Objective To screen and rescue an influenza A virus strain adapted to suspension MDCK cells in order to provide an experimental basis for the preparation and screening of cell-based influenza vaccine strains. Methods Two influenza virus strains, A/Nebraska/14/2019(H1N1) and A/Guangdong-Maonan/SWL1536/2019＿CNIC-1909(H1N1), were subcultured in suspension MDCK cells for continuous adaptive passaging. One strain stably adapted to suspension MDCK cell culture was screened by detecting hemagglutination titer and cell culture infective dose 50%(CCID_(50)). This strain was used as the parental virus for subsequent reverse genetics manipulation to obtain the influenza backbone strain reH1N1 and reassor-tant strain rcH1N1. The virus particle morphology was observed under electron microscope, and the sequence was verified by plaque purification. The purified recombinant strain rcH1N1 was continuously passaged in suspension MDCK cells,the hemagglutination titer and CCID_(50)were detected, and its genetic stability was verified. Results The hemagglutination titer and CCID_(50)of strain A/Guangdong-Maonan/SWL1536/2019＿CNIC-1909 were more stable and more adapted to suspension MDCK cell culture. The rescued reassortant strain rcH1N1 and the backbone strain reH1N1 had the morphological characteristics of influenza virus particles. The plaque forming units were 8 × 10~7 and 6 × 10~7PFU, respectively, and the PCR product sequences of each gene segment were consistent with those of the recombinant plasmid. The reassortant strain rcH1N1 obtained stable strain with high titer after five passages, the hemagglutination titer reached a maximum of 1∶512, with an lgCCID_(50)of up to 7. 57, and the genetic material of the passage strain remained stable. Conclusion The influenza A virus strain rcH1N1 adapted to suspension MDCK cells was screened and rescued, which lays a foundation for the development of cell-based influenza vaccines.

Objective To construct a gene knockout cell library based on clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9) system, and construct a cell line stably expressing Cas9 protein using human colon adenocarcinoma cells Caco-2. Methods Caco-2 cells were infected with packaged recombinant Cas9 lentivirus, and then Caco-2/Cas9 monoclonal cells were selected by medium containing blasticidin and twice limited dilution sorting,and identified by PCR and Western blot. Caco-2/Cas9 monoclonal cells were infected with lentivirus expressing both GFP gene and single guide RNA(sgRNA) targeting GFP gene. After puromycin screening, the knockout efficiency of Caco-2 cell line was calculated by flow cytometry, and the proliferation activity was detected by CCK-8 assay. Results Five Caco-2/Cas9 monoclonal cell lines, named Caco-2/Cas9-2, Caco-2/Cas9-3, Caco-2/Cas9-4, Caco-2/Cas9-5 and Caco-2/Cas9-6, were obtained, all of which were amplified for a 392 bp band of Cas9 gene. Cas9 protein was stably expressed in the cells and the knockout efficiency was 91. 27%, 20. 30%, 24. 13%, 11. 33%, 12. 27% and 8. 89%, respectively. The proliferative activity of Caco-2/Cas9-4, Caco-2/Cas9-5, Caco-2/Cas9-6 and uninfected Caco-2 cells was 2. 07, 1. 75, 1. 46 and 1. 40, respectively,with no significant difference between Caco-2/Cas9-5, Caco-2/Cas9-6 and uninfected Caco-2 cells(t = 1. 92 and 0. 37, each P > 0. 05). Conclusion A Caco-2/Cas9 monoclonal cell line, Caco-2/Cas9-6 monoclonal cells with high enzyme digestion activity of Cas9 and no significant change in proliferation activity compared with uninfected Caco-2 cells was screened out,which lays a foundation for further construction of high coverage knockout cell library and provides a platform for screening genes related to virus infection and genes with other specific functions.

Objective To optimize the culture process of Coxsackievirus A16(CV-A16) and compare the different quality attributes of CV-A16 harvest solution in order to determine the critical quality attributes(CQAs), and lay a foundation for the development of the preparation process and quality control method of CV-A16 vaccine. Methods Using Vero cells as matrix,virus titer and mature complete viral particle antigen content(1H5 antigen) as evaluation indicators, the cell growth phase(mid-log phase, late-log phase, plateau phase), MOI(0. 01, 0. 001, 0. 000 1), culture medium type(serum-free M199, DMEM,50% M199 + 50% DMEM medium), serum concentration of culture medium(serum-free, 1%, and 2% newborn bovine serum) and culture temperature(33, 35, and 37 ℃) for CV-A16 inoculation were optimized, and the optimized process was further verified in T25 cell bottle and 10-layer cell factory. Correlation analysis was performed between virus titer, antigen contents of mature complete viral particles(1H5 and 20E8 antigens), and neutralizing epitope antigen content(3H4 antigen)with immunogenicity to identify critical quality attributes(CQAs). Results The optimum culture conditions of CV-A16 were determined by single factor experiment and enlarged scale culture. Vero cells were inoculated at a MOI of 0. 001 at the end of logarithmic phase and cultured at 33 ℃ in serum-free M199 medium. The antigen contents(1H5 and 20E8 antigens) of mature complete viral particles obtained from CV-A16 harvested solution under four culture conditions(serum-free DMEM culture medium at 33 ℃ for 6 d, serum-free M199 culture medium at 33 ℃ for 6 d, serum-free DMEM culture medium at37 ℃ for 4 d, serum-free M199 culture medium at 37 ℃ for 4 d) were quite different, and the trend of change was different from that of neutralizing antibody titer. The virus titer and neutralizing epitope antigen content(3H4 antigen) of CV-A16harvested solution obtained under the four process conditions changed little, and the trend of change was the same as that of neutralizing antibody titer. Conclusion Based on the quality attribute of immunogenicity(neutralizing antibody titer), the virus titer and neutralizing epitope antigen content(3H4 antigen) can be classified as the CQA of CV-A16 virus harvest solution,and the antigen contents of mature complete viral particles(1H5 and 20E8 antigens) can be used for the analysis of virus particles in the quality control process of CV-A16 production.

Objective To analyze the immune effect of combined use of inactivated foot-and-mouth disease vaccine(IFMDV),goat pox vaccine(GPV) and live Brucella vaccine(LBV), and to provide a new and efficient immunization model for the prevention and treatment of bovine diseases in China.Methods Twenty healthy calves were immunized with IFMDV and GPV to observe the adverse reactions after immunization. The antibody levels against foot-and-mouth disease and bovine nodular skin disease in serum were determined by liquid phase blocking ELISA and competitive ELISA respectively. Twenty healthy calves were immunized with IFMDV and LBV to observe the adverse reactions after immunization, and the serum antibody levels against foot-and-mouth disease and Brucella were determined by liquid phase blocking ELISA and tube agglutination test respectively. Finally, IFMDV, LBV and GPV were combined to immunize twenty healthy calves, and the adverse reactions of the immunized calves were observed. The serum antibody levels against foot-and-mouth disease,Brucella and bovine nodular skin disease were determined by liquid phase blocking ELISA, tube agglutination test and competitive ELISA respectively.Results No serious adverse reactions were observed in IFMDV combined with GPV or LBV immunized calves. The level of GPV antibody in serum of calves immunized with IFMDV and GPV at the same time was significantly higher than that of calves immunized with GPV alone. The level of LBV antibody in serum of calves immunized with IFMDV and LBV at the same time was higher than that of calves immunized with LBV alone. In the combined immunization group with IFMDV, GPV and LBV, the antibody levels of GPV and LBV in serum of calves were both higher than those of calves with single immunization.Conclusion IFMDV combined with GPV and LBV has good safety, and can improve the immune effect of GPV and LBV.

Objective To investigate the effects of ubiquitin-like modifier activating enzyme 1(UBA1) on the proliferation and apoptosis of acute myeloid leukemia(AML) cells(HL60 and THP-1 cells) and its mechanism, so as to evaluate the possibility of using UBA1 as a molecular marker and target for diagnosis and treatment of AML. Methods The expression of UBA1 in HL60 and THP-1 cells was inhibited via shRNA interference, and stable transfection cell lines were screened. The inhibition effect was detected by qPCR and Western blot. The effect of inhibiting UBA1 on proliferation of AML cells was measured by CCK-8 assay. Flow cytometry was used to detect the effect of inhibiting UBA1 on apoptosis of AML cells. The effects on the expression of apoptosis proteins(Bax, Bc12), cell cycle regulation related proteins(CDK4, CDK6 and CyclinD1) and MAPK signaling pathway related proteins(P-ERK, P-JNK, P-P38MAPK, T-ERK, T-P38) in AML cells were determined by Western blot. Results Following UBA1 knockdown, both HL60 and THP-1 cells exhibited a significant reduction in UBA1 mRNA transcription and protein expression levels(t = 2. 065-43. 591, each P < 0. 05). Cell proliferation capacity was significantly suppressed(t = 12. 274-17. 252, each P < 0. 05), while the apoptosis rate increased significantly(t = 12. 690-18. 855, P <0. 05). The pro-apoptotic protein Bax was significantly upregulated(t = 17. 094-20. 781, P < 0. 01), whereas the anti-apoptotic protein Bcl-2 was downregulated(t = 42. 494-53. 050, P < 0. 01). The expression levels of cell cycle regulatory proteins CDK4, CDK6, and Cyclin D1 all significantly decreased(t = 12. 193-51. 666, each P < 0. 05). The expression levels of P-ERK and P-P38MAPK in MAPK signaling pathway increased significantly(t = 3. 759-10. 822, each P < 0. 05),but the expression levels of P-JNK, T-ERK and T-P38 had no statistically significant difference(t = 0. 133-1. 794, each P >0. 05). Conclusion Interference with the expression of UBA1 can inhibit the proliferation of HL60 and THP-1 cells and promote their apoptosis, of which the mechanism may be related to the increased expression of P-ERK and P-P38MAPK proteins in MAPK signaling pathway.

Objective To compare the immune responses in lung tissue of K18-hACE2 transgenic mice infected with SARS-CoV-2 prototype strain Prototype and its variants Delta and Omicron BF.7, so as to provide experimental evidence for the research and development of related drugs. Methods K18-hACE2 transgenic mice were infected with Prototype, Delta or Omicron BF.7 by nasal drip. On the 7th day after infection, the lung tissues of mice were taken aseptically for subsequent analyses. Viral RNA copy numbers and cytokine gene expression levels in the lung tissues were quantified by qPCR. Histopathological examination of lung tissue damage was performed using HE staining. Results The copy number of viral nucleic acid in lung tissue of mice in Delta group was significantly higher than that in Prototype and Omicron BF.7 groups(t = 5. 419-6. 908, each P < 0. 01), the expression levels of interferon γ(IFN γ)and interleukin-13(IL-13)in lung tissue were significantly higher than those in Prototype and Omicron BF.7 groups(t = 3. 990-7. 282, each P < 0. 05), and the expression levels of tumor necrosis factor-α(TNF-α),and IL-5 were significantly higher than those in Prototype group(t = 7. 108 and 2. 908,each P < 0. 05). In addition, obvious pathological changes related to virus infection were observed in lung tissue sections of mice in three groups. Conclusion Delta variant strain induced the strongest inflammatory response in lung tissue of K18-hACE2 transgenic mice with the highest pathogenicity, followed by prototype strain and Omicron BF.7 variant strain.

Objective To extract and identify Trueperella pyogenes(T.pyogenes) heparin high affinity proteins(THHAPs),and analyze their immunogenicity, so as to provide a reference for exploring the interaction between T.pyogenes and heparin.Methods The surface proteins of T.pyogenes were extracted using a bacterial membrane protein extraction kit. THHAPs were extracted from the surface proteins by adsorbing with heparin-agarose, washing with 1 mol/L NaCl solution, and eluting with 8 mol/L urea solution, which were then analyzed using SDS-PAGE and liquid chromatography-tandem mass spectrometry(LC-MS/MS). Bioinformatics algorithms were used to analyze the subcellular localization, signal peptides, transmembrane domains and immunogenicity of THHAPs. Fifteen female Kunming mice were immunized with THHAPs. After two weeks of the second immunization, five mice were collected for blood samples from the orbital vein and the serum was isolated, and the other 10 mice were intraperitoneally injected with 9 × 108CFU of T.pyogenes. The immunoprotective effect and antiserum hemolytic inhibitory activity were detected.Results THHAPs were observed to form a major band accounting for 98% of the protein content. A total of 1 767 pyolysin(PLO) peptides were detected, accounting for 98. 06% of the detected peptides(1767/1 802). THHAPs were composed of 1 cell wall protein, 11 secreted proteins, 1 lipoprotein, 7 membrane proteins, and 1cytoplasmic protein, 8 proteins among which might be antigens with immunoprotective properties. The mice(9/10) immunized with THHAPs were able to resist T. pyogenes infection, and their antisera exhibited hemolytic inhibitory activity.Conclusion THHAPs have been successfully extracted from the surface proteins of T. pyogenes, with PLO as their major component, which can induce a protective immune response in mice.

Objective To construct a plasmid reference for the detection of residual DNA from early region 1A(E1A) protein and simian virus 40 large T antigen(SV40LTA), and to explore a digital PCR(dPCR)-based analytical technique for copy number calibration of plasmid reference to quantify plasmid reference accurately, thereby providing a new idea for host cell DNA residual detection in biological products.Methods The plasmid pUC19-E1A-SV40LTA was constructed, and the whole gene was synthesized and then amplified to obtain sufficient reference plasmid. The digital PCR system was employed to determine the copy numbers using double fluorescence channels of E1A and SV40LTA primer-probe sets, respectively.The reference material calibrated by digital PCR was subsequently applied to qPCR to establish a method for detecting the specific E1A and SV40LTA sequences. The developed qPCR assay was systematically validated for plasmid reference linearity, accuracy, limit of quantification(LOQ), precision, specificity, applicability, and stability. Furthermore, the established qPCR system was utilized for the detection of recombinant adenovirus(rAdV) sample and recombinant adeno-associated virus(rAAV) sample.Results The copy numbers of E1A and SV40LTA targets in the plasmid reference were found to be essentially consistent through digital PCR using dual fluorescence channels. The detection values detected by E1A and SV40LTA primer-probe sets were 3. 55 × 109and 3. 48 × 109copies/μL, respectively. The coefficient of variation(CV) of these two values was 1. 42%, and the mean value of 3. 51 × 109copies/μL was taken as the calibration value of digital PCR.The qPCR system established by using digital PCR to calibrate copy number exhibited good linearity, accuracy, LOQ, precision, specificity, applicability and stability, and the recovery rates for rAdV and rAAV samples were between 70% and130%.Conclusion A reference plasmid for detecting E1A and SV40LTA residues in biological products was established,and a more sensitive and accurate digital PCR method was introduced for quantification, which can be used to detect the residual DNA of E1A and SV40LTA in gene therapy products produced with HEK293 and HEK293T as host cells.

Objective To express monoclonal antibodies(mAbs) in Pichia pastoris chassis host and explore various expression optimization strategies to improve the yield of antibodies.Methods Pichia pastoriswas utilized as the chassis cell for the recombinant expression of various mAbs using both methanol-induced system PAOX1 and ethanol-induced system E1.Molecular chaperone overexpression and media component optimization were employed to further enhance the production of mAbs. The productivity of the recombinant strains was then evaluated at a 3 L reactor scale.Results Ethanol-induced expression of Eptinezumab resulted in a yield of 12. 4 mg/L. Overexpression of molecular chaperones Ppi1 and Biph increased Eptinezumab production by approximately 22. 5% and 23. 0%, respectively. Additionally, the supplementation of1. 0 g/L ammonium sulfate, the adjustment of 0. 05 mol/L potassium phosphate buffer, and the addition of 80 mmol/L threonine individually enhanced Eptinezumab yields by approximately 57. 1%, 25. 8%, and 58. 1%, respectively. At the 3 L bioreactor scale, the Biph-overexpressing strain exhibited a remarkable increase in complete antibody production, achieving a maximum yield of 74. 1 mg/L, which was approximately 5. 9 times higher than that obtained at the flask level.Conclusion Eptinezumab can be successfully expressed in the Pichia pastoris chassis host, and the application of various expression optimization strategies has effectively enhanced the production of the antibody. This study provides novel insights and directions for the expression of monoclonal antibodies in Pichia pastoris.

Objective To investigate the genetic characteristics of viruses associated with viral diarrhea in Jilin Province, in order to provide a scientific basis for making more effective prevention and control strategies of viral diarrhea.Methods A total of 211 fecal specimens from hospitalized children under 5 years of age with diarrhea and 7 sewage samples were collected in Jilin Province from January to July 2023. Initial screening was performed using qPCR, and positive specimens were further analyzed by RT-PCR to amplify target genes. Sequencing was conducted, and phylogenetic trees were constructed to analyze genetic relationships.Results In fecal specimens, the positive rates for rotavirus(RV), Norovirus(NoV),Sapovirus(SaV), astrovirus(AstV), and adenovirus(AdV) were 10. 9%, 48. 3%, 4. 3%, 1. 4%, and 5. 7%, respectively. In sewage samples, 5 AstV and 4 NoV were detected, along with 3 samples each of RV, SaV, and AdV. Sequencing and genotyping results revealed that RV G8P [8] was the predominant circulating strain. Among NoV strains, GⅡ.4 [P16] was the most prevalent, followed by GⅡ.4 [P31]. In fecal samples, GⅠ group strains included GⅠ.3 [P13] and GⅠ.1 [P1]. One SaV strain of GⅡ.5 was identified. For AstV, three strains of HAstV-1a and one strain of HAstV-5 were detected. Among AdV strains, three were identified as HAdV-41, three as HAdV-2, one as HAdV-1, and one as HAdV-5.Conclusion The pathogenic genes of viral diarrhea in Jilin Province are diverse, the dominant strains of RV have changed, and domestic sewage contains a variety of diarrhea viruses.