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.

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

  • Available online: 05 March 1999
  • 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.

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    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.

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