Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

Functional study of the different haplotypes cDNA in the coding region of CⅡTA gene

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.     

CⅡTA基因编码区不同单倍型cDNA的功能研究

目的 研究MHCⅡ类反式激活因子(cⅡTA)基因编码区非同义单核苷酸多态性(SNP)位点C19170G(Leu45Val)和C30799G(Ala500Gly)构成的4种不同单倍型cDNA的功能.方法 将4种不同单倍型的真核表达载体和卒载体分别转染至HeLa细胞.用RT-PCR和间接细胞免疫荧光技术检测未经转染的HeLa细胞、转染4种真核表达载体及卒载体的HeLa细胞cⅡTA mRNA与3种HLAⅡ类分子(HLA-DR、DP、DQ)的表达,并用流式细胞技术对其表达的3种HLAⅡ类蛋白进行定量分析.结果 未经转染和转染空载体的HeLa细胞均尤CⅡTA mRNA和3种HLAⅡ类分子的表达,而转染4种不同单倍型真核表达载体的HeLa细胞均出现CⅡTA mRNA表达,并表达3种HLAⅡ类分子.证实了转染4种不同单倍型真核表达载体后的HeLa细胞3种HLAⅡ类分子表达水平差异无统计学意义(P均>0.05).结论 中国人CⅡTA基因编码区这两个SNP位点的多态性(2个位点氨基酸的改变)不影响CⅡTA反式激活HLAⅡ类基因表达的能力.     

Forcing Tumor Cells to Present Their Own Tumor Antigens to the Immune System： a Necessary Design for an Efficient Tumor Immunotherapy

The general principle for tumor cells to escape from immune surveillance is to prevent tumor antigens from being recognized by the immune system. Many methods have been developed to increase the immunogenecity of the tumor cells. The most efficient methods are able to force tumor cells to present their own tumor antigens to the immune system. Stimulating Th cells by converting tumor cells into MHC class II /Ii- antigen presenting cells is one of the most efficient technologies. Using antisense methods, we suppress the expression of the Ii protein that normally co-expresses with MHC class II molecules and blocks the antigenic peptide binding site of MHC class II molecules during synthesis in the endoplasmic reticulum. In such tumor cells, the“unprotected“ MHC class II molecules pick up endogenous tumor antigenic peptides, which have been transported into the ER for binding to MHC class I molecules. Simultaneous presentation of tumor antigens by both MHC class I and II molecules generates a robust and long-lasting anti-tumor immune response. MHC class II /Ii- tumor cells are potent tumor cell vaccines and also cure a significant number of animals with renal and prostate tumors. We have developed analogous human gene vectors that are suitable for most patients and cancers.     

Impact of CCAAT enhancer binding protein α on the differentiation and apoptosis of acute myeloid leukemia HL60 cells

Objective To investigate the effect of CCAAT enhancer binding protein α(C/EBPα) on differentiation and apoptosis in the acute myeloid leukemia HL60 cells in vitro and in vivo and its possible mechanism. Methods The C/EBPα expression plasmid pEGFP-C/EBPα and empty control plasmid were respectively transfected into HL60 cells with cationic liposome as transfected group and empty plasmid transfected group, and untreated HL60 cells served as control group. The cells stably expressing the C/EBPα gene were obtained by G418 selection. The morphological changes were observed under light microscope following WrightGiemsa staining. MTT assay was employed to evaluate cell proliferation, and flow cytometry(FCM) was performed to analyze cell apoptosis. Meanwhile, the expression of c-myc was respectively detected by RT-PCR and Western blot both at the mRNA and protein level. Twenty BALB/c nude mice were divided into 3 groups in a completely randomized design: 7 mice in transfected group, 7 mice in empty plasmid transfected group and 6 in control group. Three kinds of cells including pEGFP-C/EBPα-HL60 cells, pEGFP -HL60 cells and the control HL60 cells were injected into mice separately through the subcutaneous. The mice were sacrificed at 20 d after injection. The mass and size of subcutaneous xenograft tumors were measured and the cell apoptosis of subcutaneous tumor were detected by TUNEL. Results The pEGFP-C/EBPα-HL60 cell line stably expressing the C/EBPα gene was screened out. Compared to either empty plasmid transfected group or control group, the expression of C/EBPα could promote cellular differentiation of HL60. FCM showed higher apoptotic rate in transfected group[ (21.9±4.5)%,P＜0.05 ] ,while (5.4±1.4)% in control group and (5.0±1.3)% in empty plasmid transfected group. c-myc expression was significantly down-regulated by C/EBPα both at the mRNA and protein level. The mass and size of tumors in transfected group were smaller than those in empty plasmid transfected group and control group [ (5.35±1.12)g and(25±4)mm in control group, (5.12±1.31)g and ( 18±3)mm in empty plasmid transfected group ,while (3.26±0.72)g and ( 11±2)mm in transfected group, all P＜0.05]. More apoptosis cells were found in subcutaneous tumor of transfected group(both P＜0.05). Conclusion C/EBPα can not only inhibit the proliferation, but also induce massive apoptosis of HL60 cells, meanwhile C/EBPα is a tumor suppressor of acute myeloid leukemia.     

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is thekey to the cellular immune response.Non-self intracellular proteins are processed into short peptides andtransported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by severalchaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to thecell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides arecleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the targetcells the process of peptide loading and class I expression must be carefully regulated.Many of the cellularcomponents involved in antigen processing and class I presentation are known and their various functions arenow becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.     

Effect of structural domain Ⅱ of HCV 5' noncoding region on its translation initiation activity

Objective To examine the role of domain Ⅱ of hepatitis C virus(HCV) 5'noncoding region (5'NCR) in its translation initiation activity. Methods The fragment of HCV 5' NCR with deletions of 5'-proximal 118 nucleotides were amplified with PCR, then was used to substitute for the full length HCV 5'NCR of plasmid pCMVNCRIuc, a firefly luciferase(Flue) eukaryotic expression plasmid regulated by HCV 5' NCR, to generate recombinant plasmid pCN1-d3, pCMVNCRIuc, pCN1-d2 (modified pCMVNCRluc with deletions of HCV 5' NCR nt1-43) and pCN1-d3 were transfected into HepG2 cells with liposome transfection protocol, respectively, and the relative luciferase activity was measured to analyze the regulatory effect of the truncated 5'NCR on Fluc gene expression. Meanwhile the Fluc mRNA levels were detected by RT-PCR. Results The recombinant plasmid was successfully constructed. The Fluc mRNA levels of the 3 plasmids were not significantly different(P 0.05), and there were also no significant difference between the relative luciferase activity of plasmids pCN1-d2 and pCMVNCRluc(P 0.05). However, that of pCN1-d3 was decreased significantly(P < 0.01, compared with pCN1-d2 or pCMVNCRluc). Conclusion Structural domain Ⅱ of HCV 5' NCR plays an important role in itstranslation initiation activity.