This study investigated the immunogenicity of type 5 replication deficient recombinant adenovirus comprising the fused receptor binding domain protein(RBD)and capsid protein(N)of the SARS-CoV-2 Omicron strain.The overlap PCR method was used to obtain the RBDgs3N fragment.The plasmid pKAd5ES-RBDgs3N was obtained through homologous recombination,and re-combinant virus was amplifiedand harvested after transfection of HEK293 cells.Cesium chloride density gradient centrifugation was used to purify the recombinant adenovirus,and the viral titer was determined through limited dilution analysis.The fusion protein ex-pression was identified with the western blot(WB)method.Recombinant adenovirus was injected intramuscularly into BALB/c mice,and the specific immune responses of the mice were detected with indirect ELISA and enzyme-linked immunosorbent spot technology.The titer of the recombinant adenovirus pKAd5ES-RBDgs3N virus seed was 1.168×1010 IU/mL.The RBDgs3N fusion protein was suc-cessfully expressed in HEK293 cells.Single dose intramuscular immunization significantly induced production of IgG antibodies against wild type(WT)SARS-CoV-2 virus and variant(Delta and Omicron)RBDs in mice,with antibody titers of 103.677 8,103.878 5,and 104.454 9,respectively.The specific antibody titer against N protein was 104.942 2.The level of IFN-γ secretion for RBD specific cellu-lar immunity was 1 452 SFC/106 cells.The type 5 replication deficient recombinant adenovirus with RBD and N gene fusion constructed in this study elicited high levels of specific antibodies against RBD and N protein and RBD specific cellular immunity in BALB/c mice,thus providing a basis for further evaluation of the recombinant adenovirus as a potential SARS-CoV-2 vaccine.

This study investigated the immunogenicity of type 5 replication deficient recombinant adenovirus comprising the fused receptor binding domain protein(RBD)and capsid protein(N)of the SARS-CoV-2 Omicron strain.The overlap PCR method was used to obtain the RBDgs3N fragment.The plasmid pKAd5ES-RBDgs3N was obtained through homologous recombination,and re-combinant virus was amplifiedand harvested after transfection of HEK293 cells.Cesium chloride density gradient centrifugation was used to purify the recombinant adenovirus,and the viral titer was determined through limited dilution analysis.The fusion protein ex-pression was identified with the western blot(WB)method.Recombinant adenovirus was injected intramuscularly into BALB/c mice,and the specific immune responses of the mice were detected with indirect ELISA and enzyme-linked immunosorbent spot technology.The titer of the recombinant adenovirus pKAd5ES-RBDgs3N virus seed was 1.168×1010 IU/mL.The RBDgs3N fusion protein was suc-cessfully expressed in HEK293 cells.Single dose intramuscular immunization significantly induced production of IgG antibodies against wild type(WT)SARS-CoV-2 virus and variant(Delta and Omicron)RBDs in mice,with antibody titers of 103.677 8,103.878 5,and 104.454 9,respectively.The specific antibody titer against N protein was 104.942 2.The level of IFN-γ secretion for RBD specific cellu-lar immunity was 1 452 SFC/106 cells.The type 5 replication deficient recombinant adenovirus with RBD and N gene fusion constructed in this study elicited high levels of specific antibodies against RBD and N protein and RBD specific cellular immunity in BALB/c mice,thus providing a basis for further evaluation of the recombinant adenovirus as a potential SARS-CoV-2 vaccine.

Objective:To increase secretory expression of SARS-CoV-2 S-EABR protein in 293T cell line by optimizing promoter, PolyA signals, signal peptide and terminal amino acids of intracellular sequences.Methods:First, four PCDNA3.1 (-) eukaryotic vector plasmids (Mb, MS, Ab, AS) containing different combinations of elements (promoter and PolyA signals) were constructed, and the S-EABR-1 target sequence optimized according to human codons was inserted. 293T-cells were transiently transfected. After 48 hours, cell culture supernatants and cell lysates were collected, and the expression level of S protein in supernatant was detected by Western blotting and ELISA. Then, the vector with the best expression element combination was selected, and the target sequences of S-EABR-1 and S-EABR-2 (4 amino acids-HSLP were added to the tail of S-EABR-1) were inserted to compare the expression level of S protein in the supernatant. Finally, based on the combination of the above elements with the best expression effect and the insertion of the target sequence, five vector plasmids (tPA, AZ, IFNα2, HSA, GLUC) were constructed to replace the original signal peptide of SARS-CoV-2 S protein, and the expression level of S protein in the supernatant was compared. At the same time, a computer was used to simulate the molecular docking of the SRP54 subunit and the signal peptide nucleic acid sequence, and the Docking Score was used as the docking evaluation criterion to predict the binding of the two.Results:In 293T cells, the Ab combination vector secreted the highest level of S-EABR, and the yield increased by 125% compared with Mb. Based on the Ab combination vector, the level of S-EABR-2 sequence expression and secretion of S-EABR increased by about 50% compared with S-EABR-1. After further replacement with the HSA signal peptide, the level of S-EABR expression and secretion increased by about 83% compared with the original signal peptide of the S protein. In addition, computer simulation result showed that the docking score between HSA and SRP54 subunit was the highest, at 1 505.861.Conclusions:The secretory expression of codon-optimized S-EABR in 293T cells can be further improved by optimizing eukaryotic expression elements (promoter, terminator and signal peptide) and intracellular sequences. The calculated simulated docking score of the affinity between the signal peptide and the SRP54 subunit is basically consistent with the secreted expression level of S-EABR also provides a design idea and screening strategy for subsequent screening of signal peptides to improve the secreted expression of the target gene.

Objective:To express prokaryotically GII.4 human norovirus (HuNoV) VP2 protein and to prepare polyclonal antibody against VP2.Methods:Design specific primers to amplify the VP2 gene of GII.4 HuNoV, digest and connect to the prokaryotic expression vector pGEX-6P-1, transform the correctly identified recombinant plasmid into BL21 ( DE3) competent cells.Pick out and shake the monoclonal bacteria, and add IPTG to induce recombinant GST-VP2. The fusion protein was expressed, purified by GST affinity chromatography and digested to obtain GII.4 HuNoV VP2 protein. The relative molecular mass (Mr.×10 3) of the purified HuNoV VP2 protein was analyzed by SDS-PAGE. BALB/c mice were immunized with purified GII.4 HuNoV VP2 protein (0.5 mg/ml) to prepare polyclonal antibodies. Results:The VP2 protein of GII.4 HuNoV was successfully expressed and purified, with a relative molecular mass (Mr.×10 3) of about 29; the VP2 polyclonal antibody of GII.4 HuNoV was successfully prepared and its titer was as high as 1∶1 280 000. Western blot and indirect ELISA analysis showed that the polyclonal antibody could specifically bind to the VP2 antigen of GII.4 HuNoV. Conclusions:The purified GII.4 HuNoV VP2 after prokaryotic fusion expression can be used to prepare high titer polyclonal antibody.

Objective:To explore the protective effect and safety of RotaTeq vaccine on children with rotavirus gastroenteritis (RVGE) in high mortality areas in the world and guide the correct use of RotaTeq vaccine.Methods:The literature on RotaTeq vaccine in high mortality areas in the world published from February 2006 to December 2021 was searched, screened and sorted out according to the exclusion and inclusion criteria, and the data were analyzed by RevMan 5.3, Stata 14.0 and SPSS 26.0 softwares.Results:A total of 5 reports were enrolled, including 63 974 subjects, including 32 092 subjects in the vaccine group and 31 882 subjects in the placebo group. In high mortality areas, the protection rates of RotaTeq vaccine against RVGE, severe rotavirus gastroenteritis (SRVGE) and very severe rotavirus gastroenteritis (VSRVGE) were VE RVGE=35% (95% CI: 28%-41%), VE SRVGE=51% (95% CI: 33%-65%) and VE VSRVGE=64% (95% CI: 41%-78%). The protection rates of SRVGE in Asia and Africa are VE SRVGE=43% (95% CI: 28%-55%) and VE SRVGE=57% (95% CI: 17%-77%), respectively. There was no significant difference in the incidences of serious adverse events (SAEs) between RotaTeq vaccine group and placebo group ( χ2=2.05, P=0.152). Conclusions:RotaTeq vaccine has a certain protective effect on severe and above RVGE with good safety in high mortality areas in the world.

OBJECTIVETo investigate the role of P38 mitogen-activated protein kinase (P38 MAPK) signaling pathway in regulating the expression of nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) in the substantia nigra (SN) of a mouse model of Parkinson's disease (PD).

METHODSC57BL/6N mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish an subacute PD model, and the behavioral changes of the mice were observed. Immunohistochemistry and Western blotting were employed to detect the expressions of tyrosine hydroxylase (TH), NF-κB, iNOS and phosphorylated P38 (p-P38) in the midbrain before and after treatment with SB203580.

RESULTSCompared with the control mice, the PD mouse models presented with typical symptoms of PD and showed significantly increased number of p-P38-, NF-κB-, and iNOS-positive cells in the SN area (P<0.01) with significantly reduced number of TH-positive neurons (P<0.01). After SB203580 treatment, the number of p-P38-, NF-κB-, and iNOS-positive cells was reduced obviously (P<0.01) and the number of TH-positive neurons in the SN increased significantly in the PD model mice (P<0.01).

CONCLUSIONP38 MAPK signaling pathway may play an important role in modulating NF-κB and iNOS expression in the SN in the early stage of MPTP-induced subacute PD, and SB203580 can inhibit P38 signaling pathway to protect the DA neurons in PD model mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Animals ; Disease Models, Animal ; Imidazoles ; MAP Kinase Signaling System ; Mice ; Mice, Inbred C57BL ; metabolism ; NF-kappa B ; metabolism ; Neurons ; Nitric Oxide Synthase Type II ; metabolism ; Parkinson Disease ; metabolism ; Phosphorylation ; Pyridines ; Substantia Nigra ; p38 Mitogen-Activated Protein Kinases ; metabolism