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 obtain monoclonal antibodies against the glycoprotein Gn of severe fever with thrombocytopenia syndrome virus(SFTSV) based on immune repertoire sequencing and identify the biological characteristics.Methods Using purified SFTSV-Gn as bait, virus-specific B cells were captured from B cell library of immunized female BALB/c mice. Based on 10 × immune repertoire sequencing and bioinformatics analysis, the antibody sequences with potential binding activity were selected and constructed into eukaryotic expression vector pcDNA3.4. The monoclonal antibodies were expressed and purified, and identified for the biological activity.Results Fourteen antibodies were selected from 3 544 captured B cells, and the functional verification showed that 12 antibodies could bind to SFTSV-Gn glycoprotein, among which C9 and 9/10 antibodies had higher binding ability. Western blot, indirect immunofluorescence assay(IFA) and flow cytometry proved that they specifically recognized SFTSV, and the effective concentration was as low as 2 μg/mL.Conclusion The monoclonal antibodies against SFTSV-Gn glycoprotein were successfully obtained, which lays a foundation for the development of rapid diagnostic reagents for SFTSV.

Objective To prepare monoclonal antibodies against recombinant porcine trypsin(RPT) and identify the biological characteristics, so as to lay a foundation for the development of RPT detection products. Methods The PT gene was amplified by PCR and cloned into the prokaryotic expression vector pET-28a(+). The prokaryotic expression plasmid pET-28a(+)-RPT was constructed and identified by colony PCR and sequencing, which was then transformed into competent E.coli BL21(DE3), induced by IPTG, and purified by Ni-NTA affinity chromatography to obtain RPT. Mouse myeloma cells SP2/0 were fused with spleen cells of BALB/c mice immunized with RPT by hybridoma antibody preparation technique. Hybridoma cells stably secreting specific RPT monoclonal antibodies were screened and injected i.p. into the mice to prepare ascites. The positive monoclonal antibodies were screened by ELISA, and the titer, subtype, immunogenicity and specificity were identified.Results The prokaryotic expression plasmid pET-28a(+)-RPT was constructed correctly as identified by colony PCR and sequencing. RPT had a relative molecular mass of about 24 000 and mainly existed in the supernatant in soluble form, which had a purity of over 90% after purification. Two positive monoclonal cell lines, RPT-4A5C5 and RPT-4D6D11, were obtained with the antibody titers of greater than 1∶1 280 000. Both of the antibodies were IgG1 subclasses, and the light chains were Kappa chains, which reacted specifically with RPT and natural PT, but exhibited no obvious cross-reaction with recombinant carboxypeptidase B(RCPB), recombinant enterokinase(REK), sperm protein 10(SP10) and bovine serum albumin(BSA).Conclusion RPT was expressed by prokaryotic system, and monoclonal antibodies against RPT with high titer and high specificity were prepared by hybridoma technique.

Objective To develop and verify an ultra-high performance liquid chromatography-fluorescence detection(UPLCFLD) system for the measurement of N-glycans of therapeutic monoclonal antibody SIBP-A, in order to validate the method for the release and stability detection of the monoclonal antibodies.Methods The therapeutic monoclonal antibody SIBP-A was selected as the research subject, the N-glycans of which were identified by using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-Tof-MS), and then were quantitatively analyzed by UPLCFLD. The specificity, repeatability, intermediate precision, linearity and range, as well as robustness of the developed UPLCFLD method were verified. The stability tests of samples at -30 and 10 ℃ were performed, and the detection results were compared with those of samples treated at room temperature.Results Ten main types of glycan molecules were detected by UPLC-FLD, which were identified as M3B, F(6)A1, A2, F(6)A2, Man5, F(6)A2[6]G(4)1, F(6)A2[3]G(4)1, A2[6]G(4)1, A2[6]G(4)1, A2[3]G(4)1 and F(6)A2G(4)2 by mass spectrometry. The peak of SIBP-A was normal, and 80% acetonitrile solution registered no significant interference peak at the sample peak. The relative standard deviation(RSD) of the sum of galactosefree oligosaccharides' peak area percentage was 0. 8% among six parallel samples. In the three tests by two operators, the RSD of the sum of galactose-free oligosaccharides' peak area percentage was 1. 4%. There existed a linear relationship between the protein content within a sample range of 320 to 480 μg and the sum of peak areas for galactose-free oligosaccharides with the R~2of 0. 97. The RSDs of the sum of galactose-free oligosaccharide peak area percentage were determined to be0. 2%, 0. 1%, and 0. 1% at pH 4. 4, 4. 5, and 4. 6 mobile phase A, respectively, and the RSD of the percentage of overall galactose-free oligosaccharide peak area across different pH mobile phase A was found to be 0. 8%. Additionally, the RSDs of the sum of galactose-free oligosaccharide peak area percentage were 0. 5% and 0. 6% respectively after the treated samples were subjected to -30 or 10 ℃ for 24 hours in darkness.Conclusion Based on the UPLC-Q-Tof-MS technique that effectively enables the identification of N-glycans of the therapeutic monoclonal antibody SIBP-A, the developed UPLC-FLD detection method enables accurate quantitative analysis of N-glycans of SIBP-A, exhibiting excellent specificity, repeatability, intermediate precision, linearity and range, robustness, as well as stability for the detection of samples stored at-30and 10 ℃, which can be utilized for the release and stability testing of this antibody.

Objective To investigate the expression of micro RNA-19a(miR-19a) in gastric cancer tissues and serum and its effect on the proliferation, invasion and regulation of thrombospondin 1(THBS1) expression in human gastric cancer AGS cells.Methods Real-time fluorescence quantitative PCR(RT-qPCR) was used to measure the expression of miR-19a in 22 gastric cancer tissues, corresponding adjacent noncancerous tissues, serum, and the serum of 22 healthy individuals. Then,the human gastric cancer AGS cells were cultured in vitro and divided into control, inhibitor NC, mimics NC, miR-19a inhibitor, miR-19a mimics, miR-19a inhibitor + si-NC and miR-19a inhibitor + si-THBS1 groups, three repeated wells for each group. The control group was without intervention, and the other groups were transfected with inhibitor NC, mimics NC, miR-19a inhibitor, miR-19a mimics, miR-19a inhibitor + si-NC and miR-19a inhibitor + si-THBS1 by using Lipofectamine~(TM)2000 liposomes respectively. After 24 h of transfection, each group was detected for the miR-19a expression, cell viability, proliferation rate, invasion number and the related proteins expression by RT-qPCR, cell counting kit-8(CCK-8), 5-ethynyl-2'-deoxyuridine(EdU), Transwell chamber and Western blot, separately.Results The expression level of miR-19a in gastric cancer tissues was significantly higher than that in corresponding adjacent tissues(t = 5. 061, P < 0. 001), and the expression level of miR-19a in serum of patients with gastric cancer was significantly higher than that of healthy people(t = 6. 299, P < 0.001). In vitro, the expression level of miR-19a in AGS cells in the miR-19a inhibitor group was significantly lower than that in the inhibitor NC group(t = 11. 120, P < 0. 001), the expression level of miR-19a in the miR-19a mimics group was significantly higher than that in the mimics NC group(t = 11. 637, P < 0. 001), and the mRNA and protein expression levels of THBS1 in the miR-19a inhibitor + si-THBS1 group were significantly lower than those in the miR-19a inhibitor + si-NC group(t = 10. 070 and 13. 164, P = 0. 001 and < 0. 001, respectively). Compared with the inhibitor NC group, the AGS cell viability, proliferation rate, invasion number, and the protein expression levels of Cyclin D1 and proliferating cell nuclear antigen(PCNA) in the miR-19a inhibitor group significantly decreased(t = 16. 679, 10. 072, 17. 737, 11. 173, and 11. 549,respectively, each P < 0. 001), and the THBS1 protein expression level increased significantly(t = 16. 774, P < 0. 001).Compared with the mimics NC group, the AGS cells in the miR-19a mimics group had significantly higher viability, proliferation rate, invasion number, and the expression levels of Cyclin D1 and PCNA proteins(t = 15. 004, 22. 746, 13. 349, 11. 484,and 19. 022, respectively, each P < 0. 001), while the expression level of THBS1 protein significantly decreased(t = 16. 384,P < 0. 001). Compared with miR-19a inhibitor + si-NC group, the viability, proliferation rate, invasion number, and the protein expression levels of Cyclin D1 and PCNA of AGS cells in miR-19a inhibitor + si-THBS1 group significantly increased(t = 13. 180, 8. 669, 14. 493, 6. 828, and 13. 587, respectively, each P < 0. 001), while THBS1 protein expression level significantly decreased(t = 14. 824, P < 0. 001).Conclusion The miR-19a is highly expressed in gastric cancer tissues and serum of patients with gastric cancer, and knockdown of miR-19a may inhibit the proliferation and invasion of human gastric cancer AGS cells by activating the expression of THBS1.

Objective To assess the post-licensure safety and immunogenicity of freeze-dried live attenuated hepatitis A vaccine(H_2 strain) in order to popularize it as an immunization program vaccine.Methods Totally 5 000 healthy infants and young children aged 18-24 months were injected with single dose of freeze-dried live attenuated hepatitis A vaccine, and the adverse events of 0-14 d after vaccination were monitored. Among them, 200 cases were selected into immunogenic subgroups, and the serum hepatitis A virus(HAV) specific IgG antibody(anti-HAV IgG) was measured 8 weeks before and after immunization.Results A total of 5 000 participants completed inoculation, and 4 999 participants completed safety observation, among which, 424 cases of adverse reactions were reported, and the incidence of solicited adverse reactions was 8. 48%. The common local solicited adverse reactions were redness, tenderness, itching, rash and swelling at the inoculation site, while the systemic adverse reactions were mainly fever and anorexia. The severity was mainly grade 1-2, and no serious adverse events related to the vaccine occurred. Among 200 immunogenic subgroups, 188 participants were included in the per protocol set(PPS), the seroconversion rate of anti-HAV IgG antibody was 98. 91% [95% confidence interval(CI): 96. 11%-99. 87%], with a geometric mean titer(GMT) of 83. 67 mIU/mL(95% CI: 77. 05-90. 85 mIU/mL).Conclusion A single dose freeze-dried live attenuated hepatitis A vaccine(H_2 strain) presents good safety and immunogenicity for infants and young children aged 18-24 months.

Objective To sequence the whole genome and analyze the genetic characteristics of an echovirus 11(E-11)strain isolated from stool samples of patients with hand, foot and mouth disease(HFMD) in Kunming, Yunnan Province in2019, so as to provide a reference for the prevention and control of E-11.Methods The virus was isolated using Vero cells,the RNA from cytopathic products was extracted for RT-PCR and sequenced, and the whole genome sequence was analyzed by software such as MEGA 7.0, Geneious 10.0 and Simplot 3.5.1.Results The 19V30322/YN/CHN/2019 isolate was sequenced and determined to be E-11, which belongs to the D5 subtype of the same genotype D as the E-11 epidemic strain in Guangdong and Hubei, China in recent years, with a full length of 7 434 nt and encoding a polyprotein of 2 195 amino acids. 19V30322 showed 81. 8%-98. 3% nucleotide identity and 94. 5%-99. 3% amino acid similarity with the domestic isolate E-11, and recombined with Coxsackievirus A10, E-6, Coxsackievirus B4 and Coxsackievirus B5 during the evolutionary process.Conclusion The 19V3/YN/CHN/2019 isolate is the D5 subtype of E-11 and is a recombinant strain.

Objective To establish a pre-column derivatization-high performance liquid chromatography(HPLC) method for the determination of free formaldehyde in Sabin strain inactivated poliovirus vaccine(IPV)(Vero cells), validate and apply the method, so as to provide a new method for the determination of free formaldehyde in vaccines.Methods The sample was derivatized with 2, 4-dinitrophenylhydrazine and loaded onto a C18 chromatographic column(5 μm, 120 ?, 4. 6 mm ×250 mm). The detection wavelength, mobile phase ratio, flow rate, derivatization time, temperature, buffer solution, and derivatization container were optimized for the separation conditions. The specificity, linearity, repeatability, accuracy, limit of detection(LOD), limit of quantitation(LOQ) and robustness of the method were verified. The content of free formaldehyde in 20 batches of IPV(Vero cells) was detected by using the optimized method.Results Chromatographic conditions: acetonitrile and water in a 70∶30 volume ratio as mobile phase, flow rate 0. 6 mL/min, determination at a wavelength of 352 nm.Derivatization conditions: 0. 5 mL of acetonitrile solution containing 2, 4-dinitrophenylhydrazine and 0. 25 mL of pH 5. 0buffer were added, followed by a 20 min incubation in 60 ℃ water bath. This chromatographic separation conditions effectively separated 2, 4-dinitrophenylhydrazine and formaldehyde derivatives, and the acetaldehyde had no effect on the determination results. In the range of 0. 05-100 μg/mL, formaldehyde standard concentration exhibited a good linear relationship with the peak area, with the r value of 0. 999 9. The relative standard deviations(RSDs) of six test results in the repeatability test was 0. 36%. The recovery rates of formaldehyde content in nine samples were between 102. 0% and 107. 0%. The LOD and LOQ were 0. 025 and 0. 05 μg/mL, respectively. The sample remained stable for 48 h after derivation, showing good robustness. The results of the same batch of samples had good repeatability, and the formaldehyde content was between 4. 5-9. 9 μg/dose.Conclusion The established method has the advantages of wide measurement range, good linearity and high accuracy, and can accurately quantify free formaldehyde in Sabin strain IPV(Vero cells), which can be used as an auxiliary detection method for free formaldehyde content in vaccine products, and is of great significance to the lot release and quality supervision for vaccines.

Objective To establish a pre-column derivatization-high performance liquid chromatography(HPLC) method for the determination of free formaldehyde in Sabin strain inactivated poliovirus vaccine(IPV)(Vero cells), validate and apply the method, so as to provide a new method for the determination of free formaldehyde in vaccines.Methods The sample was derivatized with 2, 4-dinitrophenylhydrazine and loaded onto a C18 chromatographic column(5 μm, 120 ?, 4. 6 mm ×250 mm). The detection wavelength, mobile phase ratio, flow rate, derivatization time, temperature, buffer solution, and derivatization container were optimized for the separation conditions. The specificity, linearity, repeatability, accuracy, limit of detection(LOD), limit of quantitation(LOQ) and robustness of the method were verified. The content of free formaldehyde in 20 batches of IPV(Vero cells) was detected by using the optimized method.Results Chromatographic conditions: acetonitrile and water in a 70∶30 volume ratio as mobile phase, flow rate 0. 6 mL/min, determination at a wavelength of 352 nm.Derivatization conditions: 0. 5 mL of acetonitrile solution containing 2, 4-dinitrophenylhydrazine and 0. 25 mL of pH 5. 0buffer were added, followed by a 20 min incubation in 60 ℃ water bath. This chromatographic separation conditions effectively separated 2, 4-dinitrophenylhydrazine and formaldehyde derivatives, and the acetaldehyde had no effect on the determination results. In the range of 0. 05-100 μg/mL, formaldehyde standard concentration exhibited a good linear relationship with the peak area, with the r value of 0. 999 9. The relative standard deviations(RSDs) of six test results in the repeatability test was 0. 36%. The recovery rates of formaldehyde content in nine samples were between 102. 0% and 107. 0%. The LOD and LOQ were 0. 025 and 0. 05 μg/mL, respectively. The sample remained stable for 48 h after derivation, showing good robustness. The results of the same batch of samples had good repeatability, and the formaldehyde content was between 4. 5-9. 9 μg/dose.Conclusion The established method has the advantages of wide measurement range, good linearity and high accuracy, and can accurately quantify free formaldehyde in Sabin strain IPV(Vero cells), which can be used as an auxiliary detection method for free formaldehyde content in vaccine products, and is of great significance to the lot release and quality supervision for vaccines.