新耐药基因HA117多克隆抗体的制备与鉴定-第三军医大学学报.DOC

新耐药基因HA117多克隆抗体的制备与鉴定 陈建飞，金先庆*，丁雄辉，孙艳辉，赵利华，郭振华，王士奇。（重庆医科大学附属儿童医院儿童发育疾病研究省部共建教育部重点实验室，重庆，400014） [摘要] 目的 制备HA117多克隆抗体，以进一步研究HA117编码蛋白在肿瘤组织和正常组织中的分布特点和表达水平。方法 以HA117表达质粒pAdTrack-HA117为模板，PCR扩增HA117基因，扩增产物转入BL21（DE3）感受态细胞，进行HA117蛋白诱导表达，采用SDS检测表达产物。纯化后免疫新西兰大白兔，制备多克隆抗体。通过间接ELISA检测其效价。将转染携带HA117重组腺病毒的HEK293细胞为实验组，未转染的HEK293细胞为对照组，用细胞免疫荧光和Western blot方法检测其过表达的HA117蛋白，以验证所制备的HA117多克隆抗体的特异性。通过免疫组织化学方法检测其在淋巴瘤中的表达情况。结果 成功获得HA117多克隆抗体，间接ELISA法结果显示抗HA117血清效价为1:729 000。细胞免疫荧光和Western blot结果提示所制备的HA117多克隆抗体可以特异性识别HA117抗原蛋白。结论 获得了效价和特异性较高的HA117多克隆抗体，为进一步研究HA117编码蛋白在肿瘤组织和正常组织中的分布特点和表达水平提供了有力工具，为耐药新基因HA117的功能研究垫定了基础。 [关键词] HA117；多克隆抗体；抗体制备；鉴定 Preparation and identification of polyclonal antibody of novel resistance gene HA117 CHEN Jian-fei，JIN Xian-qing*， DING Xiong-hui，SUN Yan-hui ， ZHAO Li-hua，GUO Zhen-hua， WANG Shi-qi.(Key Laboratory of Developmental Diseases in Childhood (Chongqing Medical University), Ministry of Education) [Abstract] Objective: we produce HA117 polyclonal antibody，for further study HA117 protein’s distribution and expression in tumor tissue and normal tissueMethods: we use PCR to increase HA117 gene with pAdTrack-HA117 plasmid. PCR products are transferred to BL21 (DE3) competent cells and induced expression of the HA117 protein, detected the expression products with SDS. We immunize New Zealand rabbits with HA117 antigen to prepared polyclonal antibody, detected titer and specificity with indirect ELISA, immunofluorescence and Western blot. Results: successfully prepared HA117 polyclonal antibody. Indirect ELISA show the serum titer of anti-HA117 is1:729000. Immunofluorescence and western blot detect the HA117 polyclonal antibody with high specificity. Conclusion: Received HA117 polyclonal antibodies with high titer and specificity, For further research including distribution and expression of HA117 proteins in tumor tissue and normal tissue provides a powerful tool, for drug-resistant features of the novel gene HA117 pad for the foundation. [key words] H