CLEC4M慢病毒载体的构建及其在K-562细胞中的表达
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...
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| Published in | 临床肝胆病杂志 Vol. 30; no. 6; pp. 518 - 521 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
第四军医大学唐都医院 传染科,西安,710038
2014
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1001-5256 |
| DOI | 10.3969/j.issn.1001-5256.2014.06.010 |
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| Abstract | 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。 |
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| AbstractList | R512.63; 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。 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。 |
| Abstract_FL | Objective To construct the lentiviral vector encoding CLEC4M and prepare K -562 cells with stable overexpression of CLEC4M.Methods The gene sequence of normal CLEC4Mwas cloned by reverse transcription PCR and then inserted into GV166 vector to construct GV166-CLEC4Mlentiviral expression vector,and then lentiviral packaging was performed by transfection of293T cells.The ob-tained lentiviral liquid was used to infect human leukemia cell line K-562.Real-time PCR and Western blot were used to detect the over-expression 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.CLEC4Mwas successfully o-ver-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. |
| Author | 王媛媛 聂青和 朱婷 |
| AuthorAffiliation | 第四军医大学唐都医院传染科,西安710038 |
| AuthorAffiliation_xml | – name: 第四军医大学唐都医院 传染科,西安,710038 |
| Author_FL | WANG Yuanyuan NIE Qinghe ZHU Ting |
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| Author_xml | – sequence: 1 fullname: 王媛媛 聂青和 朱婷 |
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| DocumentTitleAlternate | Construction of lentiviral vector encoding CLEC4M and overexpression of CLEC4M in K-562 cells |
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| Keywords | 丙型肝炎病毒 树突细胞 hepertitis C virus dendritic cells 慢病毒感染 lentivirus infections |
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| Notes | hepertitis C virus;lentivirus infections;dendritic cells Objective To construct the lentiviral vector encoding CLEC4M and prepare K -562 cells with stable overexpression of CLEC4M.Methods The gene sequence of normal CLEC4Mwas cloned by reverse transcription PCR and then inserted into GV166 vector to construct GV166-CLEC4Mlentiviral expression vector,and then lentiviral packaging was performed by transfection of293T cells.The ob-tained lentiviral liquid was used to infect human leukemia cell line K-562.Real-time PCR and Western blot were used to detect the over-expression 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.CLEC4Mwas successfully o-ver-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 ho |
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| PublicationTitle | 临床肝胆病杂志 |
| PublicationTitleAlternate | Chinese Journal of Clinical Hepatology |
| PublicationTitle_FL | Journal of Clinical Hepatology |
| PublicationYear | 2014 |
| Publisher | 第四军医大学唐都医院 传染科,西安,710038 |
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| SubjectTerms | 丙型肝炎病毒 慢病毒感染 树突细胞 |
| Title | CLEC4M慢病毒载体的构建及其在K-562细胞中的表达 |
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