ABSTRACT
Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Subject(s)
Animals , Foot-and-Mouth Disease Virus/genetics , Capsid Proteins , Viral Proteins/metabolism , Foot-and-Mouth Disease/prevention & control , Tetracyclines/metabolism , Viral Vaccines , Antibodies, Viral , Mammals/metabolismABSTRACT
Objective To evaluate the immunopotentiating effect of cyclic guanosine monophos-phate-adenosine monophosphate (cGAMP) as an adjuvant on norovirus (GⅡ. 4) virus like particles (VLPs) in the development of norovirus vaccine. Methods BALB/c mice were intramuscularly immunized with norovirus (GⅡ.4) VLPs composed of capsid protein VP1 in combination with cGAMP or Al(OH)3. Norovirus VLPs-specific antibodies in serum were detected by ELISA. A synthetic histo-blood group antigen (HBGA)-VLPs blocking assay was used to analyze neutralizing antibodies against norovirus VLPs in serum samples. Results Immunization with norovirus VLPs in the presence of cGAMP induced a strong humoral immune response in BALB/c mice. Levels of specific IgG antibodies in serum induced by using cGAMP as the adjuvant were significantly higher than those induced by using Al(OH)3adjuvant when immunization of BALB/c mice with the same dosage of VLPs. The antibody level induced by 1 μg of VLPs in combination with cGAMP was equivalent to that elicited by 10 μg of VLPs combined with Al(OH)3adjuvant. Results of the synthetic HBGA-VLPs blocking assay showed that the blocking rate in cGAMP+VLPs immunization group were significantly higher than that in Al(OH)3+VLPs immunization group when using the same dosage of VLPs. No significant difference in blocking rate was observed between cGAMP+VLPs(1 μg) and Al(OH)3+VLPs (10 μg) immunization groups. Conclusion cGAMP significantly enhanced the specific humoral immune response induced by norovirus (GⅡ.4) VLPs in mice as compared with Al(OH)3adjuvant. It might be used as a novel adjuvant to replace the traditional aluminum adjuvant in the development of norovir-us vaccine.
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Objective To evaluate the immune effects of virus-like particles ( VLPs) of VP1 pro-teins derived from norovirus GⅠ. 1 and GⅡ. 4 genotypes expressed in Hansenula polymorpha expression sys-tem. Methods SDS-PAGE and Western blot assay were performed to detect the purity of GⅠ. 1 and GⅡ. 4 VP1 proteins after purification. Morphologies of the recombinant VLPs were observed under transmission electron microscopy ( TEM) . Sizes and distributions of the VLPs were analyzed by dynamic light scattering analyzer. BT50(50% of blocking titer) was detected by HBGA (histo-blood group antigen) blocking assay in BALB/c mice immunized with different regimens. Results SDS-PAGE analysis of the purified recombinant GⅠ. 1 and GⅡ. 4 VP1 proteins showed that their purity were greater than 90%. Western blot assay con-firmed the specific bands of VLPs. TEM images showed that the sizes of purified GⅠ. 1 and GⅡ. 4 VP1 VLPs were at a mean diameter of 30-50 nm with clear border and high homogeneity, which was similar to that of wild virus. BT50 significantly increased in the groups, in which Al( OH) 3 was used as adjuvant. Con-clusion Animal studies have shown that administration of GⅠ. 1 and GⅡ. 4 VP1 VLPs in the presence of Al( OH) 3 induces detectable HBGA-blocking antibody, indicating that GⅠ. 1 and GⅡ. 4 VP1 VLPs are promising candidates for norovirus vaccine.
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Objective:To study the effect of alum on immune response in mice induced by HBoV1 VP2 VLPs.Methods:BABL/c mice were randomly divided into VLPs experimental group, alum adjuvant experimental group, PBS control group and alum adjuvant control group,the experimental group mice were intramuscular immunization with HBoV1 VP2 VLPs and HBoV1 VP2 VLPs added alum,control group mice were immunization with alum or PBS buffer,then to study the effect of alum on immune response in mice induced by HBoV1 VP2 VLPs by cellular and humoral immune strength.Results: Alum adjuvant decreased cellular immune response induced by HBoV1 VP2 VLPs(P<0.001),enhance the HBoV1 VP2 VLPs immuned serum IgG titer(P<0.05)and IgG activity(P<0.01).Conclusion:Alum adjuvant can enhance humoral immune response induced by HBoV1 VP2 VLPs,but weaken cellular immune response induced by HBoV1 VP2 VLPs,when HBoV1 VP2 VLPs used as a prophylactic vaccine it should add alum adjuvant,when used as a therapeutic vaccine,it should not add alum adjuvant.
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Objective To express and characterize the virus-like particles( VLPs) of H5 subtype containing of hemagglutinin ( HA ) and matrix 1 ( M1 ) protein by using Baculovirus-insect cells .Methods Full length genes encoding HA protein from the A/Indonesia/05/2005(H5N1) strain and the M1 protein from the A/Anhui/01/2005 ( H5N1 ) strain were cloned into a baculovirus expression vector to construct pFBD-M1-HA.The expression of HA and M1 proteins were detected by Western blot and indirect immunoflu-orescence after the transfection of Spodoptra frugiperda (Sf9) insect cells with recombinant baculovirus.Pu-rified VLPs were analyzed by SDS-PAGE and visualized with transmission electron microscope.The biologi-cal activity of purified VLPs was detected by hemagglutination test.Results The HA and M1 proteins of H5 subtype expressed by baculovirus-insect cells could be self-assembled into the functional mature VLPs.The hemagglutination titer of VLPs was as high as 1024 HAU/50μl.Conclusion The H5 subtype VLPs as pre-pared in this study would pave a way for the development of a candidate recombinant A ( H5) vaccine.
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The amplified VP2 gene (PPV strain SC-1) and PCV20RF2 gene were inserted into the eukaryotic expression vector pPI-2.EGFP.Then the recombinant plasmid named pPI-2.EGFP.VP2.ORF2 was obtained.Mediated by liposome,the recombinant plasmid was transfected into Veto cells and expressed.Using immunofluorescence assay,the fluorescence of expression products were first detected at 20 h after transfection and peaked 36 h.Under electronmicroscope,virus-like particles (VLPs) can be observed in the transfected cells.To confirm the obtained VLPs to be recombinant particles,piglets were immunized using purified VLPs.The dynamic variation of blood T lymphocytes and serum antibody level of PPV and PCV2 were measured.The results showed that the ratio of CD3+,CD4+ T lymphocyte in peripheral blood lymphocyte of immunized piglets raised in a certain degree,the number of CD8+ T lymphocytes fell at 7-14 d after immunization,and then raised.Relatively high level of PP-V,PCV2 specific antibody could be detected.This indicated that the expression of recombinant plasmid pPI-2.EGFP.VP2.ORF2 was successful,the virus-like particles were formed and showed favourable immunogenicity.
ABSTRACT
HIV p24 core protein can induce both cellular and neutralizing antibody responses.HIV-1 CA-virus-like particles(VLPs)vaccines provide a promising approach for the development of an effective vaccination strategy against HIV infection.Rhizosecreion of the recombinant proteins provides a new manufacturing platform that can simplify the extraction and purification procedure.Lycium barbarum L.was transformed by Agrobacterium tumefaciens EHA105 harboring the plant expression vector pCAMBIA1305.2-MA4-CA with a GRP signal peptide and MA4-CA fusion gene.Transgenic hairy roots were induced and cultivated in hydroponic culture.Western blotting indicated that the recombinant CA proteins were present in two forms,a glycosylated monomer(37 kDa)and a dimer(50 kDa)in the roots and hydroponic medium.It appeared from the present immunohistochemical data that the recombinant CA proteins fused with GRP signal peptide were confined to the cytoplasm,cell wall and intercellular space,indicating targeting into the secretory pathway.It demonstrated for the first time the rhizosecretion of HIV-1 recombinant capsid protein in Lycium barbarum L.hairy roots,and may offer a novel method for expressing HIV-1 CA-VLPs vaccines in plants.