表達(dá)HCVcoreAnt的重組慢病毒的構(gòu)建

1、表達(dá)HCV coreAnt的重組慢病毒的構(gòu)建         11-01-07 13:13:00     作者：馬列婷，李硯，王亞文    編輯：studa20【摘要】  目的 構(gòu)建表達(dá)HCV 核心區(qū)的重組慢病毒，以探討HCV感染后該病毒核心蛋白對(duì)肝臟干細(xì)胞轉(zhuǎn)分化的影響。方法 利用PCR體系延伸互為模板的引物，通過T載體克隆法獲得HCV coreAnt基因克隆，酶切后連入pLenti6/V5DTOPO質(zhì)粒，得到pLenti6/

2、V5DHCV coreAnt，連同pLP/VSVG、pLP1和pLP2四質(zhì)粒共轉(zhuǎn)染293ET細(xì)胞,獲得了表達(dá)HCV coreAnt重組慢病毒。收集病毒上清，大量擴(kuò)增，用免疫組化法檢測(cè)目的基因在Hela細(xì)胞的表達(dá)。用類似的方法構(gòu)建了表達(dá)EGFP的重組慢病毒并測(cè)定其滴度。結(jié)果 克隆出HCV coreAnt基因，經(jīng)酶切及測(cè)序證實(shí)結(jié)果正確;使用表達(dá)HCV coreAnt重組慢病毒感染Hela細(xì)胞，免疫組化實(shí)驗(yàn)證實(shí)胞漿有HCV core表達(dá)。使用表達(dá)EGFP的重組慢病毒感染3T3細(xì)胞見到綠色熒光,說明構(gòu)建慢病毒載體有效。結(jié)論 通過分子克隆體外重組技術(shù)成功制備了表達(dá)HCV core的重組慢病毒，為進(jìn)一步研

3、究丙肝病毒致癌機(jī)制奠定了基礎(chǔ)。 【關(guān)鍵詞】  丙型肝炎病毒核心蛋白;重組慢病毒;分子克隆ABSTRACT: Objective To construct a recombinant lentiviral vector for HCV coreAnt and then study its effect on the transdifferentiation of hepatic stem cells. Methods The HCV coreAnt was obtained by PCR of two primers template with each other and Tvect

4、or cloning method, and then subcloned to pLenti6/V5DTOPO. The restriction endonuclease and T4 DNA ligase were used to construct the vector. pLenti6/V5DHCV coreAnt and the ViraPowerTM PackagingMix (containing three packaging plasmids pLP1, pLP2 and pLP/VSVG) were cotransfected into 293ET cells to pro

5、duce replication in competent lentivirus after transfection. The viral supernatant on 293T cells was collected. The expression of the lentiviral vector containing HCV coreAnt in Hela cells was measured by immunohistochemistry. Then we constructed the lentiviral vector containing green flurosecent pr

6、otein by the similar method, and the titers were determined. Results HCV coreAnt was identified and analyzed by restriction enzyme digestion and sequencing, respectively. The expression of the recombinant lentiviral vector plasmid containing HCV coreAnt in Hela cells was confirmed by immunohistochem

7、istry. The 3T3 cells transfected the lentiviral vector containing green flurosecent protein were found to show strong expression of GFP, which confirmed that the fourplasmid system of the lentiviral vector was successfully constructed. Conclusion The recombinant lentiviral vector expressing HCV core

8、Ant was successfully constructed by molecular cloning and recombination techniques in vitro, which will be beneficial to guiding further study on gene therapy of cancer.KEY WORDS: HCV core protein; recombinant lentiviral vector; molecular cloning丙型肝炎病毒(hepatitis C virus, HCV)感染大部分轉(zhuǎn)變?yōu)槌掷m(xù)感染，繼而發(fā)展為肝硬化和肝細(xì)

9、胞癌(hepatocellular carcinoma, HCC)。許多研究表明HCV的核心蛋白具有致癌潛能，且與HCC的發(fā)生發(fā)展密切相關(guān)。本試驗(yàn)通過構(gòu)建可以表達(dá)HCV coreAnt融合蛋白的真核表達(dá)載體，試圖為進(jìn)一步研究丙肝病毒產(chǎn)物核心蛋白對(duì)肝臟干細(xì)胞的轉(zhuǎn)分化作用奠定基礎(chǔ)。1 材料與方法1.1 材料 克隆載體pGEMTEasy質(zhì)粒(50ng/L)，購(gòu)自Promega公司。限制性內(nèi)切酶BamH、Himd、Xho、Taq DNA聚合酶、T4 DNA連接酶購(gòu)自華美生物工程公司。一抗為小鼠抗HCV核心蛋白單克隆抗體，購(gòu)自北京奧博森公司，HRP羊抗小鼠抗體為北京中杉公司提供。pTE28質(zhì)粒、pGEM

10、 TAnt載體、pEGMHCV core cDNA克隆質(zhì)粒、病毒穿梭質(zhì)粒pLenti6/V5DTOPO、pLP/VSVG、pLP1、pLP2、大腸桿菌DH5及293細(xì)胞系，由西安華廣生物工程公司提供。1.2 方法1.2.1 構(gòu)建pGEM THCV coreAnt載體 采用已經(jīng)構(gòu)建的HCV core cDNA克隆質(zhì)粒為模板，使用PCR技術(shù)刪除終止子，通過T載體克隆法構(gòu)建pGEMTHCV core cDNA克隆質(zhì)粒。上游引物：5GGGATCC ATG AGC ACG AAT CCTAA3，下游引物：5GAAGCTT TCA CAC TTG GTA GGC CGA AG3。PCR反應(yīng)體系：10

11、15;Taq DNA聚合酶Buffer 10L，dNTP(2.5mmol/L)及上、下游引物各1L，Taq DNA聚合酶(2.5u/L)1L，消毒去離子水將總體積加至100L。PCR條件為：94變性60s，42退火60s，72延伸90s，30個(gè)循環(huán)后，再72延伸5min。經(jīng)篩選陽(yáng)性克隆、酶切鑒定及測(cè)序后，通過常規(guī)分子生物學(xué)亞克隆方法，利用已有的pGEM TAnt載體，連接Ant和HCV core cDNA在同一開放閱讀框架(open reading fragment，ORF)，構(gòu)建pGEM THCV coreAnt載體。1.2.2 pTE28 HCV coreAnt載體構(gòu)建 堿裂解法大量制備重

12、組質(zhì)粒pGEM THCV coreAnt，用限制性內(nèi)切酶Xho徹底消化后再用BamH消化，切取HCV coreAnt用T4DNA連接酶連入帶有相同末端的已線性化pTE28表達(dá)載體，建立表達(dá)HCV core和果蠅穿膜肽Ant的融合蛋白表達(dá)克隆。用連接反應(yīng)產(chǎn)物轉(zhuǎn)化制備好的感受態(tài)細(xì)菌E.coli DH5菌株，隨機(jī)挑選單個(gè)菌落，接種于含有氨芐西林的LB培養(yǎng)基內(nèi)，37增殖培養(yǎng)后，堿裂解法小量提取質(zhì)粒。用限制性內(nèi)切酶EcoR和BamH聯(lián)合酶切，篩選出重組質(zhì)粒pTE28 HCV coreAnt。1.2.3 pLenti6/V5DHCV coreAnt的構(gòu)建 Xho和BamH聯(lián)合酶切慢病毒穿梭質(zhì)粒pLenti

13、6/V5DTOPO和pTE28 HCV coreAnt，低熔點(diǎn)凝膠回收HCV coreAnt和線性化的pLenti6/V5DTOPO，T4DNA連接酶連接，轉(zhuǎn)化DH5ct感受態(tài)細(xì)菌，挑選轉(zhuǎn)化的菌落，提取質(zhì)粒，Xho和BamH雙酶切篩選并鑒定陽(yáng)性克隆。1.2.4 重組慢病毒的包裝 采用磷酸鈣沉淀法。pLenti6/V5DHCVcoreAnt同包裝質(zhì)粒pLP1、pLP2和包膜質(zhì)粒pLP/VSVG四質(zhì)粒共轉(zhuǎn)染293ET細(xì)胞,獲得了表達(dá)HCV coreAnt重組慢病毒。轉(zhuǎn)染10d后，質(zhì)粒在細(xì)胞內(nèi)同源重組構(gòu)建出活重組慢病毒，毒斑出現(xiàn)。反復(fù)凍融含病毒栓子的培養(yǎng)基，提取病毒，收集病毒上清。1.2.5 重組蛋白的免疫細(xì)胞化學(xué)檢測(cè) 使用表達(dá)HCV coreAnt重組慢病毒感染Hela細(xì)胞，將細(xì)胞接種于鋪有蓋玻片的6孔板，培養(yǎng)2448h后，取出蓋玻片，冷丙酮固定20min。30mL/L H2O2消除細(xì)胞內(nèi)源性過氧化物酶活性。一抗為小鼠抗HCV核心蛋白單克隆抗體，二抗為