凋亡素蛋白多克隆抗体的制备及免疫学评价

Preparation and Evaluation of Polyclonal Antibodies of Apoptin

凋亡素由鸡贫血病毒中的VP3基因编码,能诱导多种肿瘤细胞发生凋亡。以真核表达载体pcDNA3.0-VP3为模板构建原核表达载体pET8a-VP3,经NdeⅠ/BamHⅠ双酶切鉴定和基因测序无误后,在IPGT诱导下表达VP3蛋白并对其进行纯化,将纯化后的VP3蛋白与弗氏完全佐剂或不完全佐剂乳化后,分别对两只新西兰大耳白兔进行皮下多点注射,间接ELISA检测免疫后血清效价,效价达到指标后第2天以心脏穿刺的方法采全血后分离抗血清。抗血清效价高的兔子进一步采用Protein A纯化总IgG,最终纯化后的抗体效价可以达到1∶243 000。用重组腺相关病毒rAAV-VP3感染细胞后对抗体的特异性进行免疫学评价。首先利用免疫荧光技术检测VP3基因在人膀胱癌细胞株T24、EJ细胞以及Vero细胞中的表达情况,观察到凋亡素在T24、EJ细胞中主要定位于细胞核,而在Vero细胞中则定位于细胞质。其次通过Western blotting检测纯化后的抗体能与细胞内腺相关病毒介导表达的凋亡素蛋白特异性结合。实验证明了制备的凋亡素蛋白多克隆抗体的有效性和特异性,为进一步阐明凋亡素抗肿瘤效应的分子机制及生物学特性奠定了基础。

Apoptin is coded by the VP3 gene from chicken anemia virus （CAV）, and the protein can induce apoptosis of a large variety of tumor cells. The preparation of polyclonal antibodies specific to VP3 protein was explored. The VP3 gene was obtained from the eukaryotic expression vector pcDNA3.0-VP3. And then, VP3 gene was cloned into the prokaryotie expression vector pET8a. The prokaryotic expression vector pET8a-VP3 was employed to express VP3 protein in E. coli BI21 which was induced by IPTG, and the protein was extracted from the cell and purified. The purified VP3 protein was applied to immunize the New Zealand rabbits combined with the Freund＇s complete adjuvant or incomplete adjuvant by multi-points subcutaneous injection. ELISA test was performed to measure the titer of the antibody after immunization. Two days after the measurement the whole blood was harvested by cardiac puncture and the serum was separated from the whole blood. The immunglobin IgG was purified by Protein A method from the antiserum. The final purified antibody titers were up to 1：243 000. The immunological evaluation of the polyelonal antibody specific binding was assayed by using recombinant adeno-associated virus rAAV-VP3 infected different cell lines. First, it was used to detect the VP3 gene expression by immunofluorescence in human bladder cancer cells T24 and E J, also in Vero cells. Apoptin was observed in T24, EJ cells, mainly located in the nucleus, whereas in Vero ceils were localized in the cytoplasm. Secondly, the specific binding of the purified antibodies to VP3 protein in different human bladder cancer cells were detected by Western blotting. The results have shown that the polyclonal antibody have displayed good effectiveness and binding specificity to apoptin, which will provide a foundation for further clarify the molecular mechanisms of anti-tumor effect and biological characteristics of apoptin.