CTP-FoxM1抗原负载DCs疫苗免疫小鼠后产生抗FoxM1阳性肝癌的免疫预防效应

发布时间：2018-07-09 14:04

  本文选题：融合蛋白CTP-FoxM1 + 树突状细胞　； 参考：《重庆医科大学》2016年硕士论文

【摘要】：目的:探讨CTP-FoxM1抗原负载DCs疫苗对C57BL/6小鼠肝癌Hepa1-6细胞皮下瘤模型的免疫预防效应,为临床应用DC疫苗防治肝癌奠定了理论基础。方法:(1)根据GenBank选出C57BL/6小鼠FoxM1的关键抗原表位序列串连为4聚体与CTP连接,将已连接好的CTP-FoxM1重组基因插入到pCold-TF载体上,进而构成pCold-TF-CTP-FoxM1质粒,并通过大肠杆菌BL21工程菌表达该蛋白,且通过镍离子亲和层析柱(Ni2+-NTA)进行纯化,得到目的蛋白后经超滤浓缩杯浓缩,Western blot鉴定目的蛋白。细胞免疫荧光和激光共聚焦观察融合蛋白CTP-Fox M1在DCs中的亚细胞定位情况。(2)CTP-FoxM1、CTP、FoxM1蛋白以及对照组PBS分别作用DCs48 h后,CCK-8试剂盒检测抗原负载DCs疫苗刺激同种异体淋巴细胞增殖能力,LDH试剂盒检测抗原负载DCs疫苗体外杀伤Hepa1-6肝癌细胞株的CTL应答效应。(3)CTP-FoxM1、CTP、FoxM1抗原负载的DCs疫苗免疫C57BL/6小鼠3次后皮下注射Hepa1-6肝癌细胞株,观察抗原负载DCs疫苗对c57bl/6小鼠肝癌的免疫预防效应。结果:(1)成功构建了pcold-tf-ctp-foxm1重组质粒;在37℃、1mmiptg诱导3h的条件下,获取了高效、可溶性ctp-foxm1融合蛋白的表达;并经ni2+-nta亲和层析柱纯化出纯度约90%的ctp-foxm1融合蛋白;westernblot进一步鉴定ctp-foxm1融合蛋白;细胞免疫荧光和激光共聚焦观察显示出ctp-foxm1融合蛋白转导进入dcs内主要定位于胞浆。(2)融合蛋白ctp-foxm1负载dcs疫苗与ctp、foxm1负载dc疫苗相比,能够更明显地刺激同源异体淋巴细胞的增殖,且增殖能力达到最佳效果是在dcs与淋巴细胞以效靶比为10:1的比例条件下。在杀伤hepa1-6肝癌细胞株ctl应答效应的体外实验中,ctp-foxm1抗原负载dcs疫苗对hepa1-6肝癌细胞株的杀伤效应明显强于其他实验组及其对照组。(3)ctp-foxm1、ctp、foxm1抗原负载dcs疫苗免疫c57bl/6小鼠后产生抗肝癌hepa1-6细胞皮下瘤模型的免疫预防效应观察中,在皮下瘤建立后的第7、9、11、13、15、17天观测,ctp-foxm1对小鼠皮下瘤的免疫预防效应明显优于其他实验组及对照组。结论:(1)经ni2+-nta亲和层析得到较高纯度的融合蛋白ctp-foxm1,并由激光共聚焦观察可知ctp-foxm1具有明显dcs的胞浆定位偏性,为后续制备ctp-foxm1负载的dc疫苗奠定基础。(2)融合蛋白ctp-foxm1负载dcs疫苗能够有效地刺激同源异体的淋巴细胞增殖,并对体外hepa1-6肝癌细胞株起特异性ctl应答效应。(3)CTP-FoxM1抗原负载DCs疫苗对C57BL/6小鼠皮下肝癌模型起到免疫预防的作用,因此,为临床应用DC疫苗防治肝癌奠定了理论基础。
[Abstract]:Objective: to investigate the immunological preventive effect of CTP-FoxM1 antigen loaded DCs vaccine on C57BL / 6 mouse hepatoma Hepa1-6 cell model, and to lay a theoretical foundation for the clinical application of DC vaccine in the prevention and treatment of hepatocellular carcinoma. Methods: (1) according to GenBank, the key epitope sequence of C57BL / 6 mouse FoxM1 was sequenced as 4 polymer and CTP ligated with CTP. The cloned CTP-FoxM1 recombinant gene was inserted into pCold-TF vector to form pCold-TF-CTP-FoxM1 plasmid, and the recombinant protein was expressed by E. coli BL21. The target protein was purified by nickel ion affinity chromatography (Ni2-NTA) and identified by ultrafiltration concentration cup concentration blot. The subcellular localization of CTP-Fox M1 in DCs was observed by cell immunofluorescence and laser confocal laser. (2) CTP-FoxM1 CTPnFoxM1 protein and control PBS were treated with DCS for 48 h and CCK-8 kit was used to detect the antigen-loaded DCs to stimulate allogeneic lymphocytes. The CTL response of antigen-loaded DCs vaccine against Hepa1-6 hepatoma cell line in vitro was detected with proliferative ability and LDH kit. (3) C57BL / 6 mice were immunized with DCs vaccine loaded with CTP-FoxM1and CTP- FoxM1 antigen for three times, then Hepa1-6 hepatoma cell line was injected subcutaneously. To observe the immune preventive effect of antigen-loaded DCs vaccine on hepatocellular carcinoma in c57bl/6 mice. Results: (1) the recombinant plasmid of pcold-tf-ctp-foxm1 was successfully constructed, and the expression of soluble ctp-foxm1 fusion protein was obtained at 37 鈩，

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