Mechanisms of STAT family mediated IDO regulation in dendritic cells / 国际肿瘤学杂志

Indoleamine 2,3-dioxygenase (IDO) is known as an endogenous immunosuppressive enzyme which plays a significant role in the process of tumor.IDO is not only found in tumor cells but also detected in dendritic cell (DC) in tumor microenvironment,which participates in the formation of tumor immune tolerance through expressing IDO enzyme.Signal transducer and activator of transcription (STAT) is the main signal protein family which participates in the IDO transcriptional regulation of DC.It is necessary to detail the signaling pathway in regulating IDO expression,which will help us develop high specific and more active IDO inhibitors and provide new options for anti-cancer targeted therapy.

Screening and cloning of the target genes transactivated by HCV FTP2 protein using suppression subtractive hybridization technique / 中华传染病杂志

Objective To clone and identify human genes transactivated by homo sapiens HCV FTP2 by constructing a cDNA subtractive library with suppression subtractive hybridization tech- nique.Methods Suppression subtractive hybridization(SSH)and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV FTP2.The mRNA was iso- lated from HepG2 cells transfected pcDNA3.1(-)-HCV FTP2 and pcDNA3.1(-)empty vector re- spectively,and SSH method was employed to analyze the differentially expressed DNA sequence be- tween the two groups.After digestion with restriction enzyme Rsa I,small-size cDNAs were ob- tained.Then tester cDNA was divided into two groups and ligated to the specific adaptor I and adap- tor 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 DH5?.Futhermore,the cDNA was se- quenced and analyzed in GenBank with Blast search after PCR.Results The subtractive library of genes transactivated by HCV FTP2 was constructed successfully.The amplified library contains 71 positive clones.Colony PCR shows that 56 clones contain 200～1000 hp inserts.Sequence analysis was performed in 24 clones randomly,and the full length sequences were obtained with bioinformatics method.Altogether 20 coding sequences in total were obtained,consisting of 19 known and 1 un- known.Conclusion The obtained sequences may be target genes transactivated by HCV FTP2,and some genes coding proteins involved in cell cycle regulation,metabolism and cell apoptosis.

Suppression subtractive hybridization for cloning of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus / 中华传染病杂志

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.

Genotype-specific functional differences of the hepatitis B virus X protein / 中华传染病杂志

pcDNA3.1/Hygro(-)+pCMV?. Conclusions Transactivation competence of genotype B HBx was higher than that of genotype C HBx, while the antiproliferative and apoptosis effects of genotype B HBx was lower than of genotype C HBx. B and C genotype-specific functional differences of HBx may closely co-related with the pathogenicity of HBV.

Cloning and identification of human gene transactivated by hepatitis C virus core protein 1 / 解放军医学杂志

Objective To screen and clone the target genes transactivated by hepatitis C virus (HCV) core protein. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-core and pcDNA3.1(-) empty vector, respectively, and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequences of the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. One of them was confirmed to be a new gene without homology with known genes in this database.Then, electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene, in conjunction with Kozak rule and the existence of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene with mRNA from HepG2 cell as the template. The coding sequence for the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named TAHCCP1.The nucleotide sequence of the new gene and its corresponding protein-encoding amino acid, which was 2 001nt and composing 667aa, have been determined. The sequence of the TAHCCP1 gene has been registered in GenBank with its accession number AY038359. Conclusion TAHCCP1 gene transactivated by HCV core protein was cloned and identified successfully by a combination of molecular biological technology and bioinformatics technique. The results are expected to pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the development of new therapies for chronic hepatitis C.

MOLECULAR MECHANISM OF PATHOGENESIS OF HEPATITIS C VIRUS / 解放军医学杂志

The pathogenesis of hepatitis C virus (HCV) is different from that of DNA virus and retrovirus. The epitope inducing neutralizing antibody in high variable region 1 (HVR1) is the reason for escape of host immune surveillance, evolution of chronic infection, and also difficulty of producing wide spectrum vaccine. Mimotope selected by using phage display technique is an alternative to solve this problem. The study on the binding protein to non coding region of HCV RNA is helpful to uncover the regulation of replication and translation of HCV genome. A study of the specific receptor of HCV is important to elucidate the mechanism of how HCV enter the hepatocyte. Yeast two hybrid is a useful technique to study the protein protein interactions between HCV and hepatocyte. Suppressive subtraction hybridization is an efficient method for identification of genes of interest transactivated by HCV structural and non structural proteins. All these studies will finally resulted in the full understanding of HCV pathogenesis, and will supply unique opportunity for the development of a new therapy for HCV infection.