摘要
目的构建含抗－ＨＢｓ的人源性噬菌体抗体库。方法用ＲＴ－ＰＣＲ技术，从自然感染的抗－ＨＢｓ阳性的两名献血员的外周血淋巴细胞中，扩增出人抗体轻链Ｋ基因和重链Ｆｄ基因，将二基因先后克隆人ＰＣｏｍｂ３Ｈｓｓ载体中，转化大肠杆菌，再用辅助噬菌体感染。结果构建了库容量为１５×１０５的轻链基因抗体库，轻链基因插人率为５７％和库容量为５×１０５的人Ｆａｂ体库，轻、重链基因插人率为５０％。经辅助噬菌体感染，得到噬菌体滴度为５×１０１４ＣＦＵ／ｍｌ的人源性噬菌体抗体库。结论成功构建了合抗－ＨＢｓ的人源性噬菌体抗体库，为进一步筛选ＨＢｓＡｇ的人Ｆａｂ噬菌体抗体奠定了基础。
Objeetive To construct a human recombinant immunoglobulin librafy containing HBsAb.Methods The mRNA isolated from peripheral blood lymphocytes of two blood donors with HBsAb werereversely transcribed to the fifst strand cDNA with oligo-(dT) primer. Human immunoglobulin k lightchains and heayy chains Fd gmes were amplified respectively by PCR with designed oligo nucleotideprimers and the first strand cDNA t6mplates. The k light chain gmes were first cloned into PComb3Hssvector to coflstruct a human recombiflant light chain library. The heavy chain gunes were subwnuentlyinserted into the corresponding sites of k-Pcomb3Hss plasmid to g6nerate a combinatorial K-Fd-PComb3Hss plasmid. The Plaslnid transformed into E.Coli.XLI-Blue and the E.Coli.XLI--Blue infected byhelp phage. R.sults The constructed light chains library size was 1.5 x 105, and the recombinantfreguency of x chain genes was 57% and the human combinatorial Fab immunoglobulin library was 5 x105 members and the recombinant frequency of Fab gmes was 50%. Finally, the titer of pharos in thephals anybody library containning HBsAb was 5 x 1014 CFU/ml. Conclusion Our work in constructinghuman immunoglobulin combinatorial library containning HBsAb will be beneficial to further study onejecting Fab phals antibodies of HBsAg from tile library.
出处
《中华肝脏病杂志》
CAS
CSCD
1999年第3期159-161,共3页
Chinese Journal of Hepatology
