肾癌肿瘤相关抗原G250及G250蛋白多糖（PG）区基因的克隆、表达及鉴定

Cloning, expression and identification of the renal cell carcinoma-associated antigen G250 and G250 PG region

目的探讨肾癌肿瘤相关抗原G250及G250蛋白多糖（PG）区基因的克隆，蛋白表达及鉴定。方法从肾癌细胞786-0中提取mRNA，采用逆转录-聚合酶链反应（RT-PCR）扩增出G250及G250PG区cDNA。将获得的cDNA片段插入pET28a（＋）表达载体，构建重组质粒pET-28a（＋）-G250／pET-28a（＋）-G250PG，转化DH5α大肠杆菌。通过PCR鉴定筛选出正确的重组子，并用其转化BL21 Codon Plus菌。采用IPTG诱导工程菌进行诱导表达，SDS-PAGE及Western blot鉴定。结果克隆的基因经测序证实，G250／G250 PG区序列正确。构建重组质粒pET-28a（＋）-G250／pET-28a（＋）-G250 PG，并在原核系统中表达G250／G250 PG重组蛋白，目的蛋白均具有较好的抗原性和特异性。结论成功完成G250／G250 PG区基因克隆及表达，为在G250／G250 PG蛋白纯化基础上进行抗体制备和G250功能研究提供了实验依据。

Objective To clone, express and identify the tumor-associated antigen G250 and G250 proteoglycan （PG） region. Methods The mRNA was extracted from human renal carcinoma cell line 786 - 0. Gene fragments encoding G250 and G250 PG were obtained by RT-PCR, and cloned into prokaryotic expression vector pET28a （ ＋ ） to construct the recombinant plasmid pET-28a（ ＋ ）-G250/pET- 28a（ ＋ ） -G250 PG. The recombinant vectors were transfected into E. coli DH5α and PCR assay was performed to find out the right recombinant clones. The right clones were transfected into the expression host （ E. coli BL21 Codon Plus） ,and the engineering bacteria were induced by IPTG. The results were acquired by SDS-PAGE and Western blot. Results DNA sequence analysis showed that the sepuence edconding G250 / G250 PG region was the same as that in GenBank. Gene of G250 and G250 PG region was expressed in E. coli BL21 Codon Plus successfully. Western blot analysis showed that the two kinds of recombinant protein could be specially recognized by their corresponding antibodies. They both had better antigenicity and specificity. Conclusion This study provides experimental basis for the purification of G250/ G250 PG protein and the further study on G250 function and preparation of antibody.