OBJECTIVESTo construct and screen a primarily phage display library of HCV C and E1 genes evolved with an artificial pattern.

METHODSTwo genes of about 1 kb with different genotypes were evolved by DNA shuffling. The re-assembled HCV C and E1 genes were cloned into a phage vector. After being rescued with helper phage M13KO7, a phage display library was constructed. Then the library was screened with anti-C and E1 McAb. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out on twenty individual phage clones selected randomly to detect their binding and reactive activity with high-titer HCV-positive sera. Normal sera were used as controls.

RESULTSThe phage display library of HCV C and E1 genes which evolved with an artificial pattern was constructed. Their capacity amounted to 1.64 x 10(6), and 86 percent of the clones contained C and E1 genes. After four rounds of panning, the phage library was specifically enriched. Twelve positive clones were successfully screened.

CONCLUSIONThe capacity and diversity of the constructed library are enough for screening. The results demonstrate the superiority of the specific binding and reactive activity and affinity of the 12 phage clones from the HCV positive sera.

DNA, Viral ; genetics ; Gene Library ; Hepacivirus ; genetics ; Peptide Library ; Viral Core Proteins ; genetics ; Viral Envelope Proteins ; genetics

Glycoproteins ; genetics ; metabolism ; Herpesviridae ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

OBJECTIVETo investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).

METHODSYeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.

RESULTSAmong twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.

CONCLUSIONSGenes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Carrier Proteins ; genetics ; Cloning, Molecular ; Hepacivirus ; genetics ; Hepatocytes ; metabolism ; Humans ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; Viral Proteins ; genetics

Influenza B virus ; genetics ; pathogenicity ; Phylogeny ; RNA, Viral ; genetics ; Viral Nonstructural Proteins ; genetics

Baculoviridae ; genetics ; metabolism ; Phylogeny ; Viral Proteins ; chemistry ; classification ; genetics ; metabolism

Animals ; Herpesviridae ; genetics ; metabolism ; Humans ; Viral Proteins ; chemistry ; genetics ; metabolism

Recently, the interactions between hepatitis C virus (HCV) genes and the host cell factors were the focus of this field. Cell factors in the different biochemical pathway were approved to be interfered when HCV infection. To make sure which HCV gene(s) was the major factor during the interaction process, ten eukaryotic expression plasmids containing different functional genes of HCV: Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B were transfected into the CHO-K1 cells respectively. Then ten stable cell lines expressing different HCV functional proteins were constructed under the selective pressure of G418. DNA and mRNA of the HCV genes were both detected by PCR and RT-PCR respectively in the corresponding stable cell lines, freezation and anabiosis would not lose the HCV genes. Besides, the El, E2 and NS5B proteins were detected by Western-blot which demonstrated that the HCV genes have formed stable expression in the host cells. The activity of UDP-glucose ceramide glucosyltransferase (UGCG) in the stable cell lines increased in different degree by TLC assay. For example, the activity of UGCG in CHO-K1-E2 and CHO-K1-p7 was doubled according to the control cells,and in CHO-K1-NS2 and CHO-K1-NS5A was about 1.6 times compared with the control cells. The establishment of the stable cell lines containing different single HCV gene will provide foundation for investigating the interactions between the virus and the host factors, and for the filtration of antiviral medicine.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Glucosyltransferases ; biosynthesis ; metabolism ; Hepacivirus ; genetics ; metabolism ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Viral Proteins ; biosynthesis ; genetics

OBJECTIVETo clone the glycoprotein G gene and its specific fragment with high conservation and antigenicity by culturing and amplifying herpes simplex virus type 2 and extracting its whole genome.

METHODSWe obtained a great deal of suspension with HSV-2 virus after infecting the cultured Hela cells with HSV-2 virus, extracted the whole genome of the virus by the phenol-chloroform method, and amplified the US4 gene coding gG-2 by PCR. Then we selected the specific target fragment according to the amino acid sequence alignment of the gG-2 gene and cloned it with the designed primers with restricted endonuclease sites.

RESULTSWe successfully obtained a lot of suspension with HSV-2 virus, and cloned the gG-2 gene from the whole genuine and its specific target fragment. Sequencing showed that both the sequences were identical with those printed in the GenBank.

CONCLUSIONIt is feasible to obtain the virus genome and specific fragment of the gG-2 gene from virus-infected cells, especially for HSV-2 virus with relatively stable hereditary trait. It has prepared the ground for further constructing the expression plasmid of the specific fragment, expressing related proteins and identifying their antigenicity.

Antigens, Viral ; genetics ; Cloning, Molecular ; DNA, Viral ; HeLa Cells ; Herpesvirus 2, Human ; genetics ; Humans ; Viral Envelope Proteins ; genetics ; Virus Cultivation

OBJECTIVETo investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.

METHODSThe PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.

RESULTSThe transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.

CONCLUSIONSoluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.

Escherichia coli ; metabolism ; Genetic Vectors ; Hepacivirus ; Recombinant Proteins ; genetics ; metabolism ; Viral Core Proteins ; biosynthesis ; genetics ; metabolism ; Viral Nonstructural Proteins ; metabolism

According to previous studies of SARS-CoV (Severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in SARS-CoV genome, which were interspersed the structural genes of SARS-CoV and considered to be unique to the SARS-CoV genome. The predicted unknown proteins (PUPs) encoded by the accessory genes might play important roles in the SARS-CoV infection. Three of those genes, called X4, X5 and ORF10, were synthesized and introduced into E. coli to induce expression. SDS-PAGE and Western blotting revealed that the three genes have been expressed in E. coli. The induction of SARS PUPs genes expression in different temperatures, induction times, IPTG concentrations and A values of E. coli cells were performed. The optimal induction condition of SARS-CoV PUPs genes was characterized according to the orthorgonal analysis. The ratio of recombinant proteins of PUPs to total proteins is as follows: X4, 20%; X5, 27.8%; ORF10, 68.5% under the optimum conditions.
Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genes, Viral ; Genetic Vectors ; Genome, Viral ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; SARS Virus ; genetics ; Temperature ; Time ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism