Capsid Proteins ; genetics ; metabolism ; Simplexvirus ; genetics ; metabolism

The HIV-1 capsid protein (CA) plays an essential role in viral core assembly and maturation. Proteolytic cleavage at the MA-CA junction of the retroviral gag polyprotein refolds the amino-terminal end of capsid into a beta-helix structure that is stabilized by a salt bridge between the protein's processed amino-terminus and a conserved acidic residue. The refolded capsid aminoterminus then creates a new CA-CA interface, allowing assembly of the mature capsid core. Recently, researches focus on assembly of CA in vitro and development of CA vaccine. CA vaccine will provide widely immune protection because CA is comparatively conserved. Experiments demonstrate that fusing as few as four matrix residues onto the amino-terminus of capsid redirects protein assembly from cylinder to spheres in vitro. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. The production of antigens for vaccines in plants indicates that plant-based transgenic expression represents a viable means of producing CA vaccine for the development of HIV vaccine and for use in HIV diagnostic procedures and it has the potential as a safe and cost-effective alternative to traditional production systems.
AIDS Vaccines ; immunology ; Capsid ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; HIV-1 ; genetics ; immunology ; metabolism ; Virion ; genetics ; immunology ; metabolism

OBJECTIVEHuman papillomavirus 6 (HPV 6) causes genital warts, a common sexually transmitted disease. L1-capsids protein is a highly promising vaccine candidate that has entered phase II clinical trial. But the existing methodologies for producing L1-capsids in insect cells is expensive for use in developing countries. As methylotrophic yeast,the Pichia pastoris expression system offers economy,and high expression levels. Over-expression of HPV6-L1 protein in P. pastoris was the purpose of this study.

METHODSThe whole L1 gene with preferred codons for P. pastoris was rebuilt and A-T rich regions were abolished, Cloning into pPIC3.5K,electroporation of KM71, in vivo screen of multiple inserts by G418 resistance, PCR analysis of pichia integrants, BMGY/BMMY are used for induction and expression of L1 proteins.

RESULTSThree clones were found to produce L1 protein which can be identified with Western blot. Compared with L1 protein from E.coli, pichia-produced L1 has some glycosylation. Reacting strongly with MabH6B10.5 in indirect immunofluorescence assay indicated that L1 protein expressed in pichia cell holds its native conformational epitopes which is important for vaccine use. A total 125 microg pure L1 protein could be obtained from 1L cultures through ion-exchange and Ni-NTA chromatography.

CONCLUSIONHPV type 6 L1 protein expressed in Pichia pastoris will facilitate the HPV vaccine development and structure-function study.

Capsid Proteins ; biosynthesis ; genetics ; isolation & purification ; Cloning, Molecular ; Genes ; Papillomaviridae ; genetics ; Pichia ; genetics ; metabolism ; Viral Proteins

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.
Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Recombinant baculovirus containing sigmaC gene of Avian reovirus was constructed using Bac-To-Bac Baculovirus expression system, and recombinant sigmaC protein was expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, sigmaC gene of Avian reovirus was cloned and inserted into donor plasmid pFastBacHTA to obtain recombinant donor plasmid pFsigmaC. Plasmid pFsigmaC was transformed into E. coli DH10Bac for integration into bacmid vector and the recombinant bacmid plasmid BacmidsigmaC was obtained. Recombinant baculovirus rBacsigmaC was obtained by transfection of the sf9 cells with BacmidsigmaC. Western blot and indirect immunofluorescence assay (IFA) were carried and the results showed that the recombinant sigmaC protein with 37 kDa molecular weight was expressed successfully.
Animals ; Baculoviridae ; genetics ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Orthoreovirus, Avian ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Spodoptera ; Transfection

OBJECTIVETo construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells.

METHODSHuman group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection. The expression baculoviruse was constructed by Tn7 and Cre-LoxP recombinant and transfected into BmN cells. The gene expression was determined by detecting 6-His tag fused into VP7 C-terminus, and the assembled VLPs were observed by transmission electron micrography.

RESULTSThree genes of rotavirus were cloned and BmMultiBac was constructed. The genes were expressed and the rotavirus-like particles assembled in BmN cells successfully as verified by ELISA and electron microscope.

CONCLUSIONWe have successfully constructed the recombinant baculovirus co-expressing the 3 structural genes of rotavirus, which provide the basis for producing protein complex containing multiple subunits and investigation of the structure of the macromolecules.

Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; virology ; Capsid Proteins ; genetics ; Cell Line ; Gene Expression ; Genetic Vectors ; Rotavirus ; genetics ; metabolism

The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.
Capsid Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Genetic Vectors ; genetics ; Human papillomavirus 16 ; genetics ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Recombinant gene expression using adeno-associated viruses (AAVs) has become a valuable tool in animal studies, as they mediate safe expression of transduced genes for several months. The liver is a major organ of metabolism, and liver-specific expression of a gene can be an invaluable tool for metabolic studies. AAV-DJ is a recombinant AAV generated by the gene shuffling of various AAV serotypes and shares characteristics of AAV2 and AAV8. AAV-DJ contains a heparin-binding domain in its capsid, which suggests that a heparin column could be used for the purification of the AAV. Given that AAV-DJ has been only recently available, relatively little is known about the optimal preparation/purification and application of AAV-DJ. Here, we present a simple large-scale preparation method that can generate 3×10(13) viral particles for in vivo experiments and demonstrate liver-specific gene expression via systemic injection in mice.
Animals ; Capsid ; Capsid Proteins/*genetics ; Dependovirus/*genetics ; *Gene Expression ; *Genetic Vectors ; Genome, Viral/genetics ; Hep G2 Cells ; Humans ; Liver/*metabolism ; Mice ; Mice, Inbred C57BL

OBJECTIVETo clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.

METHODSGene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.

RESULTSpET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.

CONCLUSIONWe successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.

Blotting, Western ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin M ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Core Proteins ; genetics ; immunology ; metabolism

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*