目的：构建抗人前列腺特异抗原(PSA)单链抗体(scFv)/人p53四聚功能域融合基因，并进行真核表达和活性测定。 方法：利用递归PCR法扩增人IgG3上游铰链区与人p53四聚功能域融合基因，克隆入pUC19载体中构建pUC19/IgG3/p53克隆载体。将抗PSA scFv克隆入pUC19/IgG3/p53载体中，构建抗PSA scFv/人p53四聚功能域融合基因。经酶切鉴定及序列测定证实后，将融合基因克隆入真核表达载体pSecTag2 B中，转染HeLa细胞进行表达，表达产物纯化后利用流式细胞仪进行活性测定。结果：获得了抗PSA scFv/人p53四聚功能域融合基因，基因全长891 bp，可编码297个氨基酸，与已发表的抗PSA scFv、人IgG3上游铰链区和人p53四聚功能域基因cDNA序列一致。表达产物经SDS-PAGE和Western印迹实验证实为约35 kD的特异蛋白条带，纯化后经流式细胞仪检测可以特异性地结合PC-3细胞，亲和力高于scFv。结论：获得了可与PC-3细胞特异结合的抗PSA scFv四聚体，为进一步临床应用奠定基础。

Objective: To construct anti-human prostate specific antigen (PSA) single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express fusion protein in HeLa cells.Methods: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-PSA scFv was then cloned into pUC19/IgG3/p53 to construct anti-PSA scFv /human p53 tetramerization domain fusion gene which was then subcloned into the pSecTag2-B expression plasmid. Then the pSecTag2-B plasmids concluding the fusion gene were transfected HeLa cells. The expression products were analyzed by both SDS-PAGE and Western blot, then were purified with Ni2+-NTA superflow affinity chromatography. The binding affinity for PC-3 cells was measured by flow cytometry.Results:The anti-PSA scFv/human p53 tetramerization domain fusion gene consisted of 891bp encoding 297 amino acid residues, and was the same as that reported before. The expression products of the tetrameric anti-PSA scFv, which relative molecular mass (Mr) was about 35 000, were confirmed by SDS-PAGE and Western blot. After purified with Ni2+-NTA superflow affinity chromatography, the tetrameric anti-PSA scFv showed significantly stronger binding to PC-3 cells than scFv.Conclusion: The tetrameric anti-PSA scFv which could bind to PC-3 cells has been successfully gained for the potential use in clinical studies.

R737.31

国家自然科学基金（39900180）资助、全军重点实验室研究基金（1997-71-22）资助