Objective To investigate the transactivating effect of hepatitis C virus(HCV)core protein on inducible nitric oxide synthase(iNOS)gene promoter and the molecular biological mecha- nisms of HCV pathogenesis.Methods Polymerase chain reaction(PCR)technique was employed to amplify the sequence of iNOS promoter by using HepG2 genomic DNA as template,and the product was cloned into pGEM-T vector.The iNOSp gene was cut from T-iNOSp by KpnⅠand XhoⅠ,and then was cloned into pCAT3-Basic,the constructed vector was named as pCAT3-iNOSp,pCAT3-iN- OSp was transfected into the LO_2 cell line.LO_2 cell was also cotransfected with pcDNA3.1(-)-core and pCAT3-iNOSp by FuGENE 6 transfection reagents.The LO_2 cells transfected with pCAT3-Basic was used as negative control.The activity of CAT in LO_2 cells was detected by an ELISA kit after 48 hours,which reflected the transactivating function of HCV core protein to iNOS gene promoter.Re- sults The expressive vector pcDNA3.1(-)-core and report vector pCAT3-iNOSp had been construc- ted and confirmed by restriction enzyme digestion and sequencing.The expression of CAT in LO_2 cells transfected with pCAT3-iNOSp and peDNA3,1(-)-core was 11 times as higher as that of pCAT3-bas- ic,and 6 times as higher as that of pCAT3-iNOSp.Conclusion It is suggested that HCV core protein can transactivate iNOS gene promoter.

Objective To construct a subtractive cDNA library of genes transactivated by XTP3 protein with suppression subtractive hybridization technique in order to clone genes associated with transactivation. Methods mRNA was isolated from HepG2 cells transfected by pcDNA3.1(-)-XTP3 and pcDNA3.1(-) empty vector respectively, and then cDNA was synthesized. After restriction enzyme Rsa Ⅰ digestion, small sizes cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2 respectively. After being hybridized with driver cDNA twice and underwent two times of nested PCR, tester cDNA was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.Coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The amplified library contained 30 positive clones. Colony PCR showed that 23 clones contained 200-1 000bp inserts. Sequence analysis was performed also. It was found that 20 kinds of known and 2 kinds of novel cDNA sequences may be target genes transactivated by XTP3 protein. Conclusion The subtractive library of genes transactivated by XTP3 protein was constructed successfully. It provides a foundation for the further research on pathogenesis of the viral proteins.

Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with glycyrrhizin (GL), and to clone genes associated with its immunological regulation, and to further elucidate the molecular immune mechanism of GL. Methods The mRNA was isolated from Jurkat cells treated with either GL or 0.9 percent sodium chloride as a control, then cDNA was synthesized. Suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The cDNA was sequenced and analyzed in GenBank with Blast search after the amplification of the subtractive library by PCR. Results The amplified library contained 28 positive clones. Colony PCR analysis showed that there were 22 clones containing 200-1 000 bp inserts. Sequence analysis was performed, and the full length sequences were obtained with bioinformatics method. Altogether 11 kinds of encoding sequences were achieved, including interleukin-12, interleukin-18, and thymosin ?1, etc. Conclusions A subtractive cDNA library of genes differentially expressed in Jurkat cells treated with GL using SSH technique was constructed successfully, and it might give some new clues for the study of the immune regulation mechanism of GL.

Objective To investigate the biological functions of HBeAg-binding protein 1(HBEBP1), suppression subtractive hybridization(SSH) technique was used to screen genes regulated by HBEBP1. Methods HBEBP1(GenBank number:AF529372) was screened and identified by yeast two-hybrid system 3 and co-immunoprecipitation technique. The HBEBP1 coding DNA fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) technique from HepG2 cell genome. The expressive vector of pcDNA3.1(-)-HBEBP1 was constructed by routine molecular biological methods. The HepG2 cells were transfected with pcDNA3.1(-) and pcDNA3.1(-)-HBEBP1, respectively by using FuGENE6 transfection reagent, then the mRNA was isolated. SSH method was employed to analyze the differentially expressed DNA sequences between the two groups. After restriction enzyme Rsa I digestion, small sized cDNAs were obtained. Then tester cDNAs were divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNAs were hybridized with driver cDNAs twice and underwent polymerase chain reaction (PCR) twice, they were then subcloned into pGEM-Teasy plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5?. The cDNAs were sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes up-regulated by HBEBP1 was constructed successfully. The amplified library contained 85 positive clones. Colony PCR showed that these clones contained 200-1 000bp inserts. Sequence analysis was performed in 26 clones at random, and the full length sequences were obtained with bioinformatics method. Altogether 15 coding sequences were obtained. Conclusions The obtained sequences may be target genes up-regulated by HBEBP1, among which some genes coding proteins were involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some new clues for the study of the biological functions of HBEBP1 and HBeAg.

Liver fibrosis can be caused by chronic liver injury arising from various etiological factors and it is a reversible process. The activation of the hepatic stellate cell( HSC) is the central event in liver fibrosis,since we know that cytokines secreted from Kupffer cell participate in HSC proliferation,apoptosis and ECM metabolism. In this paper we focus on the relation-ship between HSCs,Kupffer cell,cytokines and the course of hepatic fibrosis. Elucidating this relationship will benefit research on the role of Kupffer and HSCs in hepatic fibrosis.

Objective To clone amastin coding genes from different strains of Leishmania parasites. Methods Using amastin cDNA sequence as the reference, dbEST data base established by National Center Biotechnology Information (NCBI), USA, was searched by BLAST tool. A 309 bp DNA fragment of Leishmania major was found and used as the probe for the screening of a DNA library. The amastin gene of Leishmania major Abdou was cloned and sequenced. Specific primers were designed and amastin genes for Leishmania mexicana WR972, Leishmania brizeliensis and Leishmania amazonensis joseph were amplified by polymerase chain reaction. Results The amastin genes from 4 strains of Leishmania parasites were cloned and sequenced. It was found that all 4 amastin genes contained unique open reading frame of 552 bp and encoded amastin protein of 183 amino acid residues. Conclusion The amastin genes of 4 strains of Leishmania parasites were successfully cloned.

To construct a cDNA subtractive library of genes transactivated by hepatitis C virus core protein with suppression subtractive hybridization technique. mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-)-core and pcDNA3 1(-) empty vector,respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNA were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR, and then it was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain JM109. The cDNA were sequenced and analyzed in GenBank with Blast search after PCR. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR product showed that 213 clones contained 100~ 1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes before. The full length sequences were obtained with bioinformatics method,which had been accepted by GenBank. It suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.

Objective To construct a cDNA subtractive library of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus(MHBs t) with suppression subtractive hybridization technique for cloning genes associated with transactivation. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-Mt167 and pcDNA3.1(-) empty vectors, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small-size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes transactivated by MHBs t was constructed successfully. The amplified library contained 94 positive clones. Colony PCR showed that these clones contained 200-800bp inserts. Sequence analysis was performed in 50 clones,and the full length sequences were obtained with bioinformatics method. 23 coding sequences were obtained in total, which consisted of 19 known and 4 unknown ones.Conclusions The obtained sequences may be target genes transactivated by MHBs t, among which some genes coding proteins may involve in cell cycle regulation, immune response and tumour genesis.

In order to screen genes transregulated by core protein of hepatitis C virus, cDNA microarray technology was employed to detect the gene expression change between HepG2 cells transfected with pcDNA3 1(-) core and the empty vector, respectively. Among 1152 genes, there were 95 genes with different expressions, of which 45 genes were upregulated and 50 genes were downregulated in HepG2 cells transfected with core protein expression plasmid. These genes transregulated by HCV core protein included human genes encoding proteins involved in cell proliferation, differentiation, apoptosis, signal transduction and immune regulation. Therefore, the results provided some new clues for further clarifying the molecular biology mechanism of pathogenesis and tumorigenesis of HCV core protein

Hepatitis C virus (HCV) non structure 3 (NS3) protein and hepatitis B virus (HBV) X protein expressing plasmids were constructed with the vector pcDNA3 1(-). The plasmids were transfected into HepG2 cells and the viral proteins expressed in HepG2 cells were identified using Western blotting methods. Then the two recombined plasmids were transfected into HepG2 cells and were cotransfected into HepG2 cells with reporter plasmid pCAT3 promoter. The activity of CAT enzyme was detected by a CAT ELISA assay kit, which reflected the transactivating function of two proteins on SV40 early promoter. The findings indicated that the expression of two viral proteins were successfully detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV NS3 protein transactivated the CAT enzyme expressed at a value 3 5 fold higher than the control, while HBX protein transactivated at a value 4 4 times. It arrived at 8 5 times when transfected with two plamids simultaneously. The activating effect was increased in relation to the amount of plasmids. It was suggested that the two kinds of viral proteins had a transactivating effect on SV40 early promoter, and they acted synergistically. These results might contribute to explaining the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection