多价EZH2肿瘤相关抗原肽的制备及其抗原性  

Preparation and antigenicity of polyvalent EZH2 tumor associated antigen peptide

作　　者：杨文[1] 马小猛 李倩[1] 吕亚丰 杨建林[1] 王艳林[1] 

机构地区：[1]三峡大学医学院肿瘤微环境与免疫治疗湖北省重点实验室,湖北宜昌443002

出　　处：《中国生物制品学杂志》2015年第10期1033-1038,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(81372265);湖北省高等学校优秀中青年创新团队计划项目(T201203)

摘　　要：目的人工设计制备多价EZH2肿瘤相关抗原肽,并鉴定其抗原特性。方法设计合成编码多价EZH2肿瘤相关抗原肽的DNA序列,克隆入原核表达载体p ET-30a(+)中,构建含4个EZH2肿瘤相关抗原肽段(Aa120-128、Aa165-174、Aa291-299、Aa735-742)的重组多价抗原肽原核表达质粒p ET-30a(+)-EZH2-antigen,转化E.coli BL21(DE3),经IPTG诱导表达后,用Ni-NTA树脂在变性条件下对重组多肽进行亲和层析纯化,Western blot分析重组多价EZH2肿瘤相关抗原肽的抗原特性。用重组多价EZH2肿瘤相关抗原肽免疫大耳白兔,制备多克隆抗体,间接ELISA法检测血清中抗体效价,Western bolt分析多抗的特异性。结果重组原核表达质粒p ET-30a(+)-EZH2-antigen经酶切及测序证实构建正确;表达的重组多价EZH2肿瘤相关抗原肽表观相对分子质量约4 300,主要以包涵体形式存在;纯化的多肽纯度不低于90%,浓度为0.5 mg/ml,且能被兔抗EZH2多抗和兔抗His标签多抗有效识别和结合;纯化的多价EZH2肿瘤相关抗原肽能在动物体内高效诱导抗体生成,抗血清效价为1∶64×104,且能特异性识别和结合PC3人前列腺癌细胞中表达的EZH2蛋白。结论成功制备了一种多价EZH2肿瘤相关抗原肽,该多价抗原肽相对分子质量虽小,但抗原肽密度高,可能具有更强的肿瘤相关抗原的活性,具有重要的应用前景。Objective To artificially design and prepare polyvalent EZH2 tumor associated antigen peptide and identify its antigen characteristics. Methods The DNA sequence encoding polyvalent EZH2 tumor associated antigen peptide was designed and synthesized, then cloned into prokaryotic expression vector p ET-30a（＋）. The constructed recombinant plasmid p ET-30a（＋）-EZH2-antigen containing four EZH2 tumor associated antigen peptides（Aa120-128, Aa165-174,Aa291-299 and As735-742）was transformed into E. coli BL21（DE3）and induced with IPTG. The expressed recombinant polyvalent EZH2 tumor associated antigen peptide was purified by affinity chromatography with Ni-NTA resin under denaturing condition, and analyzed for antigen characteristics by Western blot, with which rabbits were immunized. The obtained polyclonal antibody was determined for titer in sera by ELISA, and analyzed for specificity by Western blot.Results Restriction analysis and sequencing proved that recombinant prokaryotic expression plasmid p ET-30a（＋）-EZH2-antigen was constructed successfully. The expressed recombinant polyvalent EZH2 tumor associated antigen peptide, with a apparent relative molecular mass of about 4 300, mainly existed in a form of inclusion body and reached a purity of not less than 90% and a concentration of 0. 5 mg / ml after purification, which was recognized and bound with rabbit antiEZH2 polyclonal antibody and rabbit anti-His tag polyclonal antibody effectively. The purified polyvalent EZH2 tumor associated antigen peptide induced antibody effectively in animals and recognized and bound the EZH2 protein expressed in human prostate cancer PC3 cells specifically, of which the antiserum titer was 1 ∶ 64 × 104. Conclusion A polyvalent EZH2 tumor associated antigen peptide with low relative molecular mass, high density and potentially high activity was successfully prepared, which was of good prospect in application.

关 键 词：EZH2 抗原肽 肿瘤 免疫治疗 

分 类 号：R733.62[医药卫生—肿瘤] R392-33[医药卫生—临床医学]