摘要
目的 构建抗人γ 精浆蛋白 (γ Sm)重链可变区 (VH)单域抗体 /人 p5 3四价功能域融合基因 ,并进行空间构象分析。方法 选用人IgG3上游铰链区作为抗体和人p5 3四价功能域之间连接的linker。利用回归PCR法扩增人IgG3上游铰链区与人p5 3四价功能域融合基因 ,并在融合基因 5’端和 3’端引入SalⅠ与HindⅢ酶切位点 ,将融合基因克隆入 pUC1 9载体中构建pUC1 9/IgG3 /p5 3克隆载体。将抗人γ SmVH 单域抗体克隆入 pUC1 9/IgG3 /p5 3载体中 ,构建抗人γ SmVH 单域抗体 /人 p5 3四价功能域融合基因。经酶切鉴定及序列测定证实后 ,利用计算机软件进行空间构象分析。结果 获得了抗人γ SmVH 单域抗体 /人 p5 3四价功能域融合基因 ,基因全长 5 2 8bp ,可编码 1 76个氨基酸 ,与已发表的抗人γ SmVH 单域抗体、人IgG3上游铰链区和人p5 3四价功能域基因cDNA序列一致。计算机软件分析结果显示 ,融合基因表达产物可自动装配成四聚体抗体 ,并具有四条长的、柔性好的多肽 ,可确保每个抗体空间构象的独立性。结论 构建成功四价抗人γ SmVH 单域抗体 ,为进一步临床应用奠定基础。
Objective To construct anti-human γ-seminoprotein(γ-Sm) V H single domain antibody/human p53 tetramerization domain fusion gene and analyze its space conformation. Methods Human IgG3 upper hinge was selected to be the linker between V H single domain antibody and human p53 tetramerization domain gene.The human IgG3 upper hinge/human p53 tetramerization domain fusion gene was obtained by recursive polymerase chain reaction(PCR),and was inserted into pUC19 to construct cloning plasmid pUC19/IgG3/p53.The anti-human γ-Sm V H single domain antibody was then cloned into pUC19/IgG3/p53 to construct anti-human γ-Sm V H single domain antibody/human p53 tetramerization domain fusion gene.The space conformation of the fusion gene express product was predicted by using the 3D-PSSM software.Results The anti-human γ-Sm V H single domain antibody/human p53 tetramerization domain fusion gene consisted of 528 bp encoding 176 amino acid residues,and was the same as that reported before.The analysis result of the space conformation of the fusion gene express product was that:The fusion gene express product was capable of self-associating to a tetramer antibody with four long and flexible linkers.?Conclusion? Anti-human γ-Sm V H single domain antibody/human p53 tetramerization domain fusion gene has been successfully constructed for the use in clinical studies.
出处
《西安医科大学学报》
CSCD
北大核心
2001年第6期505-509,共5页
Journal of Xi'an Medical University(Chinese)
基金
国家自然科学基金资助 ( 3990 0 180 )
