人源性EGFL6的重组表达及多克隆抗体制备研究  

Recombinant expression and polyclonal antibody preparation of human EGFL6

作　　者：杨晶[1] 王金胜[1] 张辉挺[2] 王宏[1] 宋其芳[1] 黄建芳[1] 邓宁[1] 

机构地区：[1]暨南大学生命科学技术学院暨南大学抗体工程研究中心广东省分子免疫与抗体工程重点实验室,广州510632 [2]暨南大学附属第一医院肝胆外科,广州510000

出　　处：《免疫学杂志》2013年第6期517-521,526,共6页Immunological Journal

基　　金：中央高校基本科研业务费专项资金资助(21612112);国家863项目(2009AA02Z112)

摘　　要：目的研究发现人源性类表皮生长因子域EGFL6可促进组织血管新生并在肿瘤组织细胞中表达上调,预示其与肿瘤发生发展密切相关,本研究通过对EGFL6基因克隆,表达和纯化并制备其多克隆抗体,为深入研究EGFL6相关功能奠定基础。方法用实时荧光定量PCR方法检测肿瘤及正常组织细胞系中EGFL6基因表达水平,并扩增EGFL6基因片段,采用基因重组技术构建EGFL6原核表达载体,经诱导表达后利用镍离子柱亲和纯化EGFL6重组蛋白,免疫昆明小鼠制备抗EGFL6多克隆抗体,ELISA、Western blot方法检测抗体灵敏度和特异性。结果 RT-PCR检测发现EGFL6在肿瘤细胞系A375及SKOV3中高表达。成功构建表达载体pET32a(+)-EGFL6,实现可溶性EGFL6蛋白的诱导表达,纯化和抗体制备。SDS-PAGE和Western blot证实,重组EGFL6蛋白质与预期结果一致,抗血清能够特异地识别重组片段蛋白,重组全长蛋白及细胞系中天然EGFL6蛋白。结论 qRT-PCR检测得知EGFL6在肿瘤细胞系A375及SKOV3中表达量较高,并成功实现了EGFL6的重组表达,抗体制备及初步应用研究。Recent research showed that EGFL6 is closely related to tumor genesis and development because of its angiogenesis promotion and overexpression in most tumor tissues and cells. In this study, EGFL6 gene fragmentwas cloned and expressed in E. coli, and anti-EGFL6 polyclonal antibody was prepared after the purification of recombinant target protein, which will make a foundation for EGFL6 function study. Real-time fluorescentquantitative PCR was applied to detect EGFL6 gene expression in tumor and normal cell lines. The EGFL6 gene was cloned and constructed in prokaryotic expression vector through gene recombination technology. Then therecombinant EGFL6 was expressed with IPTG inducing and purified with Ni-NTA affinity chromatography. Kunming mice were vaccinated with the purified recombinant EGFL6 and the anti-EGFL6 polyclonal antibodieswere prepared. The sensitivity and specificity of polyclonal antibody were analyzed by ELISA and Western blot. qRT-PCR detection results showed that EGFL6 was highly expressed in tumor cell strains A375 and SKOV3, whichimplied that the EGFL6 expression vector pET32a （＋）-EGFL6 was constructed successfully; SDS-PAGE and Western blot results showed that EGFL6 protein was expressed at the size of 38 KD with soluble form; ELISA andWestern blot results showed that anti-EGFL6 polyclonal antibodies with high sensitivity and specificity were prepared in Kunming mice. All results indicated that EGFL6 could express at high level in E. coli. And the specificpolyclonal antibodies against EGFL6 were successfully obtained by mouse vaccination and can be applied to identify and analyze EGFL6 expression in blood and tumor tissue.

关 键 词：肿瘤细胞 EGFL6 实时荧光定量RT—PCR 重组表达 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]