Objective: To evaluate the immune persistence following intradermal(ID) vaccination with diphtheria-tetanusacellular component pertussis and Sabin-derived inactivated poliovirus vaccine(DTacP-sIPV). Methods: 40 wistar rats were randomly assigned into four groups.Two test groups were injected intradermally with fractional-doses(1/5 and 1/10dose) of DTacP-sIPV(1/5D ID and 1/10D ID group);The positive control group was intramuscularly injected with full dose of DTacP-sIPV(full-dose IM group);The negative control group was injected with PBS intradermally.Wistar rats were immunized 3 times at 0,1 and 2 months and the blood samples were collected via tail vein 12 months after the last immunization and the serum samples were isolated.The titer of neutralizing antibody against poliovirus was detected by micro-neutralization test,and the titers of IgG antibodies against diphtheria toxin(DT),tetanus toxin(TT),pertussis toxin(PT),filamentous hemagglutinin(FHA) and pertactin(PRN) in rat serum were detected by indirect ELISA.The geometric mean titer(GMT)and positive rate of antibody were calculated.The rats were challenged with aerosolized B.pertussis for 30 min 12 months after the last immunization and determined for the white blood cell(WBC) count and colony-forming unit(CFU) in lung,trachea and nose at day 2,5 and 14 after challenge. Results: Compared with the full-dose IM group,there was no significant difference in the positive rates of poliovirus type Ⅰ,Ⅱ and Ⅲ neutralizing antibodies between 1/5D ID and 1/10D ID groups(each P> 0.05) and the positive rates of all types of antibodies in the control group were 0.The positive rates of IgG antibodies against DT,TT,PT,FHA and PRN in 1/5D ID,1/10D ID and full-dose IM groups were all 100%,and those in control group were all 0.Compared with 2 d after challenge,the WBC counts of rats in control group increased significantly 5 d after aerosol challenge with B.pertussis(F=3.48,P <0.05),and then began to decrease,while those in other groups remained stable with time(F=0.14～1.30,P> 0.05).After aerosol challenge,the CFU in lungs of rats in control group was significantly higher than that in the other three groups(F=19.00～206.00,P<0.05),and B.pertussis was still detected 14 d after challenge;Except for the control group,the bacterial load in lungs of rats in the other three groups reached the peak 5d after challenge,the B.pertussis was basically cleared on the 14d,and there was no significant difference among the groups at each time point(F=1.14～1.25,P> 0.05).The bacterial load of trachea and nose in the control group was slightly higher than that in other groups at each time point,but the difference was not significant(F=0.71～3.54,P> 0.05).Except for the control group,the bacterial load in the trachea and nose of the other three groups were similar,and no significant difference was observed(F=0.75～3.41,P>0.05). Conclusion ID immunization with1/5 dose of DTacP-sIPV induced persistent protective antibodies against various components of the vaccine in rats.This study provided an experimental basis for the formulation of immunization strategy of ID immunization with fractional dose of DTacP-sIPV.

Objective To explore the physical and chemical properties of electric charge modified liposomes and the immune enhancement effect as influenza vaccine adjuvants.Methods Four kinds of electric charge-modified liposomes were prepared with soybean lecithin,cholesterol,N-1-(2,3-dioleyloxy) propyl-N,N,N-trimethyllam-moniumchloride(DOTAP)and 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)(DOTAP and DOPC mass ratio 1:4,2:3,3:2 and 4:1).The tetravalent influenza vaccine was mixed with the four liposomes according to the volume fraction of 1.4:1.0,which was prepared into cationic modified influenza vaccine liposome freeze-dried powder by freeze-thaw freeze-drying method.The liposome freeze-dried powder was redissolved with PBS,the particle size and Zeta potential were measured by laserparticle size analyzer,and the encapsulation efficiency of liposome was measured by Lowry method.231 female KM mice were randomly divided into negative control group(PBS),positive control group(tetravalent vaccine bulk),cationic liposome group(four electric charge modified influenza vaccine liposomes) and neutral liposome group(non-electric charge modified influenza vaccine liposomes),which were injected intraperitoneally at 7,14 and 28 d,0.2 mL per mouse.The cellular immune effect was evaluated by MTT assay and T lymphatic surface labeling,and the humoral immune effect was evaluated by hemagglutinin inhibition(HI) at 3,7,14,28,42 and 56 d of immunization.Results When the mass ratio of DOTAP to DOPC was 4:1,the particle size and Zeta potential of cationic modified liposomes reached the maximum,which were 529.65 nm and 12.05 mV respectively,and the encapsulation efficiency was also high,which was 81.82%.Stimulus index(SI) and CD4~+/CD8~+value of spleen cells of mice in the four cationic liposome groups were significantly higher than those in the negative control group(F=4.651～25.866,each P<0.05),among which,the two indexes in 4:1 cationic liposome group were higher than those in the other three groups;mice in this group produced early humoral immune effect 3 d after immunization,and the antibody titer reached the highest 42 d after immunization and maintained a high level during the whole immunization cycle.Conclusion Cationic liposomes with different electric charge modifications improved the immune effect of influenza vaccine,the immune enhancement effect was the best and the immune effect lasted the longest when the mass ratio of DOTAP to DOPC was 4:1.

Objective To express the capsid proteins VP1 and VP3 of hepatitis A virus(HAV) in prokaryotic cells and evaluate their immunogenicity.Methods VP1 and VP3 gene fragments were amplified by PCR,cloned into vector pETG28a to construct recombinant expression plasmids pET-G28a-VP1 and pET-G28a-VP3,which were transformed into competent E.coli BL21(DE3),induced by IPTG,and then analyzed by 12% SDS-PAGE.The target protein was purified by ion exchange chromatography,renatured and combined with several adjuvants to immunize mice.The mice were divided into VP1-MF59,VP1-AH,VP1-AP,VP1-AP-10CpG,VP1-AP-50CpG,VP3-AP,VP3-VP1-AP and PBS control groups,five for each group.Serum IgG antibody titers of mice in various groups were detected by ELISA,and serum neutralizing antibody titers of mice in VP1-AP,VP3-VP1-AP and PBS control groups were detected by rapid fluorescence focus immunosuppression experiment.Results Colony PCR and sequencing showed that the recombinant plasmids pET-G28a-VP1 and pET-G28a-VP3were constructed correctly.The recombinant proteins VP1 and VP3,with relative molecular masses of about 37 000 and26 000 respectively,mainly existed in the form of inclusion bodies,the expression levels were 18.6% and 32.4%,and the purity was 86.3% and 84.7%,respectively.The recombinant proteins VP1 and VP3 reacted specifically with rabbit anti-HAV antiserum and mouse anti-HAV-VP3 antiserum respectively.The serum IgG antibody titer of mice in VP1-AP-50CpG group was significantly higher than that in VP1-AP group(q=22.05,P <0.01),and the serum IgG antibody titer of mice in VP3-VP1-AP group was significantly higher than that in VP1-AP group and VP3-AP group(q=22.05 and 22.49 respectively,each P <0.01).Compared with PBS control group,the serum neutralizing antibody titer of mice in VP1-AP and VP3-VP1-AP group increased significantly(q=7.79 and 25.11 respectively,P<0.01) Conclusion Prokaryotic HAV capsid proteins VP1 and VP3 showed high purity and good immunogenicity,and the addition of CpG in the preparation was beneficial to enhance the immunogenicity of antigens.This study laid a foundation of the development of HAV recombinant subunit vaccine.

Objective To analyze the evolutionary characteristics of GZ19 strain of G Ⅱ.4 norovirus(NoV) in China,and clarify its ability and mode of binding to receptors of histo-blood group antigens(HBGAs).Methods According to the sequence of ORF2 region in GZ19 strain,the evolutionary tree was constructed and the amino acid sequences at HBGA binding sites(HBSs) and key blocking epitopes were analyzed.P particles were expressed by prokaryotic expression system and purified.The obtained protein was identified by SDS-PAGE and indirect ELISA,and analyzed for the receptor binding characteristics of P particles by saliva binding and oligosaccharide binding assays.Results The GZ19 strain belonged to G Ⅱ.4Sydney [P31] lineage,of which the amino acid sequences of receptor binding sites and blocking epitopes were relatively conservative.It showed high homology with other G Ⅱ.4 Sydney [P31] strains in recent five years,while significant difference from G Ⅱ.4 Sydney 2012 original strain and G Ⅱ.4 Sydney [P16] strains.P particles only combined with A,B,O,AB secretory saliva and H-di oligosaccharide.Conclusion GZ19 strain represented the current evolutionary direction of G Ⅱ.4Sydney [P31] NoV.The successful expression of P particles and analysis of the binding characteristics with HBGA receptors laid a foundation of the research of epidemic evolution dynamics and vaccine development of G Ⅱ.4 NoVs in China.

Objective To investigate the biological distribution of B1 antisense RNA(Blas RNA) of mouse short interspersed nuclear element in blood and tissues of normal mice after vein injection and detect the cell uptake efficiency of B1 as RNA using cultured normal mouse embryo cells after transfection.Methods Six 8～12-week-old BALB/c mice,three males and three females,were injected with 20 μg Blas RNA via tail vein,and blood samples were collected at different times after injection.54 BALB/c mice of 8～12-weeks were injected with 20 μg Blas RNA via tail vein,of which six mice,three males and three females,were euthanized at different times after injection,and various tissues,including the heart,liver,spleen,lung,kidney and thymus were harvested.Blas RNA was transfected into cultured mouse embryonic cells,and a certain amount of cells were taken at different time after transfection.The biological distribution of B1as RNA in mouse blood and different tissues and the persistence of Blas RNA in cultured embryo cells were detected by RT-qPCR.30naturally senescent BALB/c mice(≥ 14 months old) were divided into three groups:treatment group(20 μg Blas RNA injected via tail vein,once a week),irrelevant RNA control group(20 μg LacZ3F3R RNA injected via tail vein,once a week) and saline control group(injected with the same volume of saline),with 10 mice in each group,and a young control group(normal young 8～12-week-old BALB/c mice,five males and five females) was set.Four weeks after administration,mice in each group were euthanized,the liver tissues were taken,and the expression levels of aging-related genes(Sirtl,p21,p16~(Ink4a),p15~(Ink4b),p19~(Arf)) were detected by RT-qPCR.Results After tail vein injection,Blas RNA was available in the blood of mice for approximately 30 min,persisted for approximately 2～4 h in most detected tissues and persisted for approximately 48 h in lungs.The efficiency of cellular uptake of Blas RNA was approximately 400 molecules per mouse embryo culture cell 45 min after transfection with B1as RNA.Compared with the saline control group,Blas RNA treatment significantly down-regulated the mRN A expression of p21,p16~(In4a),p15~(In4b) and p19~(Arf) genes(t=10.01,4.461,4.420 and 5.309 respectively,each P <0.05),and significantly up-regulated the mRNA expression of Sirt1 gene(t=4.579,P <0.05).Conclusion Blas RNA was efficiently taken up by cells after transfection.After intravenous injection,Blas RNA stayed in the blood and tissues for a certain period of time and regulated the expression of aging-related genes in aged mice so as to make them approach to the expression level of young normal mice.

Objective To investigate the expression of C-C chemokine ligand 5(CCL5) in head and neck squamous cell carcinoma(HNSCC),and explore the effect of CCL5 on the biological characteristics of laryngeal carcinoma cells.Methods Gene Expression Profiling Interactive Analysis(GEPIA) database was used to investigate the expression of CCL5 in HNSCC.The laryngeal carcinoma cells TU177 were transfected with siRNA(siRNA group),and the control(NC) group was set up.The cell proliferation,migration,cycle and apoptosis of each group were detected by CCK8 assay,cell scratch test and flow cytometry respectively.RT-PCR and Western blot were used to detect the knock-down efficiency of CCL5 and the mRNA transcription and protein expression of multidrug resistance protein 2(MRP2) and bcl-2-associated x protein(Bax).Results The expression of CCL5 in HNSCC was higher than that in normal tissues(P <0.05).Compared with NC group,siRNA showed higher knock-down efficiency(t=12.898 and 22.656 respectively,each P <0.01);siRNA interference with CCL5 inhibited the proliferation and migration of laryngeal carcinoma cells,and promoted the late apoptosis of laryngeal carcinoma cells and the expression of apoptosis protein Bax(t=2.600～11.667,each P <0.05).Conclusion CCL5 was highly expressed in HNSCC,while siRNA interference with CCL5 inhibited the proliferation,migration and promoted apoptosis of laryngeal carcinoma cells TU177 by up-regulating the expression of Bax,which laid a foundation of the possibility of CCL5 as a new target for the treatment of laryngeal carcinoma.

Objective To analyze the topology of IFN-induced transmembrane(IFITM) protein in porcine peripheral blood lymphocytes(PBMCs) and detect the change of IFITM mRNA transcription in PBMCs after porcine reproductive and respiratory syndrome virus(PRRSV) infection in vitro.Methods PRRSV,porcine circovirus 2(PCV2) and Japanese encephalitis virus(JEV) negative anticoagulant blood of piglets were collected aseptically and isolated for PBMCs.Porcine IFITM CDS sequence was amplified by PCR,sequenced and analyzed for topology.PBMCs were infected with PRRSV in vitro.Cell samples were collected at 12,24,36 and 48 h after infection,detected for PRRSV infection by RT-PCR,and detected for mRNA transcription level changes of IFITM1,IFITM2 and IFITM3 by RT-PCR.Results The porcine PBMCs were successfully isolated and the full-length sequence of IFITM CDS derived from PBMCs was cloned.The porcine IFITM protein might have two topological structures.PBMCs inoculated with PRRSV for 24 h produced obvious cytopathic effect.PRRSV was replicated in PBMCs.The transcription levels of IFITM1,IFITM2 and IFITM3 mRNA in PBMCs were significantly up-regulated at the early stage of PRRSV infection,and reached the peak at 12h after infection,and then gradually decreased;The transcription level of IFITM1 mRNA increased at 36 h after virus infection and then declined rapidly.Conclusion PRRSV infection in vitro significantly up-regulated the transcription level of IFITM mRNA in PBMCs,indicating that IFITM was involved in the antiviral immune response of PBMCs.This study provided a reference for revealing the natural immune response against PRRSV in vivo.

Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.

Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17) deletion on the production of reactive oxygen species(ROS) and mitochondrial function in nasopharyngeal carcinoma(NPC) cells.Methods Three groups of ADAM1 7 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1) and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP) by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA) kit and superoxide dismutase(SOD) kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034) and MDA content increased(t=7.415,P=0.038) significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM1 7 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in mitochondrion,reduced MMP,inhibited cell proliferation in vitro,and promoted apoptosis.

Objective To culture human sapovirus(HuSaV) GⅠ.1 in vitro and prepare polyclonal antibody against the capsid protein VP1.Methods HuSaV GⅠ.1 positive stool specimens preserved in diarrhea department of National Institute for Viral Disease Control and Prevention were inoculated with HuTu-80 cells supplemented with different bile acid salts[glycine chenodeoxycholic acid(GCDCA) and glycine cholic acid(GCA)],and the infection,proliferation and passage of the virus were determined by PCR and RT-qPCR.The VP1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1.The constructed recombinant expression plasmid pGEX-6P-1-VP1 was transformed into E.coli BL21(DE3) and induced by IPTG.Two female New Zealand white rabbits were immunized with the purified recombinant VP1 protein for 4 times.The blood samples were collected 18,28,38 and 48 d after immunization,and the serum titers were detected by ELISA.Results HuTu-80 cells were effectively infected by HuSaV GⅠ.1 in the presence of bile acid salt GCA,and the proliferated virus were stably and continuously transmitted for three generations in HuTu-80 cells.The expressed recom-binant GST-VP1 protein showed a relative molecular mass of about 86 000,and about 60 000 after purification(GST tag excision).The titer of polyclonal antibody against HuSaV VP1 protein was over 1:12 800.Conclusion HuSaV was successfully isolated and cultured in vitro using HuTu-80 cells supplemented with bile acid salt,and polyclonal antibody with high titer against HuSaV VP1 protein was prepared,which laid a foundation of in-depth research of HuSaV identification,infection and pathogenesis.