Reconstruction，expression and characterization of dsFv V<sub>L</sub> of human antibody to N terminal fragment of human lipopolysaccharide binding protein

葛晓冬; 邹佳; 杨艳丽; 刘友生

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Reconstruction，expression and characterization of dsFv V_L of human antibody to N terminal fragment of human lipopolysaccharide binding protein

更新时间：2026-01-09

- Reconstruction，expression and characterization of dsFv V_L of human antibody to N terminal fragment of human lipopolysaccharide binding protein
- Journal of Army Medical University Vol. 29, Issue 14, Pages: 1364-1367(2007)
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  CLC： R392-33;R392.11;{R392.13}
- Published：2007
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葛晓冬,邹佳,杨艳丽,刘友生.抗人脂多糖结合蛋白氨基端片段抗体dsFv VL链的构建、表达和鉴定[J].陆军军医大学学报(原第三军医大学学报),2007,29(14):1364-1367.

　GE Xiao-dong,ZOU Jia,YANG Yan-li,LIU You-sheng.Reconstruction，expression and characterization of dsFv VL of human antibody to N terminal fragment of human lipopolysaccharide binding protein[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(14):1364-1367.
葛晓冬,邹佳,杨艳丽,刘友生.抗人脂多糖结合蛋白氨基端片段抗体dsFv VL链的构建、表达和鉴定[J].陆军军医大学学报(原第三军医大学学报),2007,29(14):1364-1367. DOI：

　GE Xiao-dong,ZOU Jia,YANG Yan-li,LIU You-sheng.Reconstruction，expression and characterization of dsFv VL of human antibody to N terminal fragment of human lipopolysaccharide binding protein[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(14):1364-1367. DOI：

摘要

目的将抗人脂多糖结合蛋白氨基端片段（NH-LBP）抗体的Fv L链引入半胱氨酸（Cys），并表达、纯化。方法应用mega-primer PCR方法，在抗NH-LBP单克隆Fab抗体的轻链基因骨架区中引入编码Cys的基因序列，将目的序列放入载体pET-28a(+)，于工程菌BL21star(DE3)中表达及TALON柱芯亲合纯化，通过ELISA法初步鉴定其与NH-LBP的结合力。结果 dsFv V

链的Thr

替换为Cys，扩增出基因片段长约为650 bp，经BL21star（DE3）菌表达出相对分子质量约为28×10

的目的蛋白，与NH-LBP具有一定的结合力。结论抗体轻链中成功引入了Cys。

Abstract

Objective To introduce the mutated gene coding cysteine into the gene of dsFv V

of human antibody to N terminal fragment of lipopolysaccharide binding protein (LBP) and to express

purify the mutated dsFv V

in bacterium. Methods We reconstructed and sequenced the mutated gene of V

of human mAb Fab to LBP by Mega-primer PCR based on point mutagenesis method. Some codes of FWR1 of V

had been replaced by TGT in order to code cysteine. The DNA sequence of reconstructed V

was inserted into vector pET-28a(+)

then V

was expressed by E.coli. BL21 star (DE3) and

was purified by chromatography. Finally the activity of V

to bind NH-LBP was determined by ELISA. Results The results showed that the cysteine was introduced into the position 21 amino acid of V

to replace the threonine. The gene of V

was about 650 bp and relative molecular weight of V

was 28×10

. V

could bind NH-LBP directly. Conclusion These have laid a foundation for producing the dsFv against NH-LBP.

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Reconstruction，expression and characterization of dsFv VL of human antibody to N terminal fragment of human lipopolysaccharide binding protein

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Reconstruction，expression and characterization of dsFv V_L of human antibody to N terminal fragment of human lipopolysaccharide binding protein