The artificial 5-helix can inhibit the formation of trimer-of-hairpins structure during the course of HIV-directed membrane fusion and then inhibit human immunodeficiency virus (HIV) infecting target cells. But 5-helix was apt to form inclusion body when expressed directly in prokaryotic cell and was difficult to renature, which causes inconvenience to future study. We found a proper expression vector by simulating protein structure. We simulated its proper conformation in two vectors pGEX-6P-1 and pET44b by homology modeling. The contrast of conformations showed that the energy of salvation of its fusion protein with NusA in vector pET44b was higher than its fusion protein with glutathione-S-transferase (GST) in pGEX-6P-1 and its restriction site lay on the surface of its fusion protein in vector pET44b. 5-helix gene was amplified from pGEX-6P-1-5H by PCR, and was ligated to pET44b to construct recombinant vector pET44b-PSP-5Helix after tested correctly by enzymes digestion. The recombinant vector was transformed into Escherichia coli BL21 (DE3) to express 5-helix protein at different temperatures. Aim protein was purified with Ni column and GST column, and was determined by SDS-PAGE. Then the purified 5 -Helix was used to test the inhibitive activity of pseudo HIV virus infecting GHOST-CXCR4. Results show that its fusion protein with NusA can be effectively soluble expressed and easier to be cleaved, and that the purified 5-helix can efficiently inhibit pseudo HIV virus infecting GHOST-CXCR4 and its IC50 value is (22.77 +/- 5.64) nmol/L, which lay the foundation to further discuss the application in HIV-1 infection.
Carrier Proteins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HIV Envelope Protein gp41 ; metabolism ; HIV-1 ; genetics ; Peptides ; genetics ; Repetitive Sequences, Nucleic Acid ; genetics ; Viral Fusion Proteins ; genetics ; Virus Internalization ; drug effects

Amino Acid Sequence ; Coronavirus/drug effects* ; Coronavirus Infections/drug therapy* ; Drug Design ; Humans ; Protein Structure, Secondary ; Spike Glycoprotein, Coronavirus/metabolism* ; Viral Fusion Protein Inhibitors/pharmacology* ; Viral Fusion Proteins/metabolism*

OBJECTIVETo construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.

METHODSS2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.

RESULTSThe pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.

CONCLUSIONSThe expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

Escherichia coli ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; Membrane Fusion ; drug effects ; Membrane Fusion Proteins ; biosynthesis ; isolation & purification ; Membrane Glycoproteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; SARS Virus ; genetics ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; biosynthesis ; genetics

Based on the genomic sequence of SARS-CoV strain BJ101, antigenic immunodominant genes coding for the structure proteins of SARS-CoV were predicted by bio-informatics methods, and two chimeric genes A and B with multi-immunodominants lined up by Gly-Pro-Gly linker were synthesized. The chimeric genes were cloned into plasmid pGEX-6p-1 and expressed in E. coli with IPGT inducing. BALB/c mice were immunized with the purified recombinant fusion protein. The specificity of monoclonal antibodies were tested with a commercial ELISA kit for detecting antibody against SARS-CoV. The results showed that two peptides with molecular weights of 34kD and 35kD expressed by the two chimeric genes could be recognized by SARS patient convalescent serum in Western blot. Six positive hybridoma cell lines stably secreting monoclonal antibodies were selected. The subtype of monoclonal antibody D3C5 is IgG2a, and subtypes of all other five monoclonal antibodies are IgG1. Light chains of all monoclonal antibodies are kappa. With a commercial SARS-CoV antibodies detection ELISA kit, five out of six monoclonal antibodies were positively recognized. In western blot analysis with inactived virus cultures, D3D1 specifically recognized a band of about 180 kD. To further analyse the epitopes corresponding to the monoclonal antibodies, six oligoes (S1-S6) from S gene were synthesized and expressed. The results showed that the monoclonal antibodies D3D1 and D3C5 specifically recognized expression product of S2 and S5 oligoes, respectively. The S2 and S5 oligoes are corresponding to 447-458aa and 789-799aa of SARS-CoV S protein respectively.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Epitopes ; genetics ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Hybridomas ; secretion ; Membrane Glycoproteins ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; immunology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; immunology

Escherichia coli ; genetics ; Humans ; Recombinant Fusion Proteins ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.
Animals ; Antibodies, Viral/blood ; *Chickens ; Chimera/genetics/immunology ; Coronavirus Infections/prevention & control/*veterinary/virology ; Female ; *Immunity, Innate ; Infectious bronchitis virus/genetics/*immunology ; Influenza A Virus, H5N1 Subtype/genetics/immunology ; Injections, Intramuscular/veterinary ; Mice ; Mice, Inbred BALB C ; Neuraminidase/genetics ; Poultry Diseases/*prevention & control/virology ; Recombinant Fusion Proteins/genetics/immunology ; Spike Glycoprotein, Coronavirus/genetics/*immunology ; Vaccines, Synthetic/administration & dosage/genetics/immunology ; Vaccines, Virus-Like Particle/administration & dosage/genetics/*immunology ; Viral Proteins/genetics

Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.
Membrane Fusion ; genetics ; Mumps virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Repetitive Sequences, Amino Acid ; Viral Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.
Cricetinae ; Humans ; Animals ; Mice ; Immunoglobulin M/genetics* ; Antibodies, Viral ; CHO Cells ; Cricetulus ; Hybridomas ; Recombinant Fusion Proteins

Cloning, Molecular ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

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