Objective To detect the virulence gene of E. coli from calves with diarrhea in Tongliao City，Inner Mongolia Autonomous Region，China and analyze its antimicrobial resistance as well as the distribution of antimicrobial resistant genes. Methods The sensitivities of 82 E. coli isolates from the fecal samples of calves with diarrhea to thirteen kinds of antibiotics were determined by disk diffusion test. The carrying statuses of thirteen virulence genes and twelve antimicrobial resistant genes of the E. coli isolates were determined by PCR，based on which the phylogenetic background was investigated. Results Of the 82 pathogenic E. coli isolates，48. 78%（40／82）、31. 71%（26／82）、14. 63%（12／82）and 4. 88%（4／82）belonged to phylogenic groups A，B1，B2 and D respectively，indicating that the prominent one was group A. A total of 11 virulence genes were detected in 82 isolates. The detection rates of irp2，fyuA，eaeA and STb genes were 79. 27%（65／82），63. 41%（52／82），53. 66%（44／82）and 50%（41／82）respectively，while those of other virulence genes were less than 50%，and no tsh or LT1 was detected. The 82 isolates were significantly resistant to 13 kinds of antibiotics，in which the resistant rates to tetracycline，doxycycline and amoxicillin were 100%（82／82），97. 56%（80／82）and 90. 24%（74／82）respectively. All the isolates were mutidrug resistant，most of which were resistant to eight kinds of antibiotics（16／82，19. 51%）. A total of twelve antimicrobial resistant genes were detected in the 82 isolates，in which the positive rates of genes resistant to β ‑ lactams（blaTEM），sulfonamide（sul1 and sul2），tetracycline（tetB and tetD）and aminoglycosides（aadB）were more than 70%. Conclusion The 82 pathogenic E. coli isolates mainly belonged to group A，with high detection rates of virulence gene and antimicrobial resistant gene as well as high and multiple drug resistance. The study provided a reference for the prevention and treatment of and clinical medication of E. coli‑associated diseases in calves in Tongliao Region.

Objective To evaluate the pharmacodynamics of human interferon(IFN)α1b against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron strain in vitro.Methods Total four drugs human IFNα1b bulk,human IFNα1b eye drops,human IFNα1b spray and Remdesivir were detected for cytotoxicity by CCK-8 assay.The inhibitory effect of human IFNα1b on SARS-CoV-2 Omicron strains(BA.5/BA.2/BA.1)was determined by qPCR.Results Human IFNα1b bulk of the maximum concentration(1 × 107IU/mL)and Remdesivir of the maximum concentration(150 μmol/L)did not achieve half cytotoxicity to Vero cells;The median cytotoxicity concentrations(CC_(50))of human IFNα1b eye drops and human IFNα1b sprays were 29 958 and 37 550 IU/mL,respectively,showing toxicity to Vero cells.The median effective concentrations(EC_(50))of human IFNα1b against virus strains BA.1,BA.2 and BA.5 after incubation for 2 h in advance were 9.30,13.38 and 12.33 IU/mL and those of Remdesivir were 0.314 7,0.291 0 and0.300 3 μmol/L.When incubation with virus simultaneously,the EC_(50)of human IFNα1b to BA.1,BA.2 and BA.5 were19.68,10.91 and 18.84 IU/mL and those of the control drug Remdesivir were 0.320 5,0.274 4 and 0.304 1 μmol/L,respectively.Conclusion At the cell level in vitro,human IFNα1b of very low activity showed a good inhibitory effect on SARS-CoV-2 Omicron strain,which was expected to be a clinical specific drug for the treatment of SARS-CoV-2 Omicron strain infection.

Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.

Objective To investigate the effect of caloric restriction(CR)on myocardial ischemia/reperfusion injury(MI/RI)in mice and its mechanism.Methods C57 mice were randomly divided into normal diet group(AL group,free feeding)and CR group(diet decreased by 10% every 2 weeks)for 8 weeks and monitored for weight changes.Each group was divided into sham operation group and MI/RI group,total 4 groups,AL + Sham group,AL + I/R group,CR + Sham group and CR + I/R group).The left anterior descending coronary artery was ligated for 30 minutes and then reperfused for 24 hours in mice of MI/RI group and mice in Sham group were only threaded but not ligated.The mice were determined for myocardial ischemia and infarct size by Evans blue/TTC staining,observed for the pathology of myocardium by HE staining,determined for the activities of lactate dehydrogenase(LDH),superoxide dismutase(SOD)and the contents of creatine kinase-MB(CK-MB)and malondialdehvde(MDA)in myocardium by the corresponding kits,determined for serum levels of IL-1β and IL-18 by ELISA and detected for the expression of pyroptosis-associated proteins in myocardium by Western blot.Results After 8weeks,the weights of mice in CR group[(24.54 ± 0.41)g]were significantly lower than those in AL group[(31.46 ±0.25)g](t = 14.34,P<0.05).Compared with those in AL + I/R group,the area of myocardial ischemia in CR + I/R group showed no significant difference(t = 0.783 0,P>0.05),while the area of myocardial infarction decreased significantly(t = 7.250,P<0.01);The myocardial arrangement was relatively neat,and the degree of pathological changes was obviously reduced;LDH activity,CK-MB and MDA contents decreased significantly(t = 4.331,2.875 and 5.343 respectively,each P<0.05),while SOD activity increased significantly(t = 4.211,P<0.05);Serum levels of IL-1β and IL-18 decreased significantly(t = 3.375 and 4.266 respectively,each P<0.05);The expression levels of nod-like receptor protein 3(NLRP3),gasdermin D(GSDMD),apoptosis-associated speckle-like protein(ASC)and caspase-1 significantly decreased(t = 3.412,3.420,3.480 and 2.585 respectively,each P<0.05).Conclusion CR alleviated MI/RI in mice,and its mechanism was related to the inhibition of cardiac pyroptosis.

Objective To evaluate the effects of various polysorbates(PS)on the stability of different types of monoclonal antibody(mAb)drugs.Methods Three types of monoclonal antibodies mAbA(IgG1 proantibody drug),mAbB(IgG1 mAb)and mAbC(IgG1 mAb with Fc N297A mutation)were used as model proteins,and different kinds or contents of PS were added into the mAb formulations respectively to investigate the influencing factors.The effects of PS on the stability of mAb drugs were evaluated comprehensively by detecting the changes of quality attributes,such as protein aggregates and insoluble particles.Results PS20 and PS80 showed no significant difference in inhibiting the formation of aggregates and charge variants in the three mAbs(P>0.05),while the addition of PS80 in mAbB and PS20 in mAbC significantly inhibited the increase of insoluble particles respectively(P<0.05);The content of PS20 showed a significant effect on the detection indexes of charge variants and insoluble particles in mAbC(P<0.05).Conclusion Different types of mAbs have different sensitivities to various kinds and contents of PS.Therefore,when designing the formulation of mAbs,it is necessary to select appropriate kinds and contents of PS to further improve the stability of mAb drugs.

ObjectiveTo establish and preliminarily verify a digital polymerase chain reaction(dPCR)method for the detection of genome titer of adeno-associated virus(AAV)gene therapy products.MethodsThrough dPCR detection of the same batch of AAV gene therapy products,the primers,probes,sample treatment methods and concentrations of primer and probe were optimized. The specificity,linearity,relative accuracy and repeatability of the optimized method were verified,and the limit of quantitation(LOQ)and detection range were determined.ResultsNFS-05 vector titer 5.0 crop primer probe was selected,the final primer concentration was 800 nmol/L,the final probe concentration was 400 nmol/L,and the sample pretreatment method was digestion by Proteinase K. The established method had good specificity. The correlation coefficient(R2)of the standard curve was greater than 0. 999 in the range of 3. 3 × 10~5-1. 3 × 10~8vg/mL. The relative accuracy was between 70% and 130%,the CV of repeatability was not more than 15%,and the LOQ was 3. 3 × 10~5vg/mL.ConclusionThe established dPCR method has good specificity,linearity,accuracy and repeatability,and can be applied to the determination of the genome titer of NFS-05 recombinant adeno-associated virus(rAAV)stock solution products. 关键词(KeyWords)：数字PCR;腺相关病毒;基因组滴度;基因治疗 Digital polymerase chain reaction(dPCR);Adeno-associated virus(AAV);Genome titer;Gene therapy 基金项目(Foundation): 北京市科技计划课题资助（Z221100007922015） 作者(Author): 安怡方,毕华,刘家池,魏云欢,赵琴,于雷,史新昌,裴德宁,李响,周勇,秦玺,梁成罡 AN Yifang,BI Hua,LIU Jiachi ,WEI Yunhuan ,ZHAO Qin ,YU Lei,SHI Xinchang,PEI Dening,LI Xiang,ZHOU Yong,QIN Xi,LIANG Chenggang

Objective To establish and verify an imaged capillary isoelectric focusing(iCIEF)method for analyzing the charge heterogeneity of recombinant adeno-associated virus(rAAV)capsid proteins,in order to provide a reliable detection method for characterization research,quality control and process stability monitoring of rAAV products. Methods After denaturation,the sample was analyzed by iCIEF using UV fluorescence detection imaging mode,with injection time of 55 s,voltage of 1 500 V,focusing for 1 min,and then voltage adjusted to 3 000 V for another 10 min. The excitation wavelength was set at 280 nm,and the exposure time of emission fluorescence detection was 80 s. Taking rAAV 5 empty particles(rAAV-E)as the research object,the specificity,precision,linear range,accuracy and limit of quantitation(LOQ)of the method were verified. The established method was used to detect the charge heterogeneity of rAAV 5 full particles(rAAV-F).Results The rAAV-E sample showed two main peaks(Peak1 and Peak2)between pH 6. 9 and pH 7. 1,and no virus capsid protein peak was detected in the blank control. In the six repeated detections,the average pI values of the two main peaks Peak1 and Peak2 of rAAV-E sample were 6. 91 and 7. 04 respectively with both RSDs of 0. 14%,and the average proportions of peak area were 35. 6% and 55. 8%,with RSDs of 1. 1% and 0. 4%,respectively. The RSDs of peak area proportions of peak 1 and peak 2 of rAAV-E sample at three time points was 3. 45% and 3. 38%,and the RSDs of pI were 0. 10%and 0. 11%,respectively. In the range of(4. 08-6. 12)× 1012vp/mL,the rAAV-E concentration showed a good linear relationship with the average peak area of the main peaks,and the linear regression equation was y = 54 888 x-76 556,R~2 = 0. 977. The recovery rates of the the main peak content in rAAV-E samples at 80%,100% and 120% expected concentrations were all in the range of 80%-120%. The LOQ was 1. 02 × 10~(12)vp/mL for Peak 1,and 0. 76 × 10~(12)vp/mL for Peak 2. In addition to Peak 1 and Peak 2,obvious extra peaks were observed in the acidic region(pI < 5. 85)of rAAV-F sample,which was the main characteristic of rAAV-F compared with rAAV-E,and also indicated that rAAV-F had complex charge heterogeneity distribution. Conclusion The established iCIEF method has good specificity,precision and accuracy,and can be used to analyze the charge heterogeneity of rAAV capsid proteins.

ObjectiveTo express the receptor binding domain(RBD)protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)in soluble form by prokaryotic system and evaluate its immunogenicity by immunizing mice with the purified protein,so as to provide a new idea for the preparation of SARS-CoV-2 candidate vaccine.MethodsSARS-CoV-2RBD gene fragment was amplified and ligated with pNCMO2 vector after enzyme digestion. The obtained recombinant prokaryotic expression plasmid pNC-RBD was transformed into Bacillus choshinensis competent cells,which were subjected to expanded culture and induced by lowering temperature. The target protein was purified by affinity chromatography,adsorbed by aluminum hydroxide adjuvant,and then the female BALB/c mice were immunized subcutaneously in the back using the protein,10 mice for each group,with the aluminum adjuvant-free protein as control. The humoral immunity was measured by ELISA,while the cellular immune effect by proliferation assay in vitro. The secretion of various cytokines in vitro was measured by ELISA,and the cell typing was determined by flow cytometry.ResultsThe recombinant expression plasmid pNC-RBD was constructed correctly as identified by colony PCR and sequencing. The recombinant protein had a relative molecular mass of about 22 000,which could be expressed and secreted,and the purity of recombinant protein after purification was over 95%. The titers of binding antibody and neutralizing antibody in serum of the immunized mice were about 1∶10 000 and 1∶750,respectively,which were significantly higher than those in adjuvant group(t = 2. 845 and 2. 528,respectively,each P < 0. 01). The recombinant protein stimulated lymphocyte proliferation and promoted the secretion of IL-1β and IFNγ in vitro. The results of cell typing test showed that the lymphocyte proliferation of both CD4~+ and CD8~+T cells was promoted.ConclusionThe RBD protein of SARS-CoV-2 was successfully expressed in soluble form with good immunogenicity,which lays a foundation for the development of SARS-CoV-2 genetic engineering vaccine based on RBD protein.

ObjectiveTo compare the susceptibility of human rhabdomyosarcoma RD cells,human embryonic lung diploid KMB-17 cells and Vero cells to enterovirus(EV)isolation and culture,in order to provide experimental basis for subsequent isolation of EV genotypes.MethodsSixty stool samples of patients with hand-foot-mouth disease(HFMD)diagnosed in a hospital in Kunming in 2019 were collected,and EV strains were isolated and cultured with RD,KMB-17 and Vero cells respectively. The positive samples were detected by RT-PCR,sequenced and blasted by using NCBI BLAST software to identify EV genotypes.ResultsA total of 112 EV strains were isolated,belonging to 11 genotypes respectively. Among them,26strains of Echo-11,6 strains of Echo-6 and Echo-30,2 strains of CV-A4 and CV-A10,1 strain of CV-A6,CV-A16 and Echo-18 were isolated from RD cells;42 strains of Echo-11,3 strains of Echo-6 and 2 strains of Echo-30 were isolated from KMB-17 cells;6 strains of CV-B1,5 strains of CV-B3,4 strains of Echo-11,3 strains of CV-A16 and 1 strain of CV-B2were isolated from Vero cells.ConclusionRD cells were susceptible to most EV genotypes,KMB-17 and Vero cells were the most susceptible to Echo virus and CV-B virus,respectively.

Objective To adapt the MDCK cells of adherent culture for suspension culture,establish and characterize a sus-pension cell line,so as to lay a foundation for the development of cell substrate-based influenza vaccines. Methods MDCK(NBL-2)cells of adherent culture were domesticated into suspension culture cells by shaker adaptation method,namedMDCK-S cells,and identified by 18S and species-specific PCR. The MDCK-S cells were subcultured to the 33rd passage asthe master cell bank(MCB),and passaged to the 38th generation as the working cell bank(WCB). MCB and WCB cellswere both detected for biological characteristics,contamination,tumorigenicity and oncogenicity,chromosome variability andinfluenza virus sensitivity,and observed under transmission electron microscope. Results The MDCK-S cells were confirmedto be dog-derived cells with no mutations and deletions,and there was no DNA contamination of other species. MCB andWCB cells showed good growth status,with“S”-shaped growth curves and doubling time of 27. 78 and 27. 21 h,respectively. Thecells were negative for bacteria,fungi and mycoplasma. The cell morphology,structure and cell membrane were intact,andthere were abundant microvilli on the membrane,indicating that the cell status was good,and there was little differencebetween different cell generations. MDCK-S cells formed epithelial-like subcutaneous tumors,demonstrating its tumorigenicitybut not oncogenicity. The proportion of chromosomes with 2 n = 78 ± 2 was approximately 86% to 96. 19%,and the numberof chromosomes remained stable from P29 to P50. In addition,MDCK-S cells were sensitive to influenza virus. Conclusion MDCK cells were successfully domesticated into suspension culture cells,and a suspension cell line MDCK-S was estab-lished in this study,which lays a foundation for the development of cell substrate-based influenza vaccines.