Reconstruction,expression and characterization of dsFv V_L of human antibody to N terminal fragment of human lipopolysaccharide binding protein
- VernacularTitle:抗人脂多糖结合蛋白氨基端片段抗体dsFv V_L链的构建、表达和鉴定
- Author:
Xiaodong GE
;
Jia ZOU
;
Yanli YANG
;
Yousheng LIU
- Publication Type:Journal Article
- Keywords:
N terminal fragment of human lipopolysaccharide binding protein;
disulfide stabilized Fv fragments;
cysteine
- From:Journal of Third Military Medical University
2003;0(14):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To introduce the mutated gene coding cysteine into the gene of dsFv VL of human antibody to N terminal fragment of lipopolysaccharide binding protein(LBP)and to express,purify the mutated dsFv VL in bacterium.Methods We reconstructed and sequenced the mutated gene of VL of human mAb Fab to LBP by Mega-primer PCR based on point mutagenesis method.Some codes of FWR1 of VL had been replaced by TGT in order to code cysteine.The DNA sequence of reconstructed VL was inserted into vector pET-28a(+),then VL was expressed by E.coli.BL21 star(DE3)and was purified by chromatography.Finally the activity of VL to bind NH-LBP was determined by ELISA.Results The results showed that the cysteine was introduced into the position 21 amino acid of VL to replace the threonine.The gene of VL was about 650 bp and relative molecular weight of VL was 28?103.VL could bind NH-LBP directly.Conclusion These have laid a foundation for producing the dsFv against NH-LBP.
