OBJECTIVEConstruction and recovery of chimeric rabies virus expressing HCV envelop proteins E1E2.

METHODSOn the basis of the previously established reverse genetic system CTN-GFP, HCV E1E2 genes were cloned to both replication competent and replication constrained viral vectors based on CTN181 strain and the chimeric viruses CTN-HCV E1E2 and CTNdeltaG-HCV E1E2 were recovered.

RESULTSThe result demonstrated that both the chimeric viruses were rescued successfully, had the ability to re-infect normal sensitive cell lines and express HCV E1E2 genes detected in the level of mRNA.

CONCLUSIONThe establishment of chimeric RVs expressing HCV E1E2 genes provides the evidence that it is feasible to develop novel HCV vaccines based on viral vectors in theory and in practice.

Animals ; Cell Line ; Cricetinae ; Hepacivirus ; genetics ; metabolism ; Rabies virus ; genetics ; metabolism ; Viral Envelope Proteins ; genetics ; metabolism

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

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

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

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

To character a novel chimera(1b/2a) hepatitis C virus cell culture (HCVcc) system carrying envelope E1E2 coding gene from Hebei strain of China, chimera HCVcc (cHCVcc) was developed from Huh7.5-CD81 cells after transfection with in vitro transcribed full-length 1b/2a chimera RNA, which carrying envelope E1E2 coding gene from Hebei strain of China. Then the replication, expression and infectious titer of serial passage HCVcc were assessed by Real Time RT-PCR, indirect immunofluorescence assay (IFA) and Western blotting (WB). In addition, chimeric envelope gene from HCVcc was sequenced after serial passage. We found that the number of HCV positive focus increased gradually in cell post-transfection with chimera HCVcc (1b/2a) RNA and reach a peak platform (80% to 90%) at 41 days post-transfection; the expression of HCV protein was also confirmed by WAB during serial passage. At meantime, HCV RNA copy number in the supernatant peaked at 10(4)-10(7) copies/mL and the highest infectious titer of this 1b/2a cHCVcc reinfection were tested as 10(4) ffu/mL. Sequence analysis indicated 6 of adaptive amino acid substitutes occur among chimeric envelope E1E2 during serial passages. We con:luded that a novel 1b/2a chimera HCVcc carrying envelope E1E2 coding gene from Hebei strain of China was developed and its infectious titer increased after serial passage of HCVcc. This novel cHCVcc will be an effective tool for further evaluation of anti-virus drugs and immune effects against the major genotype from Chinese.
Cell Line ; China ; Hepacivirus ; genetics ; growth & development ; metabolism ; Hepatitis C ; virology ; Humans ; Serial Passage ; Viral Envelope Proteins ; genetics ; metabolism

OBJECTIVETo expression prM/E gene of dengue virus type I in mammalia cells.

METHODSThe full-length prM/E gene of dengue virus type I strain GZ01/95 was amplified by RT-PCR, the signal peptide preceding the prM gene was added or the carboxyl-terminal 20% of DEN-1 E was replaced with the corresponding JE sequence in the meanwhile, and three of the constructions were cloned into the pcDNA5/FRT.Then they were transfected into 293T cells by lipofectamine respectively. The expression of recombinant proteins were identified by indirect immuno-fluorescence assay(IFA) as well as Western blot.

RESULTSIn the cytoplasm of 293T cells transfected with all the recombinant plasmids DNA, the expressed products for gene of dengue virus type I were confirmed by IFA. The secreted expression products for gene of dengue virus type I specific protein bands were confirmed by Western blot only existing in the cell supernatants transfected with the modified recombinant plasmids DNA.

CONCLUSIONThe prM/E protein of dengue virus type 1 were expressed in 293T cells transfected with all the three recombinant plasmids DNA. The prM/E protein was obtained secretion after transfecting the modified recombinant plasmids adding a signal peptide preceding the prM gene or replacing the carboxyl-terminal 20% of E with the corresponding JE sequence.

Cell Line ; Dengue ; virology ; Dengue Virus ; genetics ; metabolism ; Gene Expression ; Glycoproteins ; genetics ; metabolism ; Humans ; Protein Transport ; Recombinant Fusion Proteins ; genetics ; metabolism ; Viral Envelope Proteins ; genetics ; metabolism

FMDV 2A peptide was introduced as a linker between GP5 and M protein of porcine reproduction and respiratory syndrome virus (PRRSV) to allow automatic self-cleavage the polyproteins. This strategy simultaneously displayed the neutralizing action of GP5 protein and cell-mediated immunity of M protein. We put them into the expression cassette of adenovirus vector. The results of RT-PCR, IFA and Western blotting showed that GP5 and M protein were not only expressed correctly, but also self-cleavaged and assemble heterodimers formation. To detect the advantages of rAd-GP5-2A-M, we also constructed some other recombinant adenoviruses (rAd-GP5, rAd-M and rAd-GP5-M) as control. After inoculated subcutaneously into BALB/c mice, the four recombinant adenoviruses can induce PRRSV-specific antibodies and cell-mediated immune response, but the level of humoral and cell-mediated immune response against PRRSV induced by rAd-GP5-2A-M is the strongest among the four recombinant adenoviruses. All of these suggested that it is possible to develop one multi-gene engineering vaccine utilizing FMDV 2A peptide, and also provided a novel strategy for developing other viral disease vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Swine ; Vaccines, Synthetic ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; metabolism ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; immunology

Animals ; Antigens, CD ; metabolism ; CHO Cells ; Cricetinae ; Cricetulus ; Endocytosis ; Hepacivirus ; genetics ; pathogenicity ; physiology ; Tetraspanin 28 ; Viral Envelope Proteins ; genetics