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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 multivalent EZH2 tumor associated antigen peptides and identify their antigenic properties. Methods The DNA sequence coding for the multivalent EZH2 tumor associated antigen peptide was designed and synthesized and cloned into the prokaryotic expression vector p ET-30a (+). Four EZH2 tumor-associated antigen peptides (Aa120-128, Aa165-174, Aa291- 299, Aa735-742) was transformed into E.coli BL21 (DE3). After induced by IPTG, the recombinant plasmid pET-30a (+) - EZH2- Recombinant polypeptides were purified by affinity chromatography and Western blot analysis of the antigenic properties of the recombinant multivalent EZH2 tumor-associated antigen peptide. Rabbit was immunized with recombinant multivalent EZH2 tumor-associated antigen peptide to prepare polyclonal antibody. The antibody titers in serum were detected by indirect ELISA, and the specificity of polyclonal antibody was analyzed by Western bolt. Results The recombinant prokaryotic expression plasmid p ET-30a (+) - EZH2-antigen was confirmed by restriction enzyme digestion and sequencing. The apparent relative molecular weight of the expressed recombinant multivalent EZH2 tumor-associated antigen peptide was about 4 300, mainly in the form of inclusion bodies The purity of the purified polypeptide was not less than 90% and its concentration was 0.5 mg / ml. The purified polypeptide could be effectively recognized and bound by rabbit anti-EZH2 polyclonal antibody and rabbit anti-His tag polyclonal antibody. The purified polyvalent EZH2 tumor- Antibody production was efficiently induced in vivo with an antisera titer of 1:64 × 104 and specifically recognized and bound EZH2 protein expressed in PC3 human prostate cancer cells. Conclusions A polyvalent EZH2 tumor associated antigen peptide was successfully prepared. The polyvalent antigen peptide has relatively small molecular weight but high antigen peptide density, which may have stronger activity of tumor associated antigen. Therefore, it has important application prospect.