ObjectiveTo construct self-amplifying RNA(saRNA)vaccine of severe acute respiratory syndrome coronavirus2(SARS-CoV-2)Delta mutant strain(B.1.617.2)based on Coxsackievirus-A5(CV-A5)replicon and evaluate its immunogenicity.MethodsThe recombinant plasmids pDelta-S10,pDelta-S5 and pDelta-S1(10,5 and 1 amino acid residues at the upstream of S-VP1/2A cleavage site of the fusion polyprotein respectively)were constructed by In-fusion cloning of the plasmids containing the full-length genome sequence of CV-A5 and substituting the S protein gene of SARS-CoV-2 Delta mutant for the P1 structural protein gene of CV-A5 with different lengths.Three RNA molecules,Delta-S10,Delta-S5 and Delta-S1,were obtained by in vitro transcription of linearized recombinant plasmids and transfected into HEK-293T cells respectively,which were analyzed for the expression of S protein by Western blot.The RNA molecule with the highest expression of S protein was screened out and detected for the self-amplification in HEK-293T cells by qPCR.BALB/c mice(female,6 ～ 8 weeks old and five for each group)were immunized i.m.with two doses(0.5 and 2.5 μg)of the screened Delta-S packaged with lipid nanoparticles for once on day 1 and day 14 seperately.Blood samples were collected on days 14and 28,detected for serum binding antibody titers by ELISA,and detected for neutralizing antibody titers by micro neutralization method.The spleens were harvested on day 42 and detected for the level of IFNγ secreted by mouse spleen cells by enzyme linked enzyme linked immunospot assay(ELISPOT).ResultsThe recombinant RNA molecule Delta-S10showed the highest expression of S protein and self-amplified in HEK-293T cells,which of both high and low doses induced specific binding antibody against SARS-CoV-2 Delta S1 protein in mice with obvious dose effect and enhanced immune effect;The high dose of Delta-S10 induced neutralizing antibodies and cellular immune responses in mice.ConclusionThe SARS-CoV-2 Delta mutant(B.1.617.2)saRNA vaccine Delta-S10 based on CV-A5 replicon was successfully constructed,which induced humoral and cellular immune responses in mice,laying a foundation of the further study of the construction of SARS-CoV-2 saRNA vaccine by enterovirus replication elements.

Objective To compare and analyze neutralizing antibody titer and specific IgG concentration in the sera immunized with F-genotype mumps attenuated live vaccine,so as to evaluate the possibility of using them as mutual verification indexes.Methods A total of 194 serum samples were randomly selected,including 98 in vaccine group inoculated with F-genotype mumps attenuated live vaccine(49 before immunization and 49 after immunization)and 96 in placebo group(48 before immunization and 48 after immunization),which were detected for the concentration of mumps virus specific IgG and the titer of neutralizing antibody by ELISA and neutralization test respectively,and the detection results were compared.With neutralizing antibody titer as ordinate and IgG concentration as abscissa,the scatter plot was drawn,and the correlation between them was analyzed by logistic regression.With neutralizing antibody titer as standard,the specific IgG concentration of each group was analyzed by ROC curve,which was evaluated to judge the reliability of neutralizing antibody positive conversion.Results There was no significant difference in the concentration of specific IgG(t =-0.977,P > 0.05)and the titer of neutralizing antibody(Z =-1.405,P > 0.05)in the serum of the placebo group before and after immunization.However,in the vaccine group,the concentration of specific IgG(t =-9.959,P < 0.001)and the titer of neutralizing antibody(Z =-5.696,P < 0.001)in the serum after immunization were both significantly higher than those before immunization;The positive conversion rates of specific IgG before and after immunization in the placebo group and before immunization in the vaccine group were significantly higher than those of neutralizing antibody(χ2= 27.927,29.777 and 17.563respectively,each P < 0.001),while no significant difference was observed between the positive conversion rates of the two after immunization in the vaccine group(χ2= 27.927,29.777 and 17.563respectively,each P < 0.001),while no significant difference was observed between the positive conversion rates of the two after immunization in the vaccine group(χ2= 2.346,P > 0.05).There was a significant correlation between specific IgG concentration and neutralizing antibody titer in each group(r = 0.321 0 ～ 0.620 1,each P < 0.05).After immunization,the area under ROC curve was the largest(0.961),and the specificity(1.00)and sensitivity(0.93)were higher in the vaccine group.Conclusion The neutralizing antibody titer and specific IgG concentration in the serum immunized with mumps attenuated live vaccine(F-genotype)showed a good correlation,which might be used as mutual verification indicators.

ObjectiveTo identify the sites of thermal stability at Loop structure of xylanase Xyn ASP in Aspergillus saccharolyticus JOP 1030-1 and improve the thermal stability.MethodsThe amino acid sites related to thermal stability of xylanase were predicted,and beneficial mutation sites at Loop structure were screened by Fireprot online server.Singlepoint mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and double-point mutants Xyn(T81Q)and double-point mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(LQ)(A79Y/T81Q)were constructed by site-directed mutagenesis.The recombinant mutant plasmid was transformed to E.coli BL21(DE3)and induced by IPTG.The recombinant xylanase was purified by Ni-NTA protein purification kit,and determined for the optimum temperature,thermal stability,optimum p H and p H stability.ResultsBeneficial mutation sites A79Y and T81Q were screened at Loop structure,and the purified protein samples showed high purity.Compared with that of wild type Xyn ASP,the optimum temperature of mutants Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(LQ)increased by 10℃，5℃and 5℃，respectively.After heat treatment at 40℃for 30 min,wild type Xyn ASP retained only 30.2%residual relative activity,while the mutant Xyn(T81Q)retained 37.7%,Xyn(T81Q)retained 37.7%,Xyn(A79Y)and Xyn(A79Y)and Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ )still retained more than 65%.After heat treatment at 60℃for 30 min,the enzyme activity of wild type was 7.1%,while that of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(LQ)remained 26.4%.The thermal stability of mutants was improved compared with that of wild type Xyn ASP.The optimum p H of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(LQ)was 5.0,which was lower than that of wild type Xyn ASP(p H 6.0).The p H stability of Xyn(A79Y),Xyn(A79Y),Xyn(T81Q)and Xyn(T81Q)and Xyn(LQ)at pH 3.0～8.0 showed no significant change compared with the wild type.ConclusionSite-directed mutagenesis(A79Y and T81Q)was carried out at Loop structure,and xylanase mutants with obviously improved thermal stability were obtained,which laid a foundation of the later research on the structure,function and relationship of the enzyme and its industrial application.

ObjectiveTo explore the protective effect of Sox11 gene on cerebral ischemia reperfusion injury(CIRI)in mice and its mechanism,so as to provide a new target for the treatment of CIRI.MethodsThe mouse middle cerebral artery occlusion(MCAO)model and Neuro2A cell oxygen glucose deprivation reperfusion(OGDR)model were established and detected for the temporal and spatial distribution of Sox11 in the models by real-time quantitative PCR,Western blot,immunohistochemistry(IHC)and immunohistofluorescence(IHF).The altered expression of some crucial genes in the pathway of apoptosis and inflammation in OGDR model after the disruption of Sox11 expression was detected by Western blot.ResultsThe expression level of Sox11 mRNA and protein increased significantly in both MCAO and Neuro2A OGDR models(P = 0.000 1 ～ 0.038 8);After CIRI,Sox11 expression was elevated in the hippocampal dentate gyrus(DG)region of mice;After interfering with the expression of Sox11 in OGDR model,the expression of apoptosis-related protein Cleaved Caspase 3 significantly increased,while the expression of anti-apoptosis protein Bcl-2 significantly decreased,and the expression of phosphorylated NF-κB(p-NF-κB)protein related to inflammatory reaction also up-regulated significantly.Conclusion Sox11gene had a protective effect against CIRI in mice,and was involved in the regulation of apoptosis and inflammation pathways after CIRI.

Objective To express and purify Cc PT1 protein from Aspongopus chinensis in prokaryotic cell.Methods Thesynthesized Cc PT1 gene was cloned to vector p GEX-4T-1 to construct recombinant expression plasmid p GEX-4T1-Cc PT1,which was then transformed to competent E.coli Rosetta strain and induced by IPTG.The induction temperature(20 ℃ and37 ℃),final concentration of IPTG(0.25,0.5,0.75 and 1 mmol/L)and induction time(6,8,10,12 h)were opti-mized.The obtained protein was purified by GST protein purification system,which was then analyzed by 10% SDS-PAGEand identified by Western blot.GST tags were removed by Pre Scission Protease during purification.Results The recombi-nant protein GST-Cc PT1 was expressed in the form of inclusion body with a concentration of 0.026 9 mg/ml,of which therelative molecular mass was 29 800,consistent with the expectation.The optimum induction condition was induction withIPTG of final concentration of 0.75 mol/L for 12 h at 20 ℃.The purified protein was more than 90% in purity and boundspecifically to mouse monoclonal antibody against GST.After remove of GST tags,Cc PT1 protein showed a relative molecu-lar mass of about 2 830 and the yield was 11.15%.Conclusion A.chinensis Cc PT1 protein was expressed by prokaryoticexpression system,and the purity of Cc PT1 protein was high after purification,which laid a foundation of the in-depth studyof anticancer peptides of A.chinensis.

Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334～353)),RBD9(S(334～353)),RBD9(S(439～458))and RBD13(S(439～458))and RBD13(S(499～518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.

ObjectiveTo obtain recombinant H.pylori adhesin A(rHpaA)by molecular cloning,protein expression and purification,immunize BALB/c mice to prepare anti-HpaA polyclonal antibody,and analyze its antibody specificity.MethodsThe three-dimensional structure and antigenic properties of rHpaA were analyzed by bioinformatics softwares such as Phyre2 and DNAstar;Adhesin HpaA gene was obtained by PAS(PCR-based accurate synthesis)and inserted into plasmid pCzn1.The prepared recombinant plasmid pCzn1-rHpaA was transformed to E.coli Artic Express(DE3),induced by IPTG and purified by Ni-IDA affinity chromatography to obtain rHpaA protein,which was identified for reactivity by Western blot.Six male BALB/c mice were immunized with rHpaA plus Freund's adjuvant to prepare anti-HpaA polyclonal anti-body,and the antibody specificity was identified by ELISA.ResultsrHpaA showed good three-dimensional structure and antigenic properties.Restriction analysis and gene sequencing showed that the recombinant plasmid pCzn1-rHpaA contained completely correct HpaA gene sequence.The recombinant strain pCzn1-rHpaA/Arctic Express expressed the soluble target protein rHpaA,which accounted for about 68.3% of total protein in the supernatant,with a purity of 98.1%.rHpaA bound to anti-His antibodies and anti-H.pylori antibodies;The anti-HpaA polyclonal antibody specifically recognized rHpaA and H.pylori lysates.ConclusionrHpaA protein with high purity can be obtained by induction at low temperature and purification.The prepared anti-HpaA polyclonal antibody had good specificity,which laid an experimental foundation of the development of H.pylori-related diagnostic reagents.

ObjectiveTo prepare colloidal gold immunochromatographic test paper for rapid detection of Legionella pneumophila(LP)and test its performance to ensure that it meets the national clinical diagnostic standards.MethodsLP colloidal gold immunochromatographic test paper was prepared based on double antibody sandwich ELISA,and tested for the cross reactivity,anti-interference,sensitivity,hook effect,stability and other aspects.ResultsLP colloidal gold immunochromatography test paper showed no cross reaction with 22 common pathogens in respiratory tract such as Moraxella catarrhalis,and was not affected by internal and external interferences in respiratory tract;The minimum detection limit for LP was 2.00 × 105cfu/mL,with good sensitivity and no hook effect;Under the conditions of accelerated aging at 45 ℃,simulated high temperature transportation and frozen transportation,the repeatability and stability of test paper were not affected,and the stability was good in the same batch and between different batches.ConclusionThe prepared LP colloidal gold immunochromatographic test paper realized rapid detection of LP,which was simple to operate and had good application prospect and popularization value.

ObjectiveTo analyse the coverage of inactivated enterovirus 71(EV71)vaccine and the impact on hand-foodmouth disease(HFMD)epidemiological and etiological changes in Jinshan District,Shanghai,and provide evidence for improving the prevention and control strategy of HFMD in this area.MethodsThe vaccination data of inactivated EV71vaccine from 2016 to 2019 was collected in Jinshan Immunization Information Management System of Shanghai to describe the vaccination characteristics;The data of HFMD cases in Jinshan District from 2013 to 2019 were extracted from Chinese Disease Prevention and Control Information System,and the surveillance etiological information of HFMD in the same period was obtained,which was compared for the differences of HFMD incidence and pathogen positive detection rate before and after vaccination.ResultsFrom November 2016 to December 2019,a total of 63 521 doses of inactivated EV71vaccine were applied in Jinshan District,with the first shot coverage of 22.57%,the full shot coverage of 21.05% and the two-dose completion coverage of 94.65%.There were significant differences in coverage between different years,months,current addresses,age groups and registers(P < 0.05).The highest coverage of first short was in 2018(33.45%),while full short in 2017(30.78%).More doses were applied during May to August,with highest coverage in 6 to 11 months old group and most doses in 1 year old group.The coverage of children in this city was higher than that of migrant children.There was no significant difference in the incidence of HFMD before and after vaccination(χ2= 0.427,P =0.513 ),while the incidence of severe disease,the positive detection rate of EV71 and the estimated incidence of HFMD infected with EV71 decreased significantly after vaccination(χ2= 15.312,41.431 and 432.342 respectively,each P <0.001).ConclusionVaccination with inactivated EV71 vaccine reduced the occurrence of HFMD EV71 infection and severe disease in Jinshan District,while the coverage was low,so it was necessary to pay attention to HFMD etiological changes to prevent other enterovirus infections.It is suggested to strengthen publicity and information technology to improve coverage,speed up the development of combined vaccine and provide more antibody protection.

ObjectiveTo screen a high alkaline xylanase-producing strain,subject to molecular identification and characterization of enzymatic property,and optimize its fermentation condition.MethodsThe farmland soil samples from Shanxi,Henan and Zhejiang Provinces were collected aseptically,from which the high xylanase-producing strains were screened by enrichment culture,isolation and identification of 16S r DNA,and determined for enzymatic properties.The carbon source(xylan,lactose,soluble starch,sucrose,bran and glucose),nitrogen source[ammonium oxalate,peptone,yeast powder,(NH_4)_2SO_4,NH_4Cl,Na NO_3,urea,beef extract,bean powder,KNO_3,(NH_4)_2HPO_4or random mixture of two of peptone,yeast powder,beef extract,(NH_4)_2HPO_4and bean powder],metal ion[CuCl_2,MgCl_2,ZnCl_2,Al_2(SO_4)_3,CoCl_2,MnCl_2,AgNO_3,NaCl,CaCl_2,FeCl_2,BaCl_2 and FeCl_3],pH value(3.0～10.0)in medium and the fermentation temperature(25,28,30,33,35,37 and 40℃)were optimized by single factor test,while the contents of components[xylan,(NH_4)_2HPO_4and bean powder]by response surface method.The high alkaline xylanase-producing strain was fermented by using the optimized medium under the optimized condition,and determined for xylanase activity,and the result was compared with that predicated in model.ResultsA high alkaline xylanase-producing strain named as SX6-18 was screened and identified as Paemibacillus based on 16s rDNA sequence analysis.The xylanase produced by the strain maintained more than 70%of relative activity at temperatures of 25～55℃and pH 6.0～10.0.Mg(2+)promoted while Fe(2+)promoted while Fe(3+),Mn(3+),Mn(2+)and Cu(2+)and Cu(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_4)_2HPO_4 and Fe(2+)inhibited the activity of xylanase.The optimal carbon source,nitrogen source and metal ion of the medium were xylan,bean powder+(NH_4)_2HPO_4 and Fe(3+),while the optimal p H value and temperature for fermentation were 8.0 and 30℃，respectively.However,the optimal component contents were 15.00 g/L xylan,3.03 g/L(NH_4)_2HPO_4and 3.28 g/L bean powder.The activity of xylanase cultured in optimized medium under opti-mized condition for fermentation reached 701.08 U/m L,which was closed to the expected value(693.96 U/m L).ConclusionA high alkaline xylanase-producing strain SX6-18 was successfully screened,which maintained relatively high enzyme activity in alkaline condition.This study laid a foundation of application of xylanase in papermaking and washing fields.