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

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

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

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

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

Ebola virus (EBOV) causes outbreaks of a highly lethal hemorrhagic fever in humans and there are no effective therapeutic or prophylactic treatments available. The glycoprotein (GP) of EBOV is a transmembrane envelope protein known to play multiple functions including virus attachment and entry, cell rounding and cytotoxicity, down-regulation of host surface proteins, and enhancement of virus assembly and budding. GP is the primary target of protective immunity and the key target for developing neutralizing antibodies. In this paper, the research progress on genetic structure, pathogenesis and immunogenicity of EBOV GP in the last 5 years is reviewed.
Animals ; Antibodies, Viral ; immunology ; Ebolavirus ; genetics ; immunology ; physiology ; Glycoproteins ; genetics ; immunology ; metabolism ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Viral Envelope Proteins ; genetics ; immunology ; metabolism ; Virus Assembly

We constructed the recombinant adenovirus expressing the glycoprotein G2 of Hantaan virus. Firstly we obtained coding gene fragment of G2 by PCR, and subsequently inserted the gene of interest into the Adenoviral pShuttle vector pAd5-CMV. Then we co-transfected the recombinant pShuttle vector and adenovirus skeleton plasmid into HEK293 cells by Calcium phosphate precipitation method. After the recombinant adenovirus were packaged and amplified in HEK293 cells, we observed the expression of reporter gene eGFP by fluorescent microscopy, and we obtained the recombinant adenovirus containing Hantaan virus glycoprotein G2. The recombinant adenoviruses were used to infect Hela cells and the expressed protein was detected by Indirect Immuno-fluorescence and Western blotting. The construct was confirmed at several levels: first restriction enzyme analysis demonstrated that the recombinant adenovirus vector was constructed correctly, second RT-PCR showed that the G2 gene could transcribe correctly in Hela cells. Then Fluorescent microscopy proved the expression of eGFP in the infected Hela cells. Finally, Indirect Immune-fluorescence and Western-blot confirmed the expression of interested protein identified by anti-G2 monoclonal antibody. In conclusions, this study successfully constructed the recombinant adenovirus containing Hantaan virus envelope glycoprotein G2, meanwhile obtained the G2 protein, it may lay solid foundation for the structure study of G2 protein and the new vaccine of Hantaan virus.
Adenoviridae ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Kidney ; cytology ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics