Construction，Expression and Folding of dsFv Mutated Gene of H um an Antibody to HBsAg

SONG Hong-Ban; MAO Chun-Sheng; CHEN Wei; DU Yong; XU De-Zhong; WANG Hai-Chao

PDF
Cite
Share

facebook

twitter

google

linkedin

February 1999

SONG Hong-Ban, MAO Chun-Sheng, CHEN Wei, DU Yong, XU De-Zhong and WANG Hai-Chao. Construction，Expression and Folding of dsFv Mutated Gene of H um an Antibody to HBsAg[J]. Virologica Sinica, 1999, 14(1): 42-47.

Citation: SONG Hong-Ban, MAO Chun-Sheng, CHEN Wei, DU Yong, XU De-Zhong, WANG Hai-Chao. Construction，Expression and Folding of dsFv Mutated Gene of H um an Antibody to HBsAg .VIROLOGICA SINICA, 1999, 14(1) : 42-47.

抗HsAg二硫键稳定性Fv抗体(dsFv)基因的构建与表达

1.
第四军医大学流行病学教研室

摘要

采用ＰＣＲ定点突变方法，成功地构建了抗ＨＢｓＡｇｄｓＦｖ抗体的轻、重链突变基因，ＮｄｅＩ和ＥｃｏＲＩ酶切后分别插入ｐＥＴ２０ｂ表达质粒，经测序证明在重链第４４位氨基酸和轻链第１００位氨基酸已突变形成半胱氨酸（Ｃｙｓ）。ＶＨ和ＶＬ重组质粒分别转化到大肠肝菌ＢＬ２１（ＤＥ３）中，ＩＰＴＧ诱导后，经ＳＤＳＰＡＧＥ电泳表明在１２ｋＤ处有包含体蛋白表达，表达蛋白含量分别为２８％和３５％。ＶＨ和ＶＬ包含体蛋白经ＧｕＨＣｌ变性后，等量混合在复性折叠液中结合，形成了一个约２４ｋＤ并有一定活性的ｄｓＦｖ蛋白。抗ＨＢｓＡｇｄｓＦｖ抗体表达及复性的成功，为今后基因工程抗体的研究及应用奠定了基础。
- dsFv抗体
- , HBsAg
- , PCR定点突变
- , 表达

Construction，Expression and Folding of dsFv Mutated Gene of H um an Antibody to HBsAg

1.
Deparcment ofEpidemiolog，The Fanrth Military MeMical universinity，xian 710032

Abstract

The mutated gene of dsFv to HBsAg by PCR based point mutagenesis method was constructed and sequenced. The mutated genes of VH and VL were cloned into plasmid pET 20 b and sequenced with the dideoxynucleotid method. The results showed that the cysteines were introduced into position 44 aa of VH and position 100 aa of VL. The recombinaint plasmids of VH and VL which were transformed into E.coli BL21 (DE3) separately produced a 1.2 kD inclusion body protien upon induction with IPTG. The expression level of VH(and VL) protein was 28% (and 35%). VH and VL inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. Then, VH and VL were folded into a 24 kD protein anti HBs dsFv which showed strong binding to HBsAg. These have laid a fundation to apply the dsFv against HBsAg.
- DsFv
- , HBsAg
- , PCR based point mutagenesis method
- , Expression

References
Proportional views

PDF

Article Metrics

Article views(3957) PDF downloads(1136) Cited by(0)

Proportional views

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Construction，Expression and Folding of dsFv Mutated Gene of H um an Antibody to HBsAg

1. Deparcment ofEpidemiolog，The Fanrth Military MeMical universinity，xian 710032

Abstract: The mutated gene of dsFv to HBsAg by PCR based point mutagenesis method was constructed and sequenced. The mutated genes of VH and VL were cloned into plasmid pET 20 b and sequenced with the dideoxynucleotid method. The results showed that the cysteines were introduced into position 44 aa of VH and position 100 aa of VL. The recombinaint plasmids of VH and VL which were transformed into E.coli BL21 (DE3) separately produced a 1.2 kD inclusion body protien upon induction with IPTG. The expression level of VH(and VL) protein was 28% (and 35%). VH and VL inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. Then, VH and VL were folded into a 24 kD protein anti HBs dsFv which showed strong binding to HBsAg. These have laid a fundation to apply the dsFv against HBsAg.