pEGFP-N_1-IL10-HGF真核表达载体的构建及在T6细胞中的表达
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摘要
目的构建含增强型绿色荧光蛋白(Enhanced Green Fluorescence Protein,EGFP)基因和白细胞介素10(Interleukin 10,IL10)-肝细胞生长因子(Hepatocyte Growth Factor,HGF)重组融合基因的真核表达载体pEGFP-N1-IL10-HGF,验证其在T6大鼠肝星状细胞中的表达。
方法从人单核细胞中提取总RNA,RT-PCR扩增IL10基因编码区序列,以克隆载体pUC-SRα/HGF为模板PCR扩增HGF基因编码区序列,将二者纯化片段连接进入pGEM-T-Easy克隆载体,转化Top10感受态大肠杆菌,氨苄青霉素及蓝白斑筛选,酶切及DNA测序鉴定得到阳性克隆。以上述克隆载体为模板采用重组PCR技术扩增得到IL10-HGF(LH)融合基因序列,将纯化片段连接进入pGEM-T-Easy克隆载体,氨苄青霉素及蓝白斑筛选,酶切及DNA测序鉴定得到阳性克隆。双酶切定向克隆构建pEGFP-N1-LH,转化Top10感受态大肠杆菌,卡那霉素筛选,酶切及DNA测序鉴定得到阳性克隆。提取pEGFP-N1-LH及pEGFP-N1质粒;采用脂质体介导法瞬时转染T6细胞,G418筛选获得真核载体稳定表达细胞系,荧光显微镜观察增强型绿色荧光蛋白亚细胞定位, RT-PCR及Western验证LH基因转录及蛋白表达。
结果酶切与测序证实克隆质粒中含有IL10、HGF、LH基因编码区;酶切和测序证实重组真核表达质粒中含有LH融合基因编码区序列且插入方向正确;荧光显微镜观察转染了pEGFP-N1和pEGFP-N1-LH的T6细胞中可以看到增强型绿色荧光蛋白的表达,EGFP亚细胞定位在全细胞,LH主要定位在细胞质,RT-PCR及Western结果表明重组LH在T6细胞中稳定表达。
结论成功克隆了IL10、HGF及LH基因编码区序列,构建了以增强型绿色荧光蛋白为报告基因的重组真核表达载体pEGFP-N1-LH,pEGFP-N1-LH在T6细胞中稳定表达成功。
Objective To construct eukaryotic express plasmid pEGFP-N1-IL10-HGF containing human IL10-HGF(LH) recombinant fusion gene and enhanced green fluorescence protein gene ,and to confirm its expression in T6 rat hepatic stellate cells.
Methods IL10 cDNA was amplified by RT-PCR from human mononuclear cells, HGF cDNA was amplified by PCR from pUC-SRα/HGF vector, purified fragments were cloned into pGEM-T-Easy vector,recombinant clones were selected by ampicillin and blue-white spot, and then indentfied by digestion analysis and DNA sequencing. LH cDNA was amplified by recombinant PCR from pGEM-T-Easy-IL10 and pGEM-T-Easy-HGF vector, Purified fragments was cloned into pGEM-T-Easy vector,recombinant clones were selected by ampicillin and blue-white spot, and then indentfied by digestion analysis and DNA sequencing. Then the recombinant IL10-HGF ORF was cloned into pEGFP-N1 vector by double-digest direct cloning ,recombinant clones were selected by kanamycin, and then indentfied by digestion analysis and DNA sequencing. The recombinant vector was introduced into T6 cells by lipofectin transfection,The cells were selected by G418,The expression of enhanced green fluorescent protein (EGFP) was observed under fluorescence microscope, the mRNA expression level were indentified by RT-PCR, and the expression level of protein were indentified by western-blot.
Results The IL10/HGF/LH gene fragments successfully inserted into the pEGM-T-Easy were confirmed by digestion and DNA senquencing ;the LH gene fragment inserted into pEGFP-N1 vector correctly was indentified confirmed by digestion and DNA senquencing;the expression of enhanced green fluoscence protein was observed in T6 cells transfected with pEGFP-N1-LH and pEGFP-N1 under fluorescence microscope. EGFP protein was localized in whole cell, while LH is expressed mainly in the cell Plasm. The results of RT-PCR and western-blot congfirmed that the recombinant vectors expressed LH mRNA and Protein in T6 cells stably.
Conclusion IL10/HGF/LH gene were cloned successfully,The eukaryotic express vector pEGFP-N1-LH containing human LH gene was successfully constructed and expressed in T6 cells stably.
方法从人单核细胞中提取总RNA,RT-PCR扩增IL10基因编码区序列,以克隆载体pUC-SRα/HGF为模板PCR扩增HGF基因编码区序列,将二者纯化片段连接进入pGEM-T-Easy克隆载体,转化Top10感受态大肠杆菌,氨苄青霉素及蓝白斑筛选,酶切及DNA测序鉴定得到阳性克隆。以上述克隆载体为模板采用重组PCR技术扩增得到IL10-HGF(LH)融合基因序列,将纯化片段连接进入pGEM-T-Easy克隆载体,氨苄青霉素及蓝白斑筛选,酶切及DNA测序鉴定得到阳性克隆。双酶切定向克隆构建pEGFP-N1-LH,转化Top10感受态大肠杆菌,卡那霉素筛选,酶切及DNA测序鉴定得到阳性克隆。提取pEGFP-N1-LH及pEGFP-N1质粒;采用脂质体介导法瞬时转染T6细胞,G418筛选获得真核载体稳定表达细胞系,荧光显微镜观察增强型绿色荧光蛋白亚细胞定位, RT-PCR及Western验证LH基因转录及蛋白表达。
结果酶切与测序证实克隆质粒中含有IL10、HGF、LH基因编码区;酶切和测序证实重组真核表达质粒中含有LH融合基因编码区序列且插入方向正确;荧光显微镜观察转染了pEGFP-N1和pEGFP-N1-LH的T6细胞中可以看到增强型绿色荧光蛋白的表达,EGFP亚细胞定位在全细胞,LH主要定位在细胞质,RT-PCR及Western结果表明重组LH在T6细胞中稳定表达。
结论成功克隆了IL10、HGF及LH基因编码区序列,构建了以增强型绿色荧光蛋白为报告基因的重组真核表达载体pEGFP-N1-LH,pEGFP-N1-LH在T6细胞中稳定表达成功。
Objective To construct eukaryotic express plasmid pEGFP-N1-IL10-HGF containing human IL10-HGF(LH) recombinant fusion gene and enhanced green fluorescence protein gene ,and to confirm its expression in T6 rat hepatic stellate cells.
Methods IL10 cDNA was amplified by RT-PCR from human mononuclear cells, HGF cDNA was amplified by PCR from pUC-SRα/HGF vector, purified fragments were cloned into pGEM-T-Easy vector,recombinant clones were selected by ampicillin and blue-white spot, and then indentfied by digestion analysis and DNA sequencing. LH cDNA was amplified by recombinant PCR from pGEM-T-Easy-IL10 and pGEM-T-Easy-HGF vector, Purified fragments was cloned into pGEM-T-Easy vector,recombinant clones were selected by ampicillin and blue-white spot, and then indentfied by digestion analysis and DNA sequencing. Then the recombinant IL10-HGF ORF was cloned into pEGFP-N1 vector by double-digest direct cloning ,recombinant clones were selected by kanamycin, and then indentfied by digestion analysis and DNA sequencing. The recombinant vector was introduced into T6 cells by lipofectin transfection,The cells were selected by G418,The expression of enhanced green fluorescent protein (EGFP) was observed under fluorescence microscope, the mRNA expression level were indentified by RT-PCR, and the expression level of protein were indentified by western-blot.
Results The IL10/HGF/LH gene fragments successfully inserted into the pEGM-T-Easy were confirmed by digestion and DNA senquencing ;the LH gene fragment inserted into pEGFP-N1 vector correctly was indentified confirmed by digestion and DNA senquencing;the expression of enhanced green fluoscence protein was observed in T6 cells transfected with pEGFP-N1-LH and pEGFP-N1 under fluorescence microscope. EGFP protein was localized in whole cell, while LH is expressed mainly in the cell Plasm. The results of RT-PCR and western-blot congfirmed that the recombinant vectors expressed LH mRNA and Protein in T6 cells stably.
Conclusion IL10/HGF/LH gene were cloned successfully,The eukaryotic express vector pEGFP-N1-LH containing human LH gene was successfully constructed and expressed in T6 cells stably.
引文
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[3]张跃梅,王斌,印胡莲等.人白细胞介素10(IL10)基因的克隆和序列测定[J].细胞与分子免疫学杂志.1996,12(4),17-20
[4] Toshikazu Nakamura , Katsuhiko Nawa ,Akira Ichihara. Partial Purification and Characterization of Hepatocyte Growth Factor from Serum of Hepatectomized Rats[J].Biochemical and Biophysical Research Communications.1984(3),1450-1459
[5] Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S. Molecular cloning and expression of human hepatocyte growth factor[J].Letters To Nature.1989(23),440-442
[6] Noji S, Tashiro K, Koyama E, Nohno T, Ohyama K, Taniguchi S, Nakamura T. Expression of hepatocyte growth factor gene in endothelial and kupffer cells of damaged rat livers, as revealed by in situ hybridization [J]. Biochem Biophys Res Commun, 1990;173(1):42-47
[7] Ramadori G, Neubauer K, Odenthal M, Nakamura T, Knittel T, Schw?gler S, Meyer zum Büschenfelde KH. The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is down regulated during cell growth and by transforing growth factor beta[J]. Biochem Biophys Res Commun. 1992;183(2):739-742
[8] Nishino T, Kaise N, Sindo Y, Nishino N, Nishida T, Yasuda S, Masui Y. Promyelocytic leukemia cell lane, HL-60, produces human hepatocyte growth factor[J].Biochem Biophys Res Commun,1991;181(1):323-330
[9] Yoshinaga Y, Fujita S, Gotoh M, Nakamura T, Kikuchi M, Hirohashi S. Human lung cancer cell lane producing hepatocyte growth factor/ scatter factor[J]. Jpn J Cancer Res.1992;83(12):1257-1261
[10] Wolf HK, Zarnegar R, Michalopoulos G. Localization of hepatocyte growth factor in human and rat tissues: an immunohistochemical study[J]. Hepatoloty.1991;14(3):488-494
[11] Ueki T, Okamoto E, Takeuchi M, Fujimoto J. Persective on postgenome medicine: Gene therapy for liver cirrhosis[J].Nippon Rinsho.2001, 59(1): 152-156
[12] Xue F, Takahara T, Yata Y, Kuwabara Y, Shinno E, Nonome K, Minemura M, Takahara S, Li X, Yamato E, Watanabe A. Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy[J]. Gut. 2003, 52(5): 694-700
[13] Matsuno Y, Iwata H, Umeda Y, Takagi H, Mori Y, Kosugi A, Matsumoto K, Nakamura T, Hirose H. Hepatocyte growth factor gene transfer into the liver via the portal vein using electroporation attenuates rat liver cirrhosis[J]. Gene Ther.2003,10(18): 1556- 1569
[14] Iimuro Y, Fujimoto J. Strategy of gene therapy for liver cirrhosis and hepatocellular carcinoma[J]. J Hepatobiliary Pancreat Surg.2003, 10(1): 45-47
[15] Lai L,Zeff R A,Goldschneider I.A recombinant single chain IL-7/ HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12,CLPs,and pre-B cells[J]. Blood. 2006,107(5):1776-1784
[16]刘畅,孙林英,刘秀丽,等.mGM-CSF-mMIP3-3α重组质粒的构建、表达及鉴定[J].华北国防医药.2006,18(1):1-3
[17] Quinn Lu. seamless cloneing and gene fusion[J] . Trends in Biotechnology. 2005(4):199-207.
[18]王晓凌,高志云,耿媛,等.融合基因DNA疫苗pcDNA3/MDC-VP1对科萨奇病毒B组3-型的免疫效果[J].临床心血管病杂志.2006,22(7):397-399.
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