HCV感染相关干扰素L4真核表达载体的构建与表达

doi:10.3969/j.issn.1672-5069.2018.06.003

摘要/Abstract

摘要： 目的构建HCV感染相关干扰素IFNL4 蛋白融合 His 标签的真核表达载体,并将其在293-T细胞中表达后进行初步纯化鉴定。方法采用PCR法从含人IFNL4基因序列的质粒pFC14A-p179中扩增出目的片段,将其定向连接到pcDNA3.1-His 真核表达载体中,经酶切和测序鉴定正确后,以脂质体法转染293-T细胞,培养72 h后裂解细胞,采用抗His-Tag抗体行蛋白质印迹法检测目的蛋白的表达。结果成功构建了真核表达载体pcDNA3.1-IFNL4-His,转染后经Western blot法检测显示20 kDa 位置有目的蛋白条带,大小与目的蛋白相符。结论我们成功构建了HCV感染相关IFNL4与 His 标签融合的真核表达载体,体外转染293-T细胞后鉴定其工作良好,为后续对干扰素在HCV感染及抗肝纤维化治疗中的研究奠定了基础。

关键词: 丙型肝炎病毒, 干扰素λ4, 融合蛋白, 真核表达, 体外

Abstract: Objective To construct a eukaryotic vector for recombinant interferon-λ4（IFNL4） protein expression in 293-T cells in vitro. Methods The target fragment of IFNL4 was amplified by PCR from plasmid pFC14A-p179 which contained human IFNL4 gene sequence. The amplified fragment was ligated into pcDNA3.1-His-tag vector and identified by restriction enzyme digestion and sequencing. The vector was then transfected into 293-T cells. After culture for 72 hours,the cells were lysed and the recombinant protein was purified and identified by Western blot. Results The eukaryotic expression vector pcDNA3.1-p179-His was successfully constructed and the recombinant protein was expressed in 293-T cells and successfully purified. Western blot analysis showed that the target protein band was about 20 kDa. Conclusion The eukaryotic expression vector fused with IFNL4 and His-tag is successfully constructed,which might be used as a noval candidate for immune therapy.

Key words: Hepatitis C virus, Interferon-λ4, Fusion protein, Eukaryotic expression, In vitro

徐溪, 党引利, 吴朔. HCV感染相关干扰素L4真核表达载体的构建与表达[J]. 实用肝脏病杂志, 2018, 21(6): 833-836.

Xu Xi, Dang Yinli, Wu Shuo.. Construction and expression of recombinant eukaryotic expression vector of HCV infection related interferon λ4[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(6): 833-836.

参考文献

[1] Eslam M,McLeod D,Kelaeng KS,et al. IFN-λ3,not IFN-λ4,likely mediates IFNL3-IFNL4 haplotype-dependent hepatic inflammation and fibrosis. Nat Genet,2017,49(5):795-800.
[2] Manuel O,Wójtowicz A,Bibert S,et al.Swiss transplant cohort study. Influence of IFNL3/4 polymorphisms on the incidence of cytomegalovirus infection after solid-organ transplantation. J Infect Dis,2015,211(6):906-914.
[3] O'Brien TR,Prokunina-Olsson L,Donnelly RP. IFN-λ4:the paradoxical new member of the interferon lambda family. J Interferon Cytokine Res,2014,34(11):829-838.
[4] Meissner EG,Bon D,Prokunina-Olsson L,et al.IFNL4-ΔG genotype is associated with slower viral clearance in hepatitis C,genotype-1 patients treated with sofosbuvir and ribavirin. J Infect Dis,2014,209(11):1700-1704.
[5] Prokunina-Olsson L,Muchmore B,Tang W,et al.A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus. Nat Genet,2013,45(2):164-171.
[6] Ray K.Hepatitis:New gene IFNL4 is associated with impaired clearance of HCV. Nat Rev Gastroenterol Hepatol,2013,10(2):63.
[7] Lauber C,Vieyres G,Terczyńska-Dyla E,et al.Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells. Genes Immun,2015,16(6):414-421.
[8] Huang P,Yao Y,Yue M,et al.Genetic variants in interferon-λ 4 influences HCV clearance in Chinese Han population. Sci Rep,2017,7:42408.
[9] Xie X,Zhang L,Chen YZ.Association between IFNL4 rs3682
34815 polymorphism and sustained virological response in chronic hepatitis C patients undergoing PEGylated interferon/ribavirin therapy:A meta-analysis. Hum Immunol,2016,77(7):609-615.
[10] de Seixas Santos Nastri AC,de Mello Malta F,Diniz MA,et al. Association of IFNL3 and IFNL4 polymorphisms with hepatitis C virus infection in a population from southeastern Brazil. Arch Virol,2016,161(6):1477-1484.
[11] Chinnaswamy S,Bhushan A,Behera AK,et al.Roles for transcription factors Sp1,NF-κB,IRF3,and IRF7 in expression of the human IFNL4 gene. Viral Immunol,2016,29(1):49-63.
[12] Aiken T,Garber A,Thomas D,et al.Donor IFNL4 genotype is associated with early post-transplant fibrosis in recipients with hepatitis C. PLoS One,2016,11(11):e0166998.
[13] Eslam M,Hashem AM,Leung R, et al.International hepatitis C genetics consortium (IHCGC). Interferon-λrs12979860 genotype and liver fibrosis in viral and non-viral chronic liver disease. Nat Commun,2015,6:6422.
[14] Ansari MA,Pedergnana V,L CIp C,et al. Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus. Nat Genet,2017,49(5):666-673.
[15] Bordi L,Caglioti C,Garbuglia AR,et al.IFNL4 and IFNL3 associated polymorphisms strongly influence the spontaneous IFN-alpha receptor-1 expression in HCV-infected patients. PLoS One,2015,10(2):e0117397.
[16] Lu YF,Goldstein DB,Urban TJ,et al.Interferon-λ4 is a cell-autonomous type III interferon associated with pre-treatment hepatitis C virus burden. Virology,015,476:334-340.
[17] Miyamura T,Kanda T,Nakamoto S,et al.IFNL4 ss469415590 variant is associated with treatment response in Japanese HCV genotype 1 infected individuals treated with IFN-including regimens. Int J Hepatol,2014,2014:723868.
[18] Terczyńska-Dyla E,Bibert S,Duong FH,et al.Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes. Nat Commun,2014,5:5699.
[19] Onabajo OO,Porter-Gill P,Paquin A,et al.Expression of interferon lambda 4 is associated with reduced proliferation and increased cell death in human hepatic cells. J Interferon Cytokine Res,2015,35(11):888-900.
[20] Akamatsu S,Hayes CN,Ochi H,et al.Association between variants in the interferon lambda 4 locus and substitutions in the hepatitis C virus non-structural protein 5A. J Hepatol,2015,63(3):554-563.