抗HBsAg二硫键稳定性Fv抗体（dsFv）基因的构建与表达

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。

Construction, Expression and Folding of dsFv Mutated Gene of Human Antibody to HBsAg

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.