發(fā)布時(shí)間：2018-07-09 14:04

  本文選題：融合蛋白CTP-FoxM1 + 樹突狀細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:探討CTP-FoxM1抗原負(fù)載DCs疫苗對(duì)C57BL/6小鼠肝癌Hepa1-6細(xì)胞皮下瘤模型的免疫預(yù)防效應(yīng),為臨床應(yīng)用DC疫苗防治肝癌奠定了理論基礎(chǔ)。方法:(1)根據(jù)GenBank選出C57BL/6小鼠FoxM1的關(guān)鍵抗原表位序列串連為4聚體與CTP連接,將已連接好的CTP-FoxM1重組基因插入到pCold-TF載體上,進(jìn)而構(gòu)成pCold-TF-CTP-FoxM1質(zhì)粒,并通過大腸桿菌BL21工程菌表達(dá)該蛋白,且通過鎳離子親和層析柱(Ni2+-NTA)進(jìn)行純化,得到目的蛋白后經(jīng)超濾濃縮杯濃縮,Western blot鑒定目的蛋白。細(xì)胞免疫熒光和激光共聚焦觀察融合蛋白CTP-Fox M1在DCs中的亞細(xì)胞定位情況。(2)CTP-FoxM1、CTP、FoxM1蛋白以及對(duì)照組PBS分別作用DCs48 h后,CCK-8試劑盒檢測(cè)抗原負(fù)載DCs疫苗刺激同種異體淋巴細(xì)胞增殖能力,LDH試劑盒檢測(cè)抗原負(fù)載DCs疫苗體外殺傷Hepa1-6肝癌細(xì)胞株的CTL應(yīng)答效應(yīng)。(3)CTP-FoxM1、CTP、FoxM1抗原負(fù)載的DCs疫苗免疫C57BL/6小鼠3次后皮下注射Hepa1-6肝癌細(xì)胞株,觀察抗原負(fù)載DCs疫苗對(duì)c57bl/6小鼠肝癌的免疫預(yù)防效應(yīng)。結(jié)果:(1)成功構(gòu)建了pcold-tf-ctp-foxm1重組質(zhì)粒;在37℃、1mmiptg誘導(dǎo)3h的條件下,獲取了高效、可溶性ctp-foxm1融合蛋白的表達(dá);并經(jīng)ni2+-nta親和層析柱純化出純度約90%的ctp-foxm1融合蛋白;westernblot進(jìn)一步鑒定ctp-foxm1融合蛋白;細(xì)胞免疫熒光和激光共聚焦觀察顯示出ctp-foxm1融合蛋白轉(zhuǎn)導(dǎo)進(jìn)入dcs內(nèi)主要定位于胞漿。(2)融合蛋白ctp-foxm1負(fù)載dcs疫苗與ctp、foxm1負(fù)載dc疫苗相比,能夠更明顯地刺激同源異體淋巴細(xì)胞的增殖,且增殖能力達(dá)到最佳效果是在dcs與淋巴細(xì)胞以效靶比為10:1的比例條件下。在殺傷hepa1-6肝癌細(xì)胞株ctl應(yīng)答效應(yīng)的體外實(shí)驗(yàn)中,ctp-foxm1抗原負(fù)載dcs疫苗對(duì)hepa1-6肝癌細(xì)胞株的殺傷效應(yīng)明顯強(qiáng)于其他實(shí)驗(yàn)組及其對(duì)照組。(3)ctp-foxm1、ctp、foxm1抗原負(fù)載dcs疫苗免疫c57bl/6小鼠后產(chǎn)生抗肝癌hepa1-6細(xì)胞皮下瘤模型的免疫預(yù)防效應(yīng)觀察中,在皮下瘤建立后的第7、9、11、13、15、17天觀測(cè),ctp-foxm1對(duì)小鼠皮下瘤的免疫預(yù)防效應(yīng)明顯優(yōu)于其他實(shí)驗(yàn)組及對(duì)照組。結(jié)論:(1)經(jīng)ni2+-nta親和層析得到較高純度的融合蛋白ctp-foxm1,并由激光共聚焦觀察可知ctp-foxm1具有明顯dcs的胞漿定位偏性,為后續(xù)制備ctp-foxm1負(fù)載的dc疫苗奠定基礎(chǔ)。(2)融合蛋白ctp-foxm1負(fù)載dcs疫苗能夠有效地刺激同源異體的淋巴細(xì)胞增殖,并對(duì)體外hepa1-6肝癌細(xì)胞株起特異性ctl應(yīng)答效應(yīng)。(3)CTP-FoxM1抗原負(fù)載DCs疫苗對(duì)C57BL/6小鼠皮下肝癌模型起到免疫預(yù)防的作用,因此,為臨床應(yīng)用DC疫苗防治肝癌奠定了理論基礎(chǔ)。
[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，

本文編號(hào)：2109603