穩(wěn)定表達丙型肝炎病毒單鏈絲氨酸蛋白酶的HepG2細胞克隆的建立(1)

1、穩(wěn)定表達丙型肝炎病毒單鏈絲氨酸蛋白酶的HepG2細胞克隆的建立(1)                               作者：雷迎峰，尹文，薛小平，楊敬，呂欣，韋三華，胡興斌，孫夢寧，徐志凱【關(guān)鍵詞】  丙型肝炎病毒    Establishment

2、 of stably transfected HepG2 cell line expressing HCV scNS4A/NS3 protease【Abstract】 AIM: To construct a eukaryotic expression vector of HCV single chain serine protease (scNS4A/NS3) and to obtain its stably transfected HepG2 cell line. METHODS:  According to the sequence of HCV scNS4A/NS3 gene

3、from the literature, the primers amplifying the gene coding scNS4A/NS3 protease were designed. HCV RNA was extracted from the HCV positive serum and the gene coding scNS4A/NS3 protease was amplified via RTPCR. The PCR product was digested by BamH/Hind and purified by gel extraction. This fragment wa

4、s inserted into pcDNA3.1(-) with T4 ligase and transformed into E.coli JM109. The positive recombinant plasmid was selected and identified via sequence assay and restrictive enzyme digestion. The recombinant plasmid was then transfected into HepG2 cell by LipofectAMINE2000. The cells containing stab

5、le transformants were selected by the ability of resistance to G418. The stably transfected cell line was identified by RTPCR and IFA and Westernblot. RESULTS:  The eukaryotic expression vector named pcDNA3.1(-)scNS4A/NS3 was successfully constructed  and the stably transfected HepG2 cell

6、line which expressed scNS4A/NS3 protease was obtained. CONCLUSION:  The stably transfected HepG2 cell line expressing single chain serine protease facilitates the establishment of cellbased system in evaluating antiHCV serine protease drug.【Keywords】 hepatitis C virus; single chain serine prote

7、ase; lipofectamine, transfection; colone cells【摘要】 目的：構(gòu)建丙型肝炎病毒單鏈絲氨酸蛋白酶（scNS4A/NS3）的真核表達載體；建立重組質(zhì)粒穩(wěn)定轉(zhuǎn)染的HepG2細胞克隆. 方法：根據(jù)文獻報道設(shè)計擴增scNS4A/NS3編碼基因的引物，從HCV陽性患者血清中提取病毒RNA，RTPCR方法擴增出scNS4A/NS3基因片段， BamH/Hind雙酶切后連接到經(jīng)同樣酶切的真核表達載體pcDNA3.1(-)，轉(zhuǎn)化菌株JM109感受態(tài)細胞，獲得重組質(zhì)粒pcDNA3.1(-)scNS4A/NS3，經(jīng)酶切鑒定及序列測定. 將陽性重組質(zhì)粒用脂質(zhì)體法轉(zhuǎn)染Hep

8、G2細胞，經(jīng)持續(xù)G418壓力選擇和克隆化獲得穩(wěn)定轉(zhuǎn)染的細胞系，用RTPCR，IFA，Westernblot證實該穩(wěn)定細胞系可以表達單鏈絲氨酸蛋白. 結(jié)果：成功構(gòu)建了真核表達載體pcDNA3.1(-)scNS4A/NS3；建立了穩(wěn)定轉(zhuǎn)染的HepG2細胞克隆，命名為scpHepG2. 結(jié)論：獲得穩(wěn)定的scpHepG2細胞克隆可表達單鏈絲氨酸蛋白，為下一步建立以細胞為基礎(chǔ)的評價抗HCV絲氨酸蛋白酶藥物系統(tǒng)奠定基礎(chǔ).【關(guān)鍵詞】 丙型肝炎病毒；單鏈絲氨酸蛋白酶；轉(zhuǎn)染，脂質(zhì)體法；細胞克隆0引言穩(wěn)定、可靠的細胞培養(yǎng)系統(tǒng)和小動物實驗?zāi)Ｐ偷娜狈O大地制約著抗丙型肝炎病毒(HCV)研究的深入和發(fā)展. 因此以在HC

9、V RNA復(fù)制和蛋白前體加工成熟中起著關(guān)鍵作用的酶分子為靶位的測定系統(tǒng)常被用于抗HCV 藥物的篩選. 酶抑制劑作為一個有潛力的藥物分子，它不僅需要有效，也需要有良好的藥物動力學(xué)等特征，如易進入細胞、結(jié)構(gòu)穩(wěn)定. 所以建立一種可以在細胞水平上評價酶活性的細胞系統(tǒng)非常關(guān)鍵. NS3絲氨酸蛋白酶在HCV多聚蛋白前體加工成熟中起關(guān)鍵作用，是抗HCV研究的主要靶標(biāo)分子. 我們通過構(gòu)建HCV單鏈絲氨酸蛋白酶（scNS4A/NS3）的真核表達載體，獲得重組質(zhì)粒穩(wěn)定轉(zhuǎn)染的HepG2細胞克隆，為下一步建立以細胞為基礎(chǔ)的評價抗HCV絲氨酸蛋白酶藥物的系統(tǒng)奠定基礎(chǔ).1材料和方法1.1材料E.coli. JM109菌株

10、、pcDNA3.1(-)質(zhì)粒由本室保存. Taq DNA聚合酶、限制性內(nèi)切酶BamHI，Hind等購自TaKaRa公司，膠回收試劑盒購自Vgene公司，T4 DNA連接酶購自上海生物工程公司，F(xiàn)ITC和HRP標(biāo)記羊抗鼠IgG, DNA分子標(biāo)準(zhǔn)、低分子蛋白質(zhì)標(biāo)準(zhǔn)分別購自華美生物工程公司. 反轉(zhuǎn)錄試劑盒Superscript，Trizol，Lipofecatmine2000，G418購自Gibco公司. NS3mAb由本室制備3.1.2方法1.2.1HCV RNA的提取與cDNA第一鏈的合成以HCV陽性患者的血清為材料提取病毒RNA，方法參照Trizol說明書，將RNA沉淀溶于20 L不含RNas

11、e的去離子水. 反轉(zhuǎn)錄按Superscript說明書進行，模板為病毒RNA，反轉(zhuǎn)錄引物為scNS4A/NS3基因下游引物，最后獲得cDNA第一鏈.1.2.2scNS4A/NS3基因的PCR擴增參考文獻4，設(shè)計擴增scNS4ANS3基因的引物: P1,5 CGGATCCATGGCGCCTATCGGCTCAGTAGTAATCGTAGGCAGAATCATCCTGTCCGGCCGTGGTGGCCCTATTACGGCCTACTCCCAAC 3; P2, 5 CGCGGTACCTAGGCAAAACCAGAACCAGC 3. 上述引物均由上海生工公司合成. 以病毒cDNA第一鏈為模板擴增scNS4A/NS3

12、基因，體系為：模板5 L，10×Buffer 5 L ，10 mmol/L dNTPs 4 L，p1，p2各2 L，Taq 2 L，用超純水補至終體積50 L. 條件: 預(yù)變性94 5 min，94 1 min，60 50 s，72 1 min共 30個循環(huán)， 72延伸10 min. 質(zhì)粒構(gòu)建按參考文獻5進行，獲得pcDNA3.1(-)scNS4A/NS3重組質(zhì)粒. 酶切進行鑒定并交由上海生工公司測序.1.2.3細胞培養(yǎng)和G418工作濃度的確定常規(guī)培養(yǎng)HepG2細胞于12孔板中，50 mL/L CO2，37，培養(yǎng)基為RPMI 1640 (100 mL/L胎牛血清,100 mg/L青、

13、鏈霉素,2 mmol/L谷氨酰胺)，至細胞密度達90%布滿時，加入含G418的培養(yǎng)液1 mL/孔（G418終濃度分別為2001000 mg/L，8孔）. 換液時保持G418濃度穩(wěn)定不變，持續(xù)培養(yǎng)34 wk.1.2.4細胞轉(zhuǎn)染與篩選穩(wěn)定細胞克隆質(zhì)粒制備：將pcDNA3.1(-)scNS4A/NS3質(zhì)粒轉(zhuǎn)化大腸桿菌感受態(tài)細胞JM109，挑取菌落接種5 mL LB，37振搖，12 h后離心收菌. 用去內(nèi)毒素超純質(zhì)粒提取試劑盒（博大泰克公司）提取質(zhì)粒，紫外分光光度法測定DNA含量和純度. 細胞轉(zhuǎn)染：將HepG2細胞培養(yǎng)于24孔板，待細胞90%鋪滿，采用脂質(zhì)體法轉(zhuǎn)染pcDNA3.1(-)scNS4A/N

14、S3,具體操作按LipofectAMINE 2000說明書進行. 建立穩(wěn)定的細胞克?。翰捎肎418壓力選擇. 上述轉(zhuǎn)染48 h后，通過G418壓力篩選2 wk后，細胞成團，用槍頭挑取單個細胞團種在96孔板，繼續(xù)加G418壓力選擇，直到最后獲得單獨穩(wěn)定生長的細胞克隆，擴大培養(yǎng)后檢測并凍存. 獲得的細胞克隆凍存2 mo后復(fù)蘇，檢測細胞克隆的穩(wěn)定性. 1      （version 9.00）.Variance analysis of the 2×2 factoral design was conducted for: ages; heig

15、ht; weight; anesthetic effect ratings; the period from anesthesia to operation, and Apgar scores of 1 minute after the fetus was taken out; SBP, DBP, HR, and SBP×HR prior to and after anesthesia. t Test was conducted for intra-group comparison. 0.05.     1.8  Medical Et

16、hics   It met the requirements of law and ethics to supplement with an intravenous drug in case of no or poor anesthetic effects, and was agreed by the Scientific & Technological Committee of our hospital.    2  Result    2.1  General Data  Variance analysis of the 2×2 factoral design was conducted for general data