The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio-synthesized hCG chimeric peptides (CP) that contain three linear B-cell epitopes (beta5, beta9 and beta8) of beta-hCG subunit together with various foreign 'promiscuous' T-cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B-cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B-cell epitope (betaE) of beta-hCG. Two sets of DNA fragments were chemically synthesized encoding the beta5, beta9 and beta8 epitopes (betaE) 45 approximately 52, 113 approximately 116 or 133 approximately 144 of beta-hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'-terminals of the genes. SDS-PAGE analysis revealed that only Stv-betaE (-beta5, -beta9 or -beta8) fusion genes set with the TAA codon can be expressed in E. coli BL21 (DE3) pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS-4157) generated upon immunization with the loop peptide 38 approximately 57 of beta-hCG, monoclonal antibody (mAb) FB12 to beta9 epitope and mAb OT3A that specially recognizes reporter sequence 133 approximately 139 of beta8 epitope 137 approximately 144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1 L culture. The three target Stv-betaE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.
Chorionic Gonadotropin, beta Subunit, Human ; genetics ; immunology ; Epitopes, B-Lymphocyte ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Streptavidin ; genetics ; Vaccines, Synthetic ; immunology

To prepare carboxyl terminus truncated human papillomavirus type 58L1 protein, and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV58L1 gene. Further more, the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells, constructing recombinant Baculovirus, then the recombinant virus was successfully used to infect Sf-9 insect cells. After incubating at 27 degrees C for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The interested protein was purified by ProBond purification system. The purified interested protein was identified to self-assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination, indicating that the given proteins had the conformation of VLPs, collecting, HPV58L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf-9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58L1, The present study is fundmental for preparing HPV58L1 prophylactic vaccine.
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Cloning, Molecular ; Humans ; Mice ; Mice, Inbred C57BL ; Papillomaviridae ; genetics ; Papillomavirus Vaccines ; immunology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Vaccines, Synthetic ; immunology ; Viral Proteins ; biosynthesis ; genetics ; Virion ; immunology

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.
Animals ; Antigens, Viral/genetics* ; Biological Assay ; Chickens/immunology* ; Escherichia coli/genetics* ; Infectious bursal disease virus/immunology* ; Poultry Diseases ; Vaccines, Synthetic/isolation & purification* ; Viral Structural Proteins/immunology* ; Viral Vaccines/immunology*

OBJECTIVETo investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).

METHODSThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.

RESULTSThe novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.

CONCLUSIONThe chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes ; Genetic Engineering ; Hepatitis Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis E virus ; genetics ; immunology ; Pichia ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Vaccines, Synthetic