CLEC4M慢病毒载体的构建及其在K-562细胞中的表达

王媛媛; 聂青和; 朱婷

doi:10.3969/j.issn.1001-5256.2014.06.010

CLEC4M慢病毒载体的构建及其在K-562细胞中的表达

DOI: 10.3969/j.issn.1001-5256.2014.06.010

第四军医大学唐都医院传染科

基金项目:

国家自然科学基金资助项目(81170390)；

详细信息

中图分类号: R512.63
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出版历程
- 收稿日期: 2013-09-18
- 出版日期: 2014-06-20

Construction of lentiviral vector encoding CLEC4M and overexpression of CLEC4M in K- 562 cells

Department of Infectious Diseases, Tangdu Hospital

Research funding:

摘要

摘要: 目的本研究拟构建CLEC4M慢病毒表达载体,建立稳定过表达的K-562细胞株。方法应用逆转录PCR克隆出正常人CLEC4M基因序列,将序列插入GV166载体,构建GV166-CLEC4M慢病毒表达载体,之后转染293T细胞进行慢病毒包装。用获得的慢病毒液感染人白血病细胞系K-562,通过实时荧光定量PCR和Western Blot检测K-562细胞中CLEC4M的过表达情况。结果测序结果显示成功构建重组慢病毒表达质粒GV166-CLEC4M;依据实时荧光定量PCR可测定慢病毒能够有效感染K-562细胞;CLEC4M在K-562细胞中的mRNA和蛋白水平都成功的过表达。结论 CLEC4M慢病毒表达载体的构建,为进一步研究CLEC4M基因在HCV入胞的分子机制奠定了基础。
- 丙型肝炎病毒 /
- 慢病毒感染 /
- 树突细胞
Abstract: Objective To construct the lentiviral vector encoding CLEC4M and prepare K- 562 cells with stable overexpression of CLEC4M. Methods The gene sequence of normal CLEC4M was cloned by reverse transcription PCR and then inserted into GV166 vector to construct GV166- CLEC4M lentiviral expression vector, and then lentiviral packaging was performed by transfection of 293T cells. The obtained lentiviral liquid was used to infect human leukemia cell line K- 562. Real- time PCR and Western blot were used to detect the overexpression of CLEC4M in K- 562 cells. Results Sequencing showed that the recombinant lentiviral expression plasmid GV166- CLEC4M was successfully constructed. Lentiviruses could efficiently infect K- 562 cells, according to real- time PCR. CLEC4M was successfully over- expressed in K- 562 cells at mRNA and protein levels. Conclusion The construction of lentiviral vector encoding CLEC4M lays a foundation for further study of CLEC4M gene involved in HCV entry into host cells.
- hepertitis C virus /
- lentivirus infections /
- dendritic cells

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参考文献(15)

[1]MATIAS BF, de OLIVEIRA TM, RODRIGUES CM, et al.Influence of immunotherapy with autologous dendritic cells on innate and adaptive immune response in cancer[J].Clin Med Insights Oncol, 2013, 28 (7) :165-172.

[2]CARDOSO EC, PEREIRA NZ, MITSUNARI GE, et al.TLR7/TLR8 activation restores defective cytokine secretion by myeloid dendritic cells but not by plasmacytoid dendritic cells in HIV-infected pregnant women and newborns[J].PLoS One, 2013, 8 (6) :e67036.

[3]NIE QH, GAO LH, CHEN YQ, et al.Hepatitis C virus infection of human cytotrophoblasts cultured in vitro[J].J Med Virol, 2012, 84 (10) :1586-1592.

[4]WANG L, ZHOU GB, LIU P, et al.Dissection of mechanisms of Chinese medicinal formula realgar-iasfndigo naturalis as an effective treatment for promyelocytic leukemia[J].Proc Natl Acad Sci U S A, 2008, 105 (12) :4826-4831.

[5]SU YF, CHANG GD, WILLAM KK, et al.Lentiviral expression of anti-microRNAs targeting miR-27a inhibits proliferation and invasiveness of U87 glioma cells[J].Mol Med Rep, 2012, 6 (2) :275-281.

[6]HOLKERS M, MAGGIO I, LIU J, et al.Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells[J].Nucleic Acids Res, 2013, 41 (5) :e63.

[7]WU DT, SEITA Y, ZHANG X, et al.Antibody-directed lentiviral gene transduction for live-cell monitoring and selection of human iPS and hES cells[J].PLoS One, 2012, 7 (4) :e34778.

[8]SUN Y, LIU M, YANG B, et al.Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma[J].Eur Arch Otorhinolaryngol, 2008, 265 (11) :1385-1391.

[9]CHUNG YD, SINZINGER MD, BOVEE-GEURTS P, et al.Analyzing the homeostasis of signaling proteins by a combination of western blot and fluorescence correlation spectroscopy[J].Biophys J, 2011, 101 (11) :2807-2815.

[10]LEE SJ, ARORA R, BULL LM, et al.A nonneutralizing anti-HIV Type 1 antibody turns into a broad neutralizing antibody when expressed on the surface of HIV type 1-susceptible cells.II.Inhibition of HIV type 1 captured and transferred by DC-SIGN[J].AIDS Res Hum Retroviruses, 2006, 22 (9) :874-883.

[11]ZHUANG P, LI ZY, WANG XC, et al.Construction of lentiviral vector carrying miR-122 in hepatocellular carcinoma cells[J].J Clin Hepatol, 2013, 29 (7) :529-531. (in Chinese) 庄鹏, 李志莹, 王湘郴, 等.miR-122慢病毒载体的构建及其在肝癌细胞中的作用[J].临床肝胆病杂志, 2013, 29 (7) :529-531.

[12]ZOHOUN A, HADEF R, ZAHID H, et al.Seroprevalence of HBV and HCV in blood donors at the blood transfusion center of mohammed V military teaching hospital in rabat morocco[J].Med Trop (Mars) , 2011, 71 (5) :513-514.

[13]GAO LH, NIE QH, WANG YY, et al.CLEC4L-mediated HCV infection of human placental trophoblast cells[J].J Clin Hepatol, 2012, 28 (12) :941-944. (in Chinese) 高禄化, 聂青和, 王媛媛, 等.CLEC4L介导丙型肝炎病毒感染人胎盘滋养层细胞的作用机制[J].临床肝胆病杂志, 2012, 28 (12) :941-944.

[14]BOILY-LAROUCHE G, ISCACHE AL, ZIJENAH LS, et al.Functional genetic variants in DC-SIGNR are associated with mother-to-child transmission of HIV-1[J].PLoS One, 2009, 4 (10) :e7211.

[15]HERBST F, BALL CR, TUORTO F, et al.Extensive methylation of promoter sequences silences lentiviral transgene expression during stem cell differentiation in vivo[J].Mol Ther, 2012, 20 (5) :1014-1021.

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