Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

A large number of studies have demonstrated that mRNA vaccine has been characterized as a technique with good safety, strong immunogenicity and high developmental potential, which makes it have broad prospects in immunotherapy. In recent years, the stability and in vivo delivery efficiency of mRNA vaccines have been largely addressed by the progresses in mRNA engineering and delivery innovation. And some mRNA vaccines are now clinical approved or in preclinical trials. Here, we summarize current knowledge on the research advances, technology, and application in major infectious diseases in humans and animals of mRNA vaccines, with the aim to provide a reference for improving the development of novel mRNA vaccines.
Animals ; Humans ; Communicable Diseases ; Vaccines, Synthetic/genetics* ; mRNA Vaccines

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

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.
Humans ; Animals ; Cattle ; HEK293 Cells ; Insulinoma ; Diabetes Mellitus, Type 1/genetics* ; Recombinant Proteins ; Membrane Proteins ; Pancreatic Neoplasms

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The K_m and k_cat/K_m values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.
Recombinant Proteins/metabolism* ; Paenibacillus/metabolism* ; Mannose/metabolism* ; Escherichia coli/metabolism* ; Mannitol/metabolism*

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×10⁷ U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.
Swine ; Animals ; Cricetinae ; Interferon-gamma/pharmacology* ; Cricetulus ; CHO Cells ; Sincalide ; Recombinant Proteins/pharmacology* ; Antiviral Agents/pharmacology*

In order to understand the prevalence and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) in China and to develop subunit vaccine against the epidemic lineage, the genetic evolution analysis of PRRSV strains isolated in China from 2001 to 2021 was performed. The representative strains of the dominant epidemic lineage were selected to optimize the membrane protein GP5 and M nucleotide sequences, which were used, with the interferon and the Fc region of immunoglobulin, to construct the eukaryotic expression plasmids pCDNA3.4-IFNα-GP5-Fc and pCDNA3.4-IFNα-M-Fc. Subsequently, the recombinant proteins IFNα-GP5-Fc and IFNα-M-Fc were expressed by HEK293T eukaryotic expression system. The two recombinant proteins were mixed with ISA206VG adjuvant to immunize weaned piglets. The humoral immunity level was evaluated by ELISA and neutralization test, and the cellular immunity level was detected by ELISPOT test. The results showed that the NADC30-like lineage was the main epidemic lineage in China in recent years, and the combination of IFNα-GP5-Fc and IFNα-M-Fc could induce high levels of antibody and cellular immunity in piglets. This study may facilitate the preparation of a safer and more effective new PRRSV subunit vaccine.
Humans ; Animals ; Swine ; Porcine respiratory and reproductive syndrome virus/genetics* ; Porcine Reproductive and Respiratory Syndrome/prevention & control* ; HEK293 Cells ; Viral Envelope Proteins/genetics* ; Antibodies, Viral ; Viral Vaccines/genetics* ; Recombinant Proteins ; Vaccines, Subunit

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.
Animals ; Mice ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus ; Antibodies, Neutralizing ; Immunity, Humoral ; Immunization ; Vaccines, Virus-Like Particle ; Antibodies, Viral ; Viral Vaccines

Since the outbreak of corona virus disease 2019 (COVID-19), viral strains have mutated and evolved. Vaccine research is the most direct and effective way to control COVID-19. According to different production mechanisms, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines included inactivated virus vaccine, live attenuated vaccine, mRNA vaccine, DNA vaccine, viral vector vaccine, virus-like particle vaccine and protein subunit vaccine. Among them, viral protein subunit vaccine has a wide application prospect due to its high safety and effectiveness. Viral nucleocapsid protein has high immunogenicity and low variability which could be a new direction for vaccine production. We summarized the current development of vaccine research by reviewing the current progress, vaccine safety and vaccine immune efficiency. It is hoped that the proposed possible development strategies could provide a reference for epidemic prevention work in future.
Humans ; SARS-CoV-2/genetics* ; COVID-19/prevention & control* ; Protein Subunits ; Vaccines, DNA ; Nucleocapsid Proteins

The Sepsis Coagulopathy Asahi Recombinant LE Thrombomodulin (SCARLET) trial has many defects, and thus cannot be the terminator of recombinant thrombomodulin (rTM). On the contrary, it provides sufficient evidence for further research. Based on analysis focusing on the failure of SCARLET and several previous anticoagulant studies, it is most important for new studies to grasp the following two points: (1) The enrolled cases should have sufficient disease severity and a clear standard for disseminated intravascular coagulation; (2) Heparin should not be used in combination with the investigated drugs. Multiple post-hoc analyses show that no combination of heparin will not increase the risk of thromboembolism. In fact, the combination of heparin can mask the true efficacy of the investigated drug. Due to the complexity of sepsis treatment and the limitations of clinical studies, the results of all treatment studies should be repeatedly verified, rather than be determined at one stroke. Some research conclusions contrary to disease physiology, pharmacology and clinical practice may be deceptive, and should be cautious rather than be simply accepted. On the other hand, the dissenting voices in the "consensus" scene are often well discussed by the authors and should be highly valued.
Humans ; Anticoagulants/therapeutic use* ; Thrombomodulin/therapeutic use* ; Blood Coagulation Disorders ; Disseminated Intravascular Coagulation/drug therapy* ; Sepsis/drug therapy* ; Heparin/therapeutic use* ; Recombinant Proteins