VEGFR-2/KDR三区的载体构建、表达及胃癌患者抗-KDR抗体的检测  

The cloning and expression of the third loop domain of VEGFR-2/KDR and the level of anti-KDR antibody in peripheral blood from patients with gastric cancer

作　　者：陈小波[1] 黄锡全[2] 张隆[1] 谢爽[1] 孙健[1] 康向东[1] 

机构地区：[1]上海中医药大学附属普陀医院,上海200062 [2]江苏大学医学技术学院,江苏镇江212001

出　　处：《检验医学》2007年第2期155-158,共4页Laboratory Medicine

基　　金：上海中医药大学附属普陀医院基金资助项目(Ct-I-2)

摘　　要：目的扩增人血管内皮细胞生长因子受体-2/激酶插入受体(VEGFR-2/KDR)三区基因,构建表达载体,在大肠埃希菌中表达,获得纯化蛋白。检测胃癌术前患者外周血中抗-KDR抗体水平,为进一步研究KDR与抗-KDR抗体在胃癌中的作用奠定基础。方法从人血管内皮细胞株ECV304提取总RNA,逆转录后,聚合酶链反应(PCR)扩增KDR胞外段三区基因,连接到pMD18-T载体测序;酶切后将目的基因插入原核表达载体pET28a(+)构建重组质粒,将重组质粒转化到大肠埃希菌DH5α内并提取质粒,经双酶切鉴定后,再转化入表达菌株BL21(DE3),异丙基-β-D硫化半乳糖苷(IPTG)诱导表达,用Ni2+-NTA树脂纯化、复性;用获得的蛋白包被酶联免疫吸附试验(ELISA)板,用ELISA检测32例胃癌患者和35名健康对照者外周血抗-KDR抗体水平。结果构建了表达载体pET28a(+)-KDR3,并获得高效表达,表达的蛋白质相对分子质量为16 000,重组蛋白以包涵体形式存在。胃癌患者外周血中抗-KDR抗体水平显著高于健康对照组(P<0.01)。结论VEGFR-2/KDR三区基因成功克隆到表达质粒内并表达。为进一步研究KDR与抗-KDR抗体在胃癌中的作用奠定了基础,及可能为胃癌的血清学诊断提供新的方法。Objective To construct expression vector by amplifying the third loop domain of vascular endothelial growth factor receptor-2（ VEGFR-2 ）/kinase-insert domain containing receptor （KDR） in human vascular, and detect anti-KDR antibody in the peripheral blood of the patients with gastric cancer before operation for the further investigation of the action of KDR and anti-KDR antibody in the patients with gastric cancer. Methods The total RNA was extracted from the ECV304, and the cDNA was procured with reverse-transcription-polymerase chain reaction （RT-PCR）. The third loop domain of KDR was amplified with PCR from the cDNA, and inserted into pMD18-T and sequenced. Then it was cloned into pET28a（ ＋ ） expression vector, and transformed into Escherichia coli （E. coli） DHSct. The plasmid DNA was extracted, and digested by restriction endonucleases. The right insertion was retransformed into E. coli BL21 （DE3）. The cultures induced by isopropy-β-D-thiogalactoside （IPTG） was purified and renaturated on the Ni^2＋ -NTA resin. The enzyme-linked immunosorbent assay （ELISA） plate was prepared using the renaturation protein. The anti- KDR antibody levels of 32 patients with gastric cancer and 35 healthy controls were detected by ELISA. Results Both cloning and expression recombinants of the third loop domain of KDR were obtained. The rKDR3 whose molecular weight was 16 000 was expressed as the inclusion bodies. The anti-KDR antibody levels in patients with gastric cancer were significantly higher than those of control group （P 〈 0.01 ）. ConcIusions The third loop domain of VEGFR-2/KDR was cloned and expressed in E. coli, which provides basis for further study of the action of KDR and anti-KDR antibody in the patients with gastric cancer, and a probable new way for serological diagnosis of gastric cancer.

关 键 词：血管内皮细胞生长因子受体-2 克隆 表达 胃癌 抗-KDR抗体 

分 类 号：R446.1[医药卫生—诊断学]