Construction of foot-and-mouth disease virus like particles-induced expression vectors and screening of BHK-21 cell pools / 生物工程学报

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.

Evaluation of the humoral immunity in mice induced by foot-and-mouth disease virus-like particles-ZIF-8 complexes with different sizes / 生物工程学报

To further enhance the immune effect of the foot-and-mouth disease (FMD) virus-like particles (VLPs) vaccine, this study prepared FMDV VLPs-zeolitic imidazolate (framework-8, ZIF-8) complexes with different particle sizes. We used a biomimetic mineralization method with Zn2+ and 2-methylimidazole in different concentration ratios to investigate the effect of size on the immunization effect. The results showed that FMDV VLPs-ZIF-8 with three different sizes were successfully prepared, with an approximate size of 70 nm, 100 nm, and 1 000 nm, respectively. Cytotoxicity and animal toxicity tests showed that all three complexes exhibited excellent biological safety. Immunization tests in mice showed that all three complexes enhanced the titers of neutralizing and specific antibodies, and their immune effects improved as the size of the complexes decreased. This study showed that ZIF-8 encapsulation of FMDV VLPs significantly enhanced their immunogenic effect in a size-dependent manner.

Effect of amino acid site modification on stability of foot-and-mouth disease virus-like particles / 生物工程学报

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.

Construction, expression and identification of chimeric foot-and-mouth disease virus-like particles / 生物工程学报

To improve the specific recognition and presentation of virus-like particle (VLPs), and to develop immune-targeted VLPs vaccine, the gene fragment encoding OVA₂₅₇₋₂₆₄ peptide was inserted into the VP3 gene of foot-and-mouth disease virus (FMDV) between the 171th and 172th amino acids (aa) or 173th and 174th aa by reverse PCR. The recombinant proteins were expressed by using Escherichia coli and assembled into chimeric VLP (VLP(OVA)) in vitro after purification. The VLP(OVA) was measured by dynamic light scattering and transmission electron microscopy. The recombinant protein and the assembled VLPs were evaluated by Western blotting, enzyme-linked immunosorbent assay and laser scanning confocal microscopy to confirm the insertion of OVA₂₅₇₋₂₆₄ peptide into VP3 and its location. The results show that insertion of OVA₂₅₇₋₂₆₄ into the 173th and 174th aa of FMDV VP3 did not affect the assembly of VLPs. The VLP(OVA) in size was larger than VLPs, and the OVA₂₅₇₋₂₆₄ peptide was located on the surface of VLP(OVA).

Promotion of self-nucleic acid fragments on the assembly of foot-and-mouth disease virus-like particles / 生物工程学报

The special nucleic acid fragments, 5' untranslated region (5' UTR) and internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV), which interact with the capsid proteins, were selected as scaffolds to investigate the assembly efficiency of foot-and-mouth disease (FMD) virus-like particles (VLPs). The assembled product was characterized by evaluation of particle size, surface potential, gel retardation assay, nuclease digestion experiments, size-exclusion chromatography, transmission electron microscopy and circular dichroism analysis. The results confirmed that the 5' UTR and IRES of FMDV co-assembled with the FMD VLPs and facilitated the assembly efficiency of FMD-VLPs. It demonstrates that the assembly efficiency of 75S particles of VLPs-5'UTR was significantly higher than those of the VLPs (P<0.001) and VLPs-IRES group (P<0.01). Comparatively the assembly efficiency of 12S particles of VLPs-IRES was significantly higher than those of the VLPs (P<0.000 1) and VLPs-5'UTR (P<0.000 1). It showed that the 5' UTR represented more effective in facilitating the assembly of VLPs. This study proposes an optimized strategy for improving the assembly efficiency of VLPs for the development of VLPs vaccine.

Immunological analysis of the swine vesicular disease virus (SVDV) recombinant P1 protein in Guinea pigs / 中国免疫学杂志

Objective:To investigate the immunogeneicity of a subunit vaccine of capsid protein precursor(P1) of swine vesicular diseas(SVD).Methods:In this study,the guinea pigs were immunized with the home-made antigen,T-lymphocyte proliferation response,blocking ELISA and micro-neutralization assay were used to detect the effect of the immunized responses in guinea pigs.Results:The results indicated that a retroviral-based vaccine carrying the capsid protein precursor(P1) of SVD was able to elicit strong SVDV-specific humoral immune responses in guinea pigs.Conclusion:It encourages further work towards the development of a vaccine against SVDV infection.

Expression of survivin in gastric carcinoma and analysis of its correlation with COX-2 / 中国癌症杂志

Purpose:To detect the expression of a novel i nh ibitor gene of apoptosis,survivin,in gastric cancer and in gastric carcinoma MGC -803 cell line,also to analyze its correlation with the expression of COX-2. Methods:In 93 stomach carcinoma tissues and 20 normal gastric tissues , the expression o f survivin and COX-2 were examined by using the streptavidin-biotin peroxidase (SP) method. Results:In contrast to negative expression in normal gastric mucosa,survivin was express ed in 66 of 93(71%) cases of gastric cancer samples,Overexpression of survivin i n gastric carcinoma MGC-803 cell line was also found,There was a relationship b etween survivin gene expression and degrees of differentiation,lymph node metast ases and TNM stages.The expression of survivin was positively correlated with th at of COX-2(liner index of Pearson=0.227 P