Objective To compare the production of Coxsackievirus A10(CV-A10) in Vero cells cultured in WAVE bioreactor, Basket bioreactor and NBS bioreactor.Methods Vero cells were cultured in 2 L WAVE bioreactor, 15 L Basket bioreactor and 40 L NBS bioreactor filled with pre-irradiated fibracel disk, fibracel disk and Cytodex 1 microcarriers, respectively,to prepare CV-A10. The adherence efficiency, nutrient metabolites and growth characteristics of Vero cells cultured in different types of bioreactors, as well as the virus titers and antigen yields of the prepared CV-A10, were compared. Then, the CVA10 production process in a 2 L WAVE bioreactor was scaled-up to a 50 L WAVE bioreactor, and the virus titers and antigen yields of CV-A10 prepared in different scale WAVE bioreactors were compared.Results The adherence efficiency of Vero cells on fibracel disk was much higher than that on microcarriers, and the content variation curves of glucose and lactate in different types of bioreactors were consistent. The WAVE bioreactor, Basket bioreactor and NBS bioreactor could all be used for high density culture of Vero cells, while the WAVE bioreactor and Basket bioreactor with fibracel disk could achieve much higher cell density than the NBS bioreactor with Cytodex 1 microcarriers. CV-A10 could be efficiently prepared in different types of bioreactors and there was no significant difference in the virus titers and antigen yields of the prepared CV-A10. Vero cells and CV-A10 were successfully scaled up in the 50 L WAVE bioreactor, and there was no significant difference in the virus titers and antigen yields of different scale WAVE bioreactors.Conclusion The WAVE bioreactor, Basket bioreactor and NBS bioreactor can all be used for the stable culture of Vero cells and the efficient preparation of CV-A10.

Objective To evaluate the immune effect of group C meningococcal polysaccharide(GCMP) conjugates using protein D(PD) of Haemophilus influenzae type b(Hib) as vector, in order to determine the feasibility of PD protein as GCMP protein vector.Methods The recombinant PD and tetanus toxin(TT) were conjugated to GCMP by 1-cyano-4-dimethylaminopyridinium tetrafluoroborate(CDAP) activation method to prepare conjugates respectively. The female BALB/c mice were subcutaneously immunized with GCMP and GCMP conjugates, which were divided into GCMP, GCMP-PD, PD, GCMP-TT,TT and negative control group(normal saline), with 12 mice in each group for 3 times of immunization. The blood samples were collected from the rim of eye before each booster immunization and 7 d after the last immunization, and the sera were separated for the detection of antibody levels and serum bactericidal assay(SBA). In addition, the spleen samples were taken7 d after the last immunization for the detection of cellular immune levels.Results The conjugates of protein and GCMP enhanced the level of anti-GCMP IgG. After the third immunization, serum anti-GCMP IgG levels in GCMP-PD and GCMP-TT groups were significantly higher than those in GCMP group(F = 5. 294 and 14. 917, respectively, each P < 0. 05), and the immune memory was produced to induce IgG1, while the IgG1 levels in GCMP-PD group were lower than those in GCMP-TT group(F = 5. 828, P < 0. 05). The levels of anti-PD IgG in serum of GCMP-PD and PD groups increased significantly after each immunization, and the levels of anti-PD IgG in GCMP-PD group were significantly higher than those in PD group after the third immunization(F = 15. 426, P < 0. 01), while those in GCMP-TT group were lower than those in TT group(F =211. 207, P < 0. 001). The results of SBA showed that the titers of bactericidal antibody in GCMP-PD group were higher than those in GCMP group(F = 11. 797, P < 0. 05), while lower than those in GCMP-TT group(F = 8. 688, P < 0. 05). The results of flow cytometry showed that the ratio of Th1 and Th2 cell subsets in GCMP-PD group had no significant difference from that in GCMP and GCMP-TT groups(F = 0. 073 and 3. 021, respectively, each P > 0. 05).Conclusion The conjugation of recombinant PD with GCMP can not only enhance the immunogenicity of polysaccharide, improve the humoral immune response, but also trigger the cellular immune response. The bactericidal effect of the immunized serum is remarkable, suggesting that PD is feasible as GCMP protein carrier.

Objective To observe the effect of hypoxia on Sertoli cells, and explore the mechanism of hypoxia inducible factor-1α(HIF-1α) on apoptosis of Sertoli cells in testes of rats under hypoxia through VEGF/PI3K/Akt pathway.Methods Sertoli cells of rat testes were isolated and cultured, and the cells were divided into four groups: group C(normal culture without any treatment); group V(hypoxia culture for 48 h); group H(infected with adenovirus silencing HIF-1α + hypoxia culture for 48 h); NC group(infected with empty adenovirus vector + hypoxia culture for 48 h). The protein expressions of HIF-1α, VEGF, p-Akt, Akt, p-p70S6K, p70S6K, Bax and Bcl-2 were detected by Western blot. The expression and localization of Bax protein in cells were measured by immunofluorescence assay. The apoptosis rate was analyzed by flow cytometry.Results Western blot results showed that compared with group C and H, the protein expressions of HIF-1α, VEGF, p-Akt, pp70S6K and Bax in group V and NC significantly increased(F = 11. 71, 20. 34, 10. 28, 6. 096 and 18. 55, respectively, each P < 0. 05), the protein expression of Bcl-2 significantly decreased(F = 12. 92, P < 0. 05). The expression of Akt and p70S6K proteins showed no significant difference among the four groups( F = 0. 572 7 and 0. 156 9, respectively, each P > 0. 05).Immunofluorescence results showed that Bax was mainly expressed in the cytoplasm. Compared with group C and H, the expression of Bax protein in group V and NC significantly increased(F = 64. 83, P < 0. 05). The results of flow cytometry showed that compared with group C and H, the apoptosis rates of group V and NC significantly increased(F = 9. 649, P <0. 05).Conclusion Hypoxia-induced apoptosis of Sertoli cells may be caused by the activation of VEGF/PI3K/Akt signaling pathway by the increased expression of HIF-1α.

Objective To explore the separation and purification efficiency of domestic and imported agarose gel filtration chromatography media for the inactivated SARS-CoV-2 Omicron vaccine, in order to provide data support for promoting the domestic substitution of chromatography media.Methods Three batches of concentrated SARS-CoV-2 Omicron strain inactivated virus were used in the experiments, and the purification was conducted using domestic and imported agarose gel filtration chromatography media under identical experimental conditions respectively. The virus peaks were collected, and relevant quality parameters, including antigen content, protein content, residual DNA of Vero cells, residual host proteins of Vero cells, and residual bovine serum albumin content in the collected components, were measured. The specific activity and antigen recovery rates were calculated.Results After loading and purification using a 10% column volume and a linear flow rate of 30 cm/h for both media, the average antigen recovery rates for the collected components were 55. 29% and 53. 31%,respectively, with no significant difference(t = 1. 44, P > 0. 05). Moreover, there was no significant difference in specific activity, residual DNA of Vero cells and residual host proteins of Vero cells(t =-0. 25, 1. 31 and 0. 66, respectively, each P >0. 05). Only the residual bovine serum albumin content exhibited a significant difference(t =-2. 73, P < 0. 05).Conclusion The domestic and imported agarose gel filtration chromatography media were used to purify the concentrated solution of SARS-CoV-2 Omicron strain inactivated virus, and the quality parameters of the collected components had no significant difference except for the residual bovine serum albumin content. Therefore, in the purification of the inactivated SARS-CoV-2 Omicron vaccine, the domestic chromatography media could be considered as a viable alternative to imported media

Objective To screen the nanobody against Helicobacter pylori(H.pylori) urease based on the nanobody library,analyze the structure and binding site, and to identify its inhibitory effect on the activity of urease. Methods Through the display library of natural alpaca phage antibody, using urease B subunit as the target antigen, the nanobody against H.pylori urease was obtained by four rounds of washing and positive clone screening. The nanobody structure and the space binding region with urease were analyzed by structure prediction software, and the inhibitory effect of urease nanobody on urease activity was determined. Results Three nanobodies against H.pylori urease B subunit were obtained. The sequence homology of the three nanobodies was 78. 43%, and the variable regions were all hydrophobic amino acids, containing more β folds and Loop regions. Closer and wider contact was formed at the same site in the 3D models, which was formed with the sites of 301-312,371-385, 440-460 and 550-570 of urease B. When combined with urease B, all of the three nanobodies formed steric hindrance, which interfered with the formation of the dimer of urease A and B subunits, thus inhibiting the function of urease.Conclusion The obtained high affinity anti-urease nanobodies can inhibit the urease activity, which provides an experimental basis for the subsequent preparation of H.pylori urease diagnostic antibody and the research for new therapeutic targets.

Objective To develop a national reference for nucleic acid detection reagents of Yersinia pestis, so as to use it for the evaluation and quality control of Yersinia pestis nucleic acid detection reagents. Methods Five strains of Yersinia pestis[(EV strain, 0614F strain, otten strain, Tjiusidej(R) strain and Microtus vole 201 strain)] and 10 strains of negative bacteria(3 Yersinia enterocolitica strains, Brucella bovis 104M strain, Bacillus anthracis, Salmonella typhi, Francis tulage LVS strain,Yersinia pseudotuberculosis, Vibrio cholerae and Shigella) were cultured, harvested and inactivated. The stock solution of the reference was obtained and verified by ordinary PCR amplification, and a set of national references composed of 5 positive references, 10 negative references, 1 reference of minimum detection limit(EV strain positive reference was prepared by freeze-drying) and 1 repetitive reference(the same preparation method as the minimum detection limit reference) was prepared. Fluorescence quantitative PCR was used to detect the coincidence rate, uniformity and stability of the national reference products, and four laboratories were organized for collaborative calibration. Results After ordinary PCR amplification, the corresponding target gene bands were found in the stock solution of all the positive references, and no corresponding gene bands were amplified in the stock solution of negative references. The coincidence rates of the national references detected by fluorescence quantitative PCR were 100%. There was no significant difference in Ct values of 3a gene in 10 minimum detection limit references in three repeated detections(F = 1. 567, P = 0. 193). Compared with the references without freezing and thawing, there was no significant difference in Ct values of 3a gene of the minimum detection limit references and positive references after freezing and thawing for three times(t = 0. 416 and 0. 079, respectively, each P > 0. 05). Compared with the references without high temperature preservation, the Ct value of the minimum detection limit reference stored at37 ℃ for 5 d was significantly different(t = 7. 109, P = 0. 002), while there was no significant difference at 4 and 25 ℃(t = 0. 341and 0. 751, respectively, each P > 0. 05). The Ct values of positive references had no significant difference after preservation at 4, 25 and 37 ℃ for 5 d(t = 2. 442, 0. 373 and-0. 043, respectively, each P > 0. 05). The coincidence rates of positive and negative references in four laboratories were all 100%. The minimum detection limit of the minimum detection limit reference detected by two laboratories was 1 × 10~2/mL, and that by the other two laboratories was 1 × 10~3/mL. The CVs of the repetitive reference detection results in four laboratories were all less than 10. 0%. Conclusion This set of national reference for Yersinia pestis nucleic acid detection reagents developed in this study has good uniformity and freeze-thaw stability, and has good accelerated stability at 4 and 25 ℃, which can be used for the evaluation and quality control of Yersinia pestis nucleic acid detection reagents.

Objective To investigate the effect of Cynanoside H on the proliferation and metastasis of triple negative breast cancer(TNBC) MDA-MB-231 cells and its mechanism, so as to provide an experimental basis for the development of TNBC therapeutics.Methods MDA-MB-231 cells were randomly divided into one control group(without Cynanoside H) and three test groups including 2. 5, 5 and 10 μmol/L Cynanoside H dose groups. The effect of Cynanoside H on the proliferation of MDA-MB-231 cells was detected by MTT assay and clone formation assay, while the effect on the cell cycle was detected by flow cytometry, and the effect on the metastasis of MDA-MB-231 cells was measured by wound healing assay, Transwell migration and invasion assay. In addition, the effects of Cynanoside H on the expression of cell cycle(c-Myc, CDK1, CDK2and cyclin E1), metastasis(E-cadherin, Vimentin and β-catenin) and PDGFRB/JAK2/STAT3 pathway related proteins(PDGFRB, p-JAK2, JAK2, p-STAT3 and STAT3) in MDA-MB-231 cells were determined by Western blot.Results The levels of cell viability(t = 4. 598, 19. 77 and 53. 43, respectively, each P < 0. 05), growth(36 h: t = 8. 256, 11. 57 and12. 16, respectively, each P < 0. 05; 48 h: t =10. 49,22. 49 and 19. 63, respectively, each P < 0. 01; 72 h: t = 50. 20, 28. 84and 15. 83, respectively, each P < 0. 01; 96 h: t = 11. 18, 18. 35 and 20. 92, respectively, each P < 0. 01) and clone formation(t = 4. 618, 9. 821 and 12. 14, respectively, each P < 0. 05) of 2. 5, 5 and 10 μmol/L dose groups were significantly lower than those of the control group. Compared with the control group, the proportion of cells in S phase of three test group increased in a concentration-dependent manner(48 h: t = 6. 316, 8. 156 and 14. 11, respectively, each P < 0. 05; 72 h: t =7. 231, 15. 36 and 25. 16, respectively, each P < 0. 05), and the expression of c-Myc(5 and 10 μmol/L:t = 10. 39 and12. 18, P < 0. 05 and < 0. 01, respectively), CDK1(t = 3. 777, 5. 069 and 6. 974, respectively, each P < 0. 05), CDK2(5 and10 μmol/L:t = 12. 72 and 19. 43, respectively, each P < 0. 01) and cyclinE1(t = 3. 813 and 15. 23, respectively, each P <0. 01) was down-regulated at the protein level. Compared with the control group, each test group significantly inhibited wound healing(48 h: t = 6. 969, 56. 16 and 27. 73, respectively, each P < 0. 05; 72 h: t = 8. 619, 22. 12 and 32. 15, respectively, each P < 0. 05), migration(t = 9. 817, 14. 74 and 19. 39, respectively, each P < 0. 01) and invasion(t = 5. 614, 13. 85and 14. 22, respectively, each P < 0. 01) of MDA-MB-231 cells, and up-regulated the expression of E-cadherin(10 μmol/L:t = 11. 79, P < 0. 01), down-regulated the expression of Vimentin(5 and 10 μmol/L:t = 12. 05 and 13. 02, respectively,each P < 0. 01) and β-catenin(5 and 10 μmol/L:t = 4. 516 and 9. 305, respectively, each P < 0. 01) at the protein level. In addition, the test groups of Cynanoside H could significantly down-regulate the protein expression of PDGFRB(5 and10 μmol/L:t = 7. 083 and 18. 87, respectively, each P < 0. 01) and inhibit the protein levels of p-JAK2(t = 4. 050, 10. 95and 11. 05, respectively, each P < 0. 05) and p-STAT3(5 and 10 μmol/L:t = 15. 25 and 25. 89, respectively, each P <0. 01), but had no effect on the total protein levels of JAK2 and STAT3.Conclusion Cynanoside H inhibits the proliferation of breast cancer MDA-MB-231 cells by inducing S-phase cell cycle arrest and reverses the epithelial-mesenchymal transition(EMT) to inhibit cell metastasis. These effects may be mediated by down-regulating PDGFRB/JAK2/STAT3 signaling pathway.

Objective To analyse the prevalence of Coxsackievirus A6(CV-A6) causing hand, foot and mouth disease(HFMD) and the genetic characteristics of its VP1 region in Changchun area from 2019 to 2022, explore its epidemic regularity, and provide a reference for optimizing the prevention and control strategies.Methods A total of 878 samples of clinically diagnosed HFMD cases in Changchun from 2019 to 2022 were collected, and real-time RT-PCR was used for enterovirus(EV) nucleic acid detection. The VP1 gene region of CV-A6-positive samples were amplified by RT-PCR and sequenced. A phylogenetic tree was then constructed using MEGA 11, and DNAstarV8.1.3 software was used to analyze the nucleotide homology and amino acid mutations.Results Among the 878 HFMD samples, 588 EV positive were detected,with the total detection rate of 66. 97%(588/878); 245 CV-A6 positive were detected, with the CV-A6 detection rate of41. 67%(245/588). The VP1 gene was successfully amplified and sequenced from 125 CV-A6 positive samples. Phylogenetic analysis showed that all 125 CV-A6 strains detected in Changchun from 2019 to 2022 belonged to the D3a genotype.The nucleotide sequence homology among the 125 CV-A6 strains was 88. 1% to 100%, and the homology with the prototype strain(Gdula) was 81. 2% to 84. 3%. There were 36 site mutations in the amino acid sequence of VP1 region.Conclusion The dominant pathogen causing HFMD in Changchun from 2019 to 2022 was CV-A6, which was also the mainstream gene subtype D3a in the mainland of China. However, multiple transmission chains are circulating and evolving together, suggesting that the surveillance for CV-A6 should be further strengthened.

Objective To evaluate the effect of pre-exercise on immune response to influenza vaccine(InfV) in healthy adults by Meta-analysis, so as to provide evidence-based basis for the study of "exercise adjuvant" of InfV immune response.Methods PubMed, Cochrane Library, EMbase, Web of Science database, CNKI, Wanfang Data Knowledge Service Platform(Wanfang), and SinoMed were searched to screen the research literature of randomized controlled trials(RCTs) about the effect of pre-exercise on immune response to InfV in adults. Cochrane bias risk assessment tool was used to evaluate literature quality, and Stata 15.0 software was used for Meta-analysis.Results A total of 21 RCT studies on the effect of pre-exercise intervention on immune response to InfV were included, with 702 subjects in the experimental group and 698 subjects in the control group. There was a low probability of risk bias in the included studies. Meta-analysis showed that, compared with the control group, pre-exercise significantly increased the antibody titer and serum protection rate [OR = 1. 38(95% CI: 1. 20-1. 56), P < 0. 05] of influenza virus A H1N1 [MD = 0. 47(95% CI: 0. 36-0. 58), P < 0. 05], influenza virus A H3N2 [MD =0. 34(95% CI:0. 21-0. 47), P < 0. 05], and influenza virus B [MD = 0. 34(95% CI: 0. 22-0. 46), P < 0. 05] in adults after InfV vaccination. The results of subgroup analysis showed that chronic pre-exercise significantly increased the influenza virus antibody titer and serum protection rate [OR = 1. 42(95% CI: 1. 22-1. 62), P < 0. 05] in adults after InfV vaccination,including influenza virus A H1N1[MD = 0. 72(95% CI: 0. 54-0. 90), P < 0. 05], influenza virus A H3N2 [MD = 0. 52(95%CI: 0. 15-1. 09), P < 0. 05], and influenza virus B [MD = 0. 81(95% CI: 0. 53-1. 09), P < 0. 05]. The effect of acute preexercise on antibody titers of type A H3N2 [MD = 0. 14(95% CI: 0-0. 29), P > 0. 05] and influenza virus B [MD = 0. 22(95% CI: 0. 09-0. 36), P > 0. 05] after InfV vaccination still needs further verification. Age may be a factor limiting the titer of influenza virus antibodies in subjects receiving InfV vaccination after pre-exercise.Conclusion Pre-exercise intervention can effectively improve the immune response after InfV vaccination in adults. Follow-up high-quality studies are needed to identify exercise regimens that improve immune response to InfV.

Objective To compare the disrupting effects of Hansenula polymorpha cells by two kinds of high-pressure homogenization methods, step-by-step disrupting and cyclic disrupting, so as to provide experimental basis for the selection of disrupting methods in the industrial large-scale production of Hansenula polymorpha cells in the future. Methods Three batches of concentrated fermentation broth of Hansenula polymorpha cells were disrupted by two high-pressure homogenization methods:step-by-step disrupting and cyclic disrupting. The samples before and after disrupting were taken, and the cell morphology was observed under light microscope. The host protein content, cell disruption rate, protein release rate and particle size distribution of released protein were detected and compared. Results After disrupting three batches of concentrated fermentation broth of Hansenula polymorpha cells by step-by-step disrupting and cyclic disrupting, the number of intact cells decreased and cell fragments increased, while there was no obvious difference in cell morphology. In addition, there was no significant difference in host protein content(average gray value of 115. 259 and 109. 125 respectively), cell disruption rate(average value of 60. 29% and 58. 96% respectively), protein release rate(average value of 39. 55% and 37. 28% respectively), and released protein particle size distribution(average size of 90. 41 and 525. 07 nm after step-by-step disrupting, accounting for31. 37% and 68. 63% of sample protein content; average size of 109. 11 and 547. 37 nm after cyclic disrupting, accounting for29. 93% and 70. 07% of sample protein content, respectively. F = 1. 054, 1. 159, 4. 245 and 8. 875, t = 1. 036 0, 0. 504 1,0. 605 0 and 0. 139 7, respectively, each P < 0. 05). Conclusion Cyclic and step-by-step high-pressure homogenization disrupting methods have the equivalent disrupting effect on Hansenula polymorpha cells, while cyclic disrupting is simpler and can avoid pollution risks, which is more suitable for the disrupting of Hansenula polymorpha cells for industrial large-scale production