Objective To express the capsid(Cap) protein of porcine circovirus 2(PCV2) by insect-baculovirus expression system, evaluate its immunogenicity as a vaccine candidate, and explore whether it can induce humoral and cellular immune responses in BALB/c mice.Methods Following codon optimization, the PCV2 Cap gene was cloned into pFastBac Dual vector and transformed into E.coli DH10 Bac cells. After Bacmid extraction, PCR amplification was carried out with M13 universal primers. The appropriately identified Bacmid was then transfected into Sf9 cells to express Cap protein, and the protein was quantified. Forty-five female BALB/c mice were randomly divided into three groups: PBS control, Cap and commercial vaccine YI Cap group, 15 mice in each group, which were immunized subcutaneously on the back once at 14-day intervals, for a total of three times, with PBS 100 ??L, Cap 10 ??g, and commercial vaccine YI Cap 10 ??g respectively each time. The serum IgG level of mice was measured by ELISA, the cellular immune response type was analyzed by flow cytometry, and the histocompatibility of the subunit vaccine was observed by H&E staining.Results The insect-baculovirus expression system effectively expressed the target protein, and the Cap protein with enhanced green fluorescent protein(eGFP) colocalized with the cell's plasma membrane. Transmission electron microscopy revealed that the Cap protein could self-assemble to produce virus-like particles(VLPs) before constructing another recombinant vector sans the eGFP fluorescent flag. SDS-PAGE and Western blot analysis revealed the conspicuous bands at the desired area. The IgG content of Cap vaccine candidate mice reached the highest level at 42 days. The levels of IFNγ and IL-4 were(27. 75 ± 0. 99) and(17. 63 ± 1. 13) pg/mL, respectively, both of which were significantly higher than those of PBS group(F = 14. 89 and 0. 211 9,P < 0. 05 and < 0. 001, respectively). The stimulation index(SI) of splenic lymphocytes in the Cap group reached 6. 72, which was significantly higher than that in the PBS group(F = 1. 228, P < 0. 001). The Cap vaccine candidate could induce Th1-type cellular immune response and exhibited good histocompatibility.Conclusion The Cap protein was successfully expressed in the insect-baculovirus expression system. The Cap vaccine candidate can induce humoral and cellular immune responses in mice after immunization, which is a potential vaccine candidate for further development of more effective vaccines against PCV-related diseases.

Objective To construct a recombinant vector for Mycobacterium tuberculosis(Mtb) Rv2153c(MurG), express it in prokaryotic cells, and then immunize C57 BL/6 mice to assess the immunogenicity.Methods The recombinant vector pProRv2153c was constructed, expressed in prokaryotic cells and purified. The purified product was identified by SDS-PAGE and Western blot, and the levels of related cytokines in peripheral blood of Chinese Mtb infected patients induced by recombinant Rv2153c protein were measured by multiplexed microsphere-based flow cytometric assay(MFCIA). The female C57 BL/6 mice were randomly divided into PBS, Rv2153c/SAS subunit vaccine, SAS and BCG groups with 6 mice in each group. After 9 weeks of immunization, the levels of specific antibodies in peripheral blood and secreted cytokines in splenocyte supernatant were detected.Results The recombinant vector pPro-Rv2153c was constructed correctly as identified by double enzyme digestion. The expressed Rv2153c protein had a relative molecular mass of about 43 200, and had specific reaction with Monoclonal Mouse Anti-His Tag and MurG Antibody. After stimulation with recombinant Rv2153c protein, the levels of IFNγ, TNF-α and IL-6 in subjects with active pulmonary tuberculosis(ATB) were significantly higher than those in latent tuberculosis infection(LTBI) and healthy control(HC) subjects. The serum specific antibody titers in Rv2153c/SAS group were significantly higher than those in the rest of the groups, and the ratio of IgG2a to IgG1 was greater than 1. In addition, the levels of secreted IFNγ, TNF-α and IL-2 in splenocyte supernatant of Rv2153c/SAS group were also significantly higher than those of other groups.Conclusion The recombinant Rv2153c protein can be specifically recognized by peripheral blood T cells of subjects infected with Mtb in China, and can induce strong antigen-specific Th1-type cellular immune response in the combined adjuvant SAS immunized mice, confirming the possible use of Rv2153c as a candidate target antigen for novel TB vaccine.

Objective To establish influenza A and influenza B virus infection models in mice, systematically evaluate the pathogenic characteristics of different strains in vivo, so as to provide experimental tools for vaccine immunogenicity assessment and antiviral drug screening.Methods SPF-grade BALB/c mice were intranasally inoculated with gradient viral doses of seasonal epidemic virus strains A/H3N2, A/H1N1, B/Vic, and B/Yam circulating in the Northern Hemisphere 2021-2022.The body mass changes, mortality, and clinical symptoms were dynamically monitored for 7 days. Animals were euthanized on days 4 and 7 post-infection. Five lung lobes(left and right) were dissected for viral load quantification by qPCR and histopathological examination to determine the optimal modeling infection dose of each strain. The optimal dose was used for verification test, and the viral loads, pathological changes and inflammatory cytokine levels were measured 7 days after infection.Results Two days after infection with A/H1N1, B/Vic, and B/Yam strains, the body mass of mice began to decrease, and the viral loads in lung tissues reached the peak on the 4 th day after infection, accompanied by moderate interstitial pneumonia characterized by fibrinous exudates in alveolar spaces and inflammatory cell infiltration. The viral loads persisted until day 7 without decline after infection, and the pathological damage persisted. The viral load and pathological changes in different parts of the lung tissue of the same mouse were different, and individual differences were large. No significant body mass fluctuations were observed across all dose groups of A/H3N2 strain, the pulmonary viral loads remained below detection thresholds,and histopathology revealed no influenza-specific lesions, indicating inefficient replication in BALB/c mice. The optimal modeling infection doses of A/H1N1, B/Vic, and B/Yam strains were determined to be 1. 6 × 10~5, 8. 9 × 10~2, and 2. 5 × 10~4 TCID_(50),respectively. Verification experiments showed that 7 days after infection with A/H1N1, B/Vic, and B/Yam strains, mice all experienced body mass loss and corresponding clinical symptoms, and the viral loads in lung tissues were all more than6 lgcopies/??g RNA. Typical pathological changes of interstitial pneumonia were observed, accompanied by increased levels of key inflammatory cytokines such as TNF-α, IL-6, and IFNγ.Conclusion A/H1N1, B/Vic and B/Yam strains can establish stable infection models in BALB/c mice, which are suitable for vaccine efficacy assessments and preliminary drug screening. A/H3N2 strain has not been effectively replicated in mice, and ferret model can be used for related research.

Objective To investigate the biological activity of raddeanin A(RA) against nasopharyngeal carcinoma(NPC)and the molecular mechanism of anti-NPC mediated by ERK/MAPK signaling pathway.Methods CCK-8 assay was used to detect the inhibitory effect of RA on the growth of NPC cells. Bioinformatics was utilized to predict the targets of RA acting on NPC and their associated signaling pathways. The binding affinity between RA and core target was analyzed by molecular docking. Annexin V-FITC/PI, JC-1 staining, flow cytometry, combined with Western blot were used to further investigate the anti-proliferation mechanism of RA in NPC cells.Results RA effectively inhibited the proliferation of NPC cell lines 6-10B and 5-8F, with IC_(50)values of 5. 770 and 5. 068 ??mol/L, respectively. The pharmacological effects were primarily associated with cell apoptosis and the MAPK signaling pathway. The binding affinities between RA and core target proteins, such as MAPK1 and caspase 3, predicted through molecular docking, were less than-5 kcal/mol. RA induced apoptosis and mitochondrial membrane potential changes in 6-10B cells. The expression levels of apoptosis-related proteins, including cleaved PARP, cleaved caspase 3, and cleaved caspase 9, significantly increased(F = 229. 60, 136. 60 and 73. 67, P < 0. 001,< 0. 001 and < 0. 01, respectively). Additionally, the expression of the mitochondrial pathway-related protein Bax was marked-ly upregulated, while Bcl-2 expression was significantly downregulated(F = 47. 42 and 17. 54, P < 0. 001 and P < 0. 05,respectively). Furthermore, the expression levels of ERK/MAPK pathway-related proteins, including p-p90 RSK, p-ERK1/2,and p-MSK1, were significantly reduced(F = 106. 90, 27. 73 and 101. 50, P < 0. 05, < 0. 01 and < 0. 05, respectively).Conclusion RA regulates the ERK/MAPK signaling pathway, reduces mitochondrial membrane potential, triggers mitochondrial pathway to induce apoptosis, and then exerts the activity of inhibiting NPC cell proliferation.

Objective To investigate the antibody levels of measles, rubella and mumps among healthy residents in Fengtai District, Beijing, and to provide reference for further improving the level of immunization coverage.Methods A total of 242 healthy people who had continuously resided in Fengtai District for more than six months were randomly selected. Blood samples were collected for quantitative detection of IgG antibodies against measles, rubella and mumps by indirect ELISA.Results Among the 242 people surveyed, the positive rates of measles, rubella and mumps antibodies were 76. 45%, 89. 67%and 75. 62%, and the geometric mean concentrations(GMCs) were 739. 97 IU/L, 35. 87 IU/mL and 43. 01 RU/mL,respectively. There was no significant difference in the positive rates of measles, rubella, and mumps antibodies between males and females(χ~2= 0. 659, 0. 173 and 0. 046, respectively, P > 0. 05). There was no significant difference in the positive rates of measles, rubella, and mumps antibodies between this city and floating household registrations(χ~2= 1. 859, 0. 045 and 1. 098, respectively, P > 0. 05). However, the positive rates of measles, rubella, and mumps antibodies between different age groups were statistically significantly different(χ~2= 31. 324, χ~2_(fisher)= 28. 650, χ~2= 24. 817, P < 0. 05), and the positive rates of three antibodies were statistically significantly different among different immunization times(χ~2= 14. 315, χ~2_(fisher)=21. 766, χ~2= 17. 764, P < 0. 05).Conclusion The levels of measles, rubella and mumps antibodies among healthy individuals in Fengtai District remain to be further enhanced. In the future, routine vaccination services should be continuously strengthened, and women of childbearing age should be advocated to receive measles-mumps-rubella vaccine, so as to further enhance the level of immunization coverage.

Objective To analyze the current status of influenza vaccination in Liaoning Province from 2014 to 2024, and provide a basis for formulating population influenza prevention and control strategies.Methods The information related to influenza vaccine was collected, and the influenza vaccination data in Liaoning Province were derived through “China Immunization Program Information Management System” and “Liaoning Province Immunization Program Comprehensive Information Platform-Vaccination Information System” to analyze the current situation and characteristics of influenza vaccination from 2014 to 2024.Results From 2014 to 2024, the number of per capita doses of influenza vaccine in Liaoning Province showed an upward trend, reaching 348. 96 doses per 10 000 people in 2023, with a month-on-month increase of119. 40%. The proportion of influenza vaccinations in non-immunization program vaccinations increased from 6. 91% in2018 to 32. 19% in 2023. Starting from 2019, influenza vaccines with different technical routes have been included in the reporting scope. The quadrivalent influenza split vaccine accounted for 67. 40% of the total influenza vaccination of all varieties, and the proportion of vaccination in 2024 was as high as 82. 83%. Regional distribution showed that the number of influenza vaccination doses was the highest in Dalian(1 679 818 doses), followed by Shenyang(1 284 318 doses). The vaccination rate was the highest in Dalian(2. 55%), followed by Benxi(2. 17%) and Shenyang(1. 56%). Influenza vaccination time was mainly concentrated from August to December, accounting for 96. 14% of the annual vaccination times.Conclusion The number of per capita vaccinations of influenza vaccine in Liaoning Province shows a rapid growth trend,which reflects the gradual improvement of public awareness of health and the positive effects of public health interventions.However, there are still low vaccination rates and regional differences. In addition to strengthening publicity and promotion,it is suggested to further improve the overall vaccination rate of influenza vaccine through policy guidance, prevent influenza more efficiently and reduce the burden of diseases.

Objective To verify the double antibody sandwich ELISA method for the detection of residual recombinant trypsin(RT) in monovalent virus harvest fluid and monovalent bulk solution of influenza split virion vaccine(MDCK cells).Methods Double antibody sandwich ELISA was used to detect residual levels of RT in monovalent virus harvest fluid and monovalent bulk solution, and was verified for the limit of quantitation(LOQ), specificity, repeatability, intermediate precision, accuracy and robustness.Results The method exhibited good linearity within the range of 10-0. 156 ng/mL, with a correlation coefficient R~2 of 1. 000. The LOQ was 0. 156 ng/mL. After diluting the monovalent harvest by a factor of two using sample diluent and phosphate buffer, the coefficient of variation(CV) for RT content was 1%, and the RT content measured after diluting monovalent bulk solution two-fold with sample diluent and phosphate buffer was less than 0. 156 ng/mL.The spiked recovery rates of RT solutions at different concentrations ranged from 81% to 105%, with a CV ranging from 1%to 6%. The CV of RT content measured by different test personnel in monovalent harvest was 3%, and the RT content in the monovalent bulk solution measured by different operators was also less than 0. 156 ng/mL. The CV of RT residue content in monovalent harvest at different chromogenic times was 5%, and the residual RT content in the monovalent bulk solution was less than 0. 156 ng/mL, indicating this detection method had good tolerance to different chromogenic times.Conclusion The double antibody sandwich ELISA method for detecting residual RT in monovalent virus harvest solution and monovalent bulk solution of influenza split virion vaccine(MDCK cells) has good linearity and range, LOQ, specificity, repeatability, intermediate precision, accuracy and robustness, and is suitable for the detection of residual RT in influenza split virion vaccine(MDCK cells).

Objective To further optimize and validate the original cell species identification multiplex PCR method to make it applicable to different testing agencies and laboratories.Methods The primer sequences of pigs, Chinese hamsters, African green monkeys and rats, the methods of nucleic acid extraction, the ratios of primers and reference cell mixed genomic DNA, the PCR procedures of amplification and the DNA template dosage were optimized. The sensitivity and cross contamination limits of the optimized method were validated, and reviewed and verified across different laboratories.Results The optimized method was successfully used for cell identification and detection of 10 species, with a sensitivity of 50-5 000 cells and a detection level of cross contamination of 1∶100-1∶10 000. Four different laboratories could use this method to successfully detect cells.Conclusion The optimized cell species identification multiplex PCR method has good specificity and ability to detect cell cross contamination, and can be used for cell species identification in different laboratories with good robustness, which will provide effective technical means for quality control of cells used in production and testing.

Objective To establish an ELISA method for the determination of porcine trypsin residues in reassortant rotavirus vaccine, live, oral, hexavalent(Vero cell) and verify the method, so as to control the quality of the products more comprehensively.Methods The absorbance values of standard and test solutions were determined at 450 nm. The concentration value of standard was used as independent variable X and the mean value of the absorbance value was used as dependent variable Y to establish a four-parameter linear regression equation. The porcine trypsin content of the test solution was calculated using the linear regression equation. The method was verified for linear range and detection limit, accuracy, precision,robustness and specificity. Results Porcine trypsin concentration showed good linearity in the range of 1. 56-100 ng/mL(R~2> 0. 99). The coefficients of variation(CVs) of single virus harvest solution and monovalent bulk solution in repeatability verification was 5% and 8%, and the CVs of single virus harvest solution and monovalent bulk solution in intermediate precision verification was 5% and 6%, respectively. In detection limit verification, the recovery rate was 102%-112% at the porcine trypsin concentration of 1. 56 ng/mL, with the CV of 4% and the quantification limit of 1. 56 ng/mL. In specificity verification, the recovery rates of the test solution diluted with bovine serum albumin(BSA) were 101%-102%, and the recovery rates of the test solution diluted with sucrose-phosphate-glutamate(SPG) buffer and DMEM were 90%-101%,indicating the excipients had no interference with the detection. The CVs of the results at different color development time and temperatures for robustness verification were less than 15%. In accuracy verification, the recovery rates of single virus harvest solution and monovalent bulk solution were within 86%-105% and 109%-117%, respectively.Conclusion The linear range and detection limit, accuracy, precision, robustness and specificity of the established ELISA method were all in line with the acceptance criteria and were suitable for the determination and quality control of the residual amount of porcine trypsin in reassortant rotavirus vaccine, live, oral, hexavalent(Vero cell).

Objective To develop a specific quantitative real-time PCR(qPCR) method for the detection of residual E.coli host DNA in recombinant human hepatocyte growth factor(rhHGF) naked plasmid injection products, and perform verification and preliminary application to provide a reliable basis for the quality control of the drug, and at the same time provide reference for the safety evaluation of similar gene therapy products.Methods The 16 SrRNA gene of E.coli was selected as the target sequence, and specific primers and probes were designed for qPCR to measure residual DNA. The specificity, linearity and range, lower limit of quantification(LLOQ), accuracy, repeatability and intermediate precision of the established method were verified. Additionally, exogenous DNA residues in three batches of rhHGF naked plasmid bulk solutions of injections were tested.Results The method demonstrated excellent linearity within the DNA concentration range of 0. 01-100 pg/??L, R~2= 0. 999, with an LLOQ of 0. 01 pg/??L. No specific amplification was observed for DNA of CHO cells. The recovery rates for high, medium, and low concentration spiked samples were 104. 0%, 105. 1%, and 100. 0%,respectively. The RSDs of repeatability and intermediate precision verification were both less than 30%. The residual exogenous DNA in all three batches of bulk solutions was below 2 ??g/mg plasmids.Conclusion The developed qPCR method exhibits high sensitivity, strong specificity, and reliable accuracy and intermediate precision, which is suitable for detecting residual exogenous DNA in rhHGF naked plasmid injections and other E.coli-expressed biopharmaceutical products.