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

Dengue ; epidemiology ; Dengue Virus ; genetics ; isolation & purification ; Evolution, Molecular ; Genes, Viral ; Humans ; Viral Envelope Proteins ; 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 achieve secretory and extracellular production of recombinant dengue virus serotypes I-IV envelope glycoprotein domain III (DENV-1-4 EDIII) in Pichia pastoris.

METHODSEDIII genes of DENVI-IV were amplified and cloned into vector pPIC9K, respectively. These recombinant plasmids were then linearized and transferred into Pichia pastoris strain GS115. Clones highly produced in 4.0 mg/ml G418 were amplified and induced by methanol to achieve the secreted recombinant proteins. Ni-NTA agarose beads were used for purification, while SDS-PAGE and Western blotting were used for identification.

RESULTSThe recombinant plasmids pPIC9K-DENV-1-4 EDIII were constructed and successfully transferred into Pichia pastoris strain GS115. The recombinant EDIII proteins were expressed in a secretory way with the molecular weight about 12 × 10(3) and specifically identified by anti-His monoclonal antibody and anti-DENVI-IV mice sera.

CONCLUSIONDENVI-IV EDIII proteins are successfully achieved from Pichia pastoris expression system and could be used for development of dengue vaccines, diagnostic reagents and study of biological function of the E protein.

Dengue Virus ; genetics ; Genetic Vectors ; Pichia ; metabolism ; Recombinant Proteins ; genetics ; Viral Envelope Proteins ; secretion

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

Glycoprotein N is encoded by glycoprotein N (gN) gene of herpesviruses. The amino acid composition and expression level of this protein vary among difference species of herpesviruses. According to present studies, gN protein is expressed in cytoplasm of host cells, mainly in endoplasmic reticulum. The gN forms a complex with glycoprotein M in host cells. The complex is involved in the processes of viral replication and inter-cellular infection. Moreover, this protein plays a role in immune evasion from host immune system. The study will provide a theoretical basis for further study of herpesvirus gN gene and its encoded protein.
Animals ; Herpesviridae ; genetics ; metabolism ; Herpesviridae Infections ; virology ; Humans ; Viral Envelope Proteins ; genetics ; metabolism

OBJECTIVETo observe the regulating effect of hepatitis C virus (HCV) envelop (E) 2 gene immunization-induced immune responses by adenovirus mediated interleukin 12 (IL-12).

METHODSHCV E2 protein was expressed and purified from NIH 3T3 and then used as an antigen to detect antibodies against HCV E2. With 51Cr release, SP2/0 expressing HCV E2 was used as target cell to detect specific cytotoxic T lymphocytes (CTL) response; adenovirus recombined IL-12 was propagated by 293 cell. HCV E2 recombinant and adenovirus recombined IL-12 were injected into the quadriceps femoris muscles and abdominal cavities of 6-8 weeks old BALB/C mice. Sera were collected at 2, 3, and 4 weeks and detected for antibodies for E2. Spleen cells isolated at 4 weeks were analyzed for specific CTL response.

RESULTSIt was found that expression of IL-12 at an undetectable level did enhance HCV E2 gene immunization-induced CTL activity and there was no effect on its hormonal immune response.

CONCLUSIONUsing adenovirus to express interleukin 12 was helpful for regulation of HCV E2 gene immunization-induced immune response. Combined HCV E2 and IL-12 can render a strong anti-HCV CTL activity and may be of use in the development of HCV gene vaccine in the future.

Adenoviridae ; physiology ; Interleukin-12 ; biosynthesis ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Viral Envelope Proteins ; genetics ; immunology

Animals ; Herpesviridae ; genetics ; metabolism ; Herpesviridae Infections ; virology ; Humans ; Viral Envelope Proteins ; genetics ; metabolism

BACKGROUNDTo clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD.

METHODSThe authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA.

RESULTSThe constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm.

CONCLUSIONSThe authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics

Tegument is the unique structure of a herpesvirion which occupies the space between nucleocapsid and envelope. Accumulating data have indicated that interactions among tegument proteins play a key role in virion morphogenesis. Morphogenesis of gammaherpesviruses including Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) is poorly understood due to the lack of efficient de novo lytic replication in cell culture. Murine gammaherpesvirus-68 (MHV-68) is genetically related to these two human herpesviruses and serves as an effective model to study the lytic replication of gammaherpesviruses. We previously showed that ORF33 of MHV-68 encodes a tegument protein and plays an essential role in virion maturation in the cytoplasm. However, the molecular mechanism of how ORF33 participates in virion morphogenesis has not been elucidated. In this study we demonstrated that ORF38 of MHV-68 is also a tegument protein and is localized to cytoplasmic compartments during both transient transfection and viral infection. Immuno-gold labeling assay showed that ORF38 is only present on virions that have entered the cytoplasmic vesicles, indicating that ORF38 is packaged into virions during secondary envelopment. We further showed that ORF38 co-localizes with ORF33 during viral infection; therefore, the interaction between ORF38 and ORF33 is conserved among herpesviruses. Notably, we found that although ORF33 by itself is distributed in both the nucleus and the cytoplasm, in the presence of ORF38, ORF33 is co-localized to trans-Golgi network (TGN), a site where secondary envelopment takes place.
Animals ; DNA Replication ; genetics ; Gammaherpesvirinae ; genetics ; pathogenicity ; Humans ; Mice ; Viral Envelope Proteins ; genetics ; Viral Matrix Proteins ; genetics ; Virion ; genetics ; Virus Replication ; trans-Golgi Network ; genetics