2.48×107。最终得到库容量为6.4×106,滴度为7.8×1013 cfu/L的噬菌体抗体库。所构建的抗腮腺炎病毒特异性噬菌体抗体库容量中等大小,能满足从中筛选抗HN蛋白噬菌体抗体的需要。
The total RNA extracted from PBLs of volunteers infected with mumps viruses was reverse transcripted, and light chain and heavy chain Fd fragment genes of the Immunoglobulin were amplified by PGR using primers scanning κ,λand Fd. After digestion with SacI+Xbal and Spel+XhoI, the amplified fragments were cloned into phagemid pComb3 and electrotransinfected competent E.coli XL1-blue respectively. 110 μg total RNA was extracted from PBLs with high quality. About 700 bp κ,λand Fd genes were amplified respectively by RT-PCR. The PCR products and pComb3 were ligated after purification and double digestion, and electrotransinfection was conducted with final 2.48×107 transforming ratio under the condition of 2.5 kV, 200 Ω, 25 μF. By then, a 6.4×106 phage Fab library(titer 7.8×1013 cfu/L) against mumps viruses was constructed by using phage surface display technique. Human phage antibody library against mumps viruses was constructed effectively and practicably. Further scanning work is undertaking.
出处
《生物技术通讯》
CAS
2003年第6期546-548,共3页
Letters in Biotechnology
基金
2002年度广东省医学科研基金(A2002666)
深圳市科技计划项目基金
