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

Abstract

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

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.

References

[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.

Construction and expression of recombinant eukaryotic expression vector of HCV infection related interferon λ4

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	Ren Yufei, Chen Xiaoqing, Kong Weizong, et al.. Effect of salvianolic acid B on autophagy in nonalcoholic fatty liver disease cell model in vitro [J]. Journal of Practical Hepatology, 2019, 22(6): 804-807.
[2]	Guo Liping, Zhang Chenghong, Ning Baoshuo, et al.. An improved approach for seperating hepatocytes from neonatal rats in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(1): 17-20.
[3]	Zhang Xiaosan, Zhang Yiming, Yang Shujun, et al.. Impact of miR-21 on proliferation and apoptosis in HepG2 cells in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2019, 22(1): 21-24.
[4]	Li Junfeng, Zheng Sujun, Liu Shuang, et al.. Impact of UGCG siRNA on 7702 hepatocyte proliferation in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(5): 697-700.
[5]	Zhou Da, Wang Jing, Li Binghang, et al. Inhibition of proliferation and regulation of heaptic fibrosis-related genes in HSC-T6 cells by prolyloligopeptidase inhibitor in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(3): 388-392.
[6]	Li Wenjiao, Cao Haichao, Ma Lanjing, et al. Downregulation of CCAAT/enhancer-binding protein alpha promotes growth of hepatocellular carcinoma cells in vitro by increasing O-GlcNAcylation [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(3): 393-396.
[7]	Zhang Yongting, Zhu Chuanlong, Li Jun, et al. Construction of overexpression of c-met in human umbilical cord mesenchymal stem cells with lenti-vector transfection in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2018, 21(3): 397-400.
[8]	Li Binghang，Li Mengting，He Lingnan，et al.. Inhibitory effect of AcSDKP on the proinflammatory profiles induced by lipopolysaccharides in a mouse RAW264.8 macrophage cell line in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2017, 20(2): 142-147.
[9]	Zhang Kun，Hu Hai，Zhang Yuan，et al.. Changes of CTGF，fibronectin and integrin mRNA levels in Gan-fu-kang，a herbal medicine-intervened rat hepatic stellate cells in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2017, 20(2): 153-156.
[10]	Feng Ye，Shen Jie，Guo Lili，et al.. Bile immerse of new type of bile duct stent with Mg-Nd-zinc-Zr magnesium alloy material in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2017, 20(2): 211-214.
[11]	Tang Weiliang, Xie Qing. Hepatitis C virus genotype 3 infection [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(4): 394-397.
[12]	Tong Yanyan, Li Guangming, Deng Yilin, et al. Inhibition of primary cultured hepatic stellate cell activation by phenylarsine oxide in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(4): 413-417.
[13]	Xin Liangliang, Li Bing, Rong Yihui. Full-length hepatitis E virus amplification and cloning and establishment of transient transfer system in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(4): 423-427.
[14]	He Lingnan,Li Binghang,Zhou Da,et al.. Inhibitory effect of galanin on proinflammatory response induced by lipopolysaccharides in a mouse RAW264.7 macrophage cell line in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(3): 270-274.
[15]	Ding Ning, Zhang Mingxiang. Construction and identification of eukaryotic expression vector of human hepatocyte nuclear factor 4α gene in vitro [J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2016, 19(2): 156-159.