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Vaccine(2008)26,460—468Intranasal administration of a recombinant adenovirus expressing the norovirus capsidprotein stimulates specific humoral,mucosal,and cellular immune responses in miceLi Guo a,1,Jianwei Wang a,b,∗,1,Hongli Zhou a,b,Hongli Si a,c,Min Wang a, Jingdong Song b,Bingjuan Han b,Yi Shu a,Lili Ren b,Jianguo Qu b,Tao Hung a,ba National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing100052,Chinab State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,9#Dong Dan San Tiao,Beijing100730,Chinac North-western A&F University,Yangling712100,ChinaReceived18May2007;received in revised form19October2007;accepted18November2007Available online4December2007KEYWORDSNorovirus capsidprotein;Adenovirus vector;Immune responseSummary Norovirus(NV)is a major cause of acute,epidemic nonbacterial gastroenteritis inindividuals of all ages.The immunological mechanism of NV infection and the approaches usedto prevent infection remain to be elucidated.In this study,the specific immune responses ofBALB/c mice were assessed following intranasal immunization with a recombinant adenovirusvector expressing the genogroup II4(GGII/4)norovirus capsid protein.Analysis of IgM,IgG,andIgA antibodies specific for the recombinant virus-like particles(VLPs)of NV demonstrated that ahigh level of humoral immunity developed following immunization.Mucosal immune responseswere also detectable in stool,intestinal homogenates,lung homogenates,and lung lavage sam-ples.Specific cellular immune responses were observed in NV VLPs-restimulated splenocytesby ELISPOT and Th1/Th2cytokine cytometric array(CBA).Serum IgG subclass analysis showedthat a balanced Th1-and Th2-like cellular immune response was induced in BALB/c mice follow-ing immunization with recombinant adenovirus.Thesefindings demonstrate that the intranasalimmunization of a recombinant adenovirus expressing the NV capsid protein is an efficient strat-egy to stimulate systemic,mucosal,and cellular Th1/Th2immune responses in mice,and couldserve as a novel approach for designing NV vaccines.©2007Elsevier Ltd.All rights reserved.∗Corresponding author at:State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Chinese Academy of Medical Sciences,9#Dong Dan San Tiao,Beijing100730,China.T el.:+861065105188;fax:+861065105188.E-mail address:wangjw28@(J.Wang).1These authors contributed equally to this work.0264-410X/$—see front matter©2007Elsevier Ltd.All rights reserved.doi:10.1016/j.vaccine.2007.11.039Immunity against norovirus induced by recombinant adenovirus461IntroductionNorovirus(NV),or Norwalk-like virus,is a genus of the Caliciviridae family and is recognized as a major cause of acute,epidemic nonbacterial gastroenteritis in individuals of all ages[1,2].NV-related gastroenteritis outbreaks usu-ally occur in families,schools,nursing homes,hospitals,and military facilities through contact with contaminated food and water[2—4].While NV gastroenteritis is a mild self-limiting illness,it is associated with a high incidence of morbidity and hospitalization even in industrialized coun-tries.Recent studies have illustrated that∼90%of acute nonbacterial gastroenteritis outbreaks in the United States were caused by NV[5—7].In England and Wales,NV is responsible for almost50%of gastroenteritis outbreaks[8]. Finland,Sweden,Netherlands,Germany,and Japan have similar infectivity rates[9—12].In developing countries,NV-associated disease also takes an economic toll,especially in the young and very old[13,14].For these reasons,it is important to develop an effective NV vaccine especially for these at-risk populations[15].NV is composed of a7.4—8.3kb positive-sense,single-stranded RNA genome with three open reading frames (ORFs)[16,17].ORF1encodes a non-structural polyprotein that contains the3D RNA-dependent RNA polymerase region, ORF2encodes the viral capsid(VP-1),and ORF3encodes a minor basic virion protein(VP-2)[18].ORF2expres-sion by recombinant baculovirus in insect cells[19]or by the Venezuelan equine encephalitis virus replicon vector in mammalian cells[20,21]yields self-assembled virus-like particles(VLPs).These recombinant NV VLPs are morpho-logically and antigenically similar to the wild-type virus [20,21].Since cell culture systems and animal models of NVs are lacking[2],little is known about the details of the anti-NV immune response and few approaches have been designed to stimulate long-term,stable Th1/Th2-balanced immunity that would be required for a reliable prophylaxis. Recombinant VLPs may thus serve as an important source of antigen to study anti-NV immunity.Recent studies sug-gest that rNV VLPs can elicit systemic and mucosal immune responses in CD1and BALB/c mice,indicating that they may serve as a potential mucosal vaccine against NV[21—24]. However,these responses were only achieved by the addi-tion of mucosal adjuvants such as cholera toxin(CT)or the mutant E.coli heat-labile toxin,LT(R192G)[21—23].Since such adjuvants cannot be used in humans as a result of their potential toxicity,it is important to develop other immu-nization approaches to vaccine development.In this report,we determined whether intranasal immu-nization of mice with a recombinant adenovirus,rvAdGGII4, expressing the NV capsid,could elicit a strong anti-NV immune response,and illustrated a potential new approach for NV vaccine development.Materials and methodsPreparation of the recombinant adenovirus expressing NV capsid proteinThe GGII/4NV capsid protein gene from Lordsdale virus (Genbank X86557)was codon-optimized and artificially syn-thesized by adopting codons that were used in insect cells as described previously[25].The recombinant adenovirus, rvAdGGII4,was generated in HEK293cells using the AdEasy adenoviral vector system(Stratagene,Cedar Creek,TX) according to the manufacturer’s protocol.NV capsid pro-tein expression was confirmed by Western blot using an anti-NV capsid monoclonal antibody(Guo L.et al.,unpub-lished data).The recombinant adenovirus was purified by cesium chloride density gradient centrifugation[26],titered with an Adeno-X TM Rapid Titer Kit(BD Biosciences Clon-tech,Mountain View,CA)and stored at−70◦C prior to use.Preparation of NV-like particlesNV VLPs were prepared and purified as previously described, with minor modifications[27].The optimized GGII/4NV cap-sid gene was cloned into the baculovirus transfer vector, pFastBac1(Invitrogen,Carlsbad,CA)and a recombinant baculovirus was generated in Sf9cells using the Bac-to-Bac®Baculovirus Expression System(Invitrogen,Carlsbad,CA) protocol provided by the manufacturer.Recombinant bac-ulovirus infected Sf9cell supernatants were collected5days post-infection and cellular debris was removed by centrifu-gation(20min at10,000rpm).VLPs were precipitated from the clarified supernatants using PEG6000(6%),and the pel-lets were sonicated and centrifuged at35,000rpm through a40%sucrose cushion for3h.Purified VLPs were confirmed by Western blot using an anti-NV capsid monoclonal antibody [25].Protein concentrations were determined using the BCA Protein Assay Reagent Kit(Pierce,Rockford,IL)and stored at−70◦C prior to use.Animal immunization and sample collectionSix-to8-week-old female BALB/c mice,purchased from the Institute of Laboratory Animal Sciences at the Chinese Academy of Medical Sciences,were used for all immuniza-tions and maintained in Animal Biosafety Level-2facilities. After intranasal anesthetization with ether,mice were intranasally(i.n.)inoculated with1×106ifu(infectious units)(in0.1ml)of the recombinant adenovirus,rvAdG-GII4(rvAdGGII4group),1×106ifu(in0.1ml)of the empty recombinant adenovirus serotype5vector,which contained no insert(rvAd5-empty group,negative control),or0.1ml phosphate-buffered saline(PBS;PBS group,blank control) at day0.All mice were boosted at days14and28.Serum and fecal samples from all groups were col-lected at days0,14,28,and35post-inoculation(dpi),and stored at−20◦C prior to testing.At35dpi,the mice were sacrificed,and lung lavages,lung homogenates,and intesti-nal homogenates were used to measure mucosal immune responses as previously described[28]with minor modifica-tions.In brief,the lungs and20-cm sections of the small intestines excluding the Peyer’s patches were ground into homogenates using a high-speed tissue grinder,suspended in PBS(pH7.2)to produce a10%(w/v)suspension,and cen-trifuged at3000rpm for5min.The supernatants and lung lavages were stored at−70◦C prior to use.Splenocytes were also isolated and restimulated in order to measure cellular immune responses.462L.Guo et al.Analysis of NV-specific antibody production Antibodies in the sera,fecal matter,intestinal homogenates, lung homogenates,and lung lavages were measured by indi-rect ELISA.Microtiter plates(Costar,Bethesda,MD)were coated with0.1g VLPs from GGII/4NV per well at4◦C overnight.The plates were blocked with1%(w/v)BSA in PBS at37◦C for2h.The fecal samples were diluted into a10%(w/v)suspension in PBS.Serum and fecal extracts were twofold serially diluted using0.1%BSA in PBS.Intesti-nal homogenates,lung homogenates,and lung lavages were diluted1:10.Samples were added to the wells and incubated for1h at37◦C.After washingfive times with0.05%T ween 20in PBS(PBS-T),the plates were incubated at37◦C for1h using a1:5000dilution of horseradish peroxidase-conjugated goat anti-mouse IgG,IgA,IgM,IgG1,or IgG2a(Sigma, St.Louis,MO).Plates were washed and developed with 100l/well of0.1mg/ml3,3 ,5,5 -tetramethylbenzidine (TMB;Sigma,St.Louis,MO)peroxidase substrate containing 0.03%hydrogen peroxide.Color development was stopped by the addition of50l/well of2M sulfuric acid.The absorbency at450nm(A450)was determined using an ELISA plate reader(BioRad550,Hercules,CA).IFN-␥ELISPOT assayMurine spleens were removed and ground under sterile conditions using a5-ml syringe plunger,and splenocytes were isolated with Mouse Lymphoprep(Dakewe,Shenzhen, China).The freshly made splenocytes were washed twice and resuspended in R10medium[RPMI1640supplemented with10%fetal bovine serum(FBS),100IU/ml penicillin, and100g/ml streptomycin(Hyclone,Logan,UT)].IFN-␥ELISPOT assays were performed using an ELISPOT Set(BD Biosciences,San Diego,CA)as recommended by the manu-facturer.In brief,5×105freshly isolated splenocytes were added to each of two replicate wells coated with anti-mouse IFN-␥monoclonal antibody(MAb)(BD Biosciences,Catalog No.51-2525kc)and stimulated with20g/ml VLPs at37◦C in a5%CO2incubator for24h.Unstimulated splenocytes were used to measure background cytokine production.The cells were then lysed with deionized(DI)water,and the plates were incubated at room temperature with biotinylated IFN-␥antibody(BD Biosciences,Catalog No.51-1818kz)for2h, and peroxidase-labeled streptavidin(BD Biosciences,Cata-log No.51-9000209)for1h.After washing with PBS,100l of thefinal substrate solution(BD Biosciences,Catalog No. 551951)was added to each well and spot development was monitored.The plates were washed with distilled water to stop the reaction.IFN-␥spot-forming cells(SFC)were counted using an automated ELISPOT plate reader system. Results were expressed as the number of SFC/106cells. Assay for multi-cytokine productionFreshly isolated murine splenocytes were cultured in a96-well,round-bottom tissue culture plate at5×105cells/well in R10medium and stimulated with NV VLPs for48h.Super-natants were collected and IL-2,IL-4,IL-5,TNF-␣,and IFN-␥levels were quantitated using the mouse Th1/Th2Cytokine Cytometric Array Bead(CBA)Kit(BD PharMingen,San Diego,CA)according to the manufacturer’s protocol.The data were acquired with a FACSCalibur®flow cytometer(BD Bio-sciences,San Jose,CA)using2-color detection and analyzed using CBA software(BD PharMingen).Analysis of anti-adenoviral serotype5(Ad5) immunityThe anti-Ad5serum IgG,IgM,and IgA antibodies were ana-lyzed by indirect ELISA as described in Section of Analysis of NV-specific antibody production.The microtiter plates (Costar,Bethesda,MD)were coated with1×105ifu(in 0.1ml)of wild serotype5adenovirus,which was puri-fied using the CsCl2cushion ultra-centrifuge[26],at4◦C overnight.Statistical analysisSerum and fecal antibody titers were log10transformed, expressed as geometric mean titers(GMT s),and com-pared using the Student’s t-test.The Wilcoxon rank sum test was used to compare intestinal homogenates,lung homogenates,and lung lavage antibody levels between groups.ELISPOT and CBA results between groups were compared using the Student’s t-test.Nonresponders were included in all calculations.The lowest serum and fecal sample dilution(1:50)was divided by two and used as the negative sample titer(i.e.,negative samples were assigned a titer of25).All tests were two-tailed,and a value of p<0.05was considered statistically significant.ResultsHumoral immune responses in miceT o determine whether the recombinant adenovirus,rvAdG-GII4,could stimulate specific immune responses following i.n.administration,BALB/c mice were immunized three times on days0,14,and28,respectively.The control groups were treated with rvAd5-empty or PBS accord-ing to the same scheme.All preimmune serum titers of IgM,IgA,and IgG antibodies against norovirus VLPs or adenovirus were negative(<50)(data not shown).In addi-tion,each of the control mice remained negative for VLP antibodies at35dpi.After thefirst i.n.administra-tion of rvAdGGII4,however,100%of the mice had strong specific serum IgM titers(GMT=315.9).The GMT of IgM peaked(GMT=348.2)after the second immunization(28dpi) and decreased(GMT=219.1)after the third immunization (35dpi)(Fig.1A).Serum IgG responses were mounted by allfive mice at day14post-immunization with rvAdGGII4 (GMT=3816,ranging from3200to10,000).After the sec-ond(28dpi)and the third immunizations(35dpi),IgG titers continued to increase,with GMT s of155,261and632,978, respectively(Fig.1B).No mice had detectable levels of serum IgA antibodies by14dpi.Serum-specific IgA anti-body titers were detected in all groups after the second immunization(GMT=377.6,ranging from300to800)and increased after the third immunization(GMT=2009.5,rang-ing from1600to3200)(Fig.1C).IgM,IgG,and IgA responsesImmunity against norovirus induced by recombinant adenovirus463Figure 1Humoral responses in mice sera.BALB/c mice were immunized as described in Section 2.Blood samples were col-lected at 14,28,and 35dpi,respectively .Serum-specific IgM (A),IgG (B),and IgA (C)antibodies from individual mice were examined by ELISA and used to calculate the GMT for each group.For the IgM-negative,IgG-negative,and IgA-negative (<50)control samples,titers of 25were used to calculate the GMT .Days post-immunization schedule are shown on the X-axis.Error bars represent the standard errors of the means.Above each bar is the number of responders over the total number of mice tested.were undetectable in mice treated with the rvAd5-empty vector or PBS.These data indicate that rvAdGGII4stimu-lates a strong NV specific humoral response and that serum antibodies were substantially enhanced following rvAdGGII4immunization.Figure 2Fecal IgA and IgG responses in BALB/c mice.Murine fecal samples were collected at 35dpi.Levels of specific IgG and IgA antibodies in the feces were measured by ELISA and used to calculate the GMTs.The error bars show the standard errors of the mean.The number above each bar depicts the number of responders over the total number of animals tested.Serum IgG subclass analysisAnti-VLPs IgG1and IgG2a antibodies were measured in NV VLP-specific IgG positive sera (T able 1).The increase in IgG1and IgG2a levels coincided with the rise in serum IgG.Sig-nificantly high titers of both IgG1and IgG2a were observed following administration of rvAdGGII4,and there was no significant difference between these class types (p >0.05,Student’s t -test).Thus,the Th1-(IgG2a)and Th2-like (IgG1)immune responses were roughly balanced in rvAdGGII4-immunized mice.Mucosal responses in miceT o evaluate whether recombinant adenovirus could stimu-late specific mucosal responses,fecal suspensions,intestinal homogenates,lung homogenates,and lung lavages were prepared from immunized mice.Mucosal immune responses to NV VLPs were observed in all vaccinated mice fol-lowing primary i.n.administration of 1×106ifu rvAdGGII4and after each rvAdGGII4boost immunization.All immu-nized mice (5/5)had NV VLP-specific fecal IgG and IgA responses (GMT =470.43and 819.07,respectively),while the rvAd5-empty and PBS groups remained nega-tive (titers <50)(Fig.2).IgA and IgG levels in intestinal homogenates,lung homogenates,and lung lavages were evaluated at a single dilution (1:10),and results were expressed as A 450values.Strong IgA and IgG responses were induced in the intestinal homogenates (p <0.05for IgA,p <0.001for IgG,respectively),lung homogenates (p <0.001for IgA and IgG,respectively)and lung lavage samples (p <0.001for IgA and IgG,respectively)(Fig.3).TheseTable 1Analysis of serum Ig antibody isotypes to norovirus VLPsImmunogenDays post-immunizationGeometric mean titer IgG1/IgG2aIgG1IgG2a rvAdGGII435201189.380546.30.539rvAd5-empty 35<50<50PBS 35<50<50464L.Guo etal.Figure3Levels of specific IgA(A)and IgG(B)antibodies in the intestines and lungs of BALB/c mice immunized with rvAdGGII4. At days14and28following the primary immunization,each group of mice was administered the same amount of antigen, and intestinal homogenates,lung homogenates,and lung lavage were obtained from each animal1week later.Levels of specific IgA and IgG antibodies were examined by ELISA.The results are represented as the mean OD(450nm)that was detected in individual mice at a1:10dilution.Error bars represent the standard errors of the mean.data suggest that rvAdGGII4elicits strong mucosal immune responses.IFN-␥ELISPOT responses and cytokine assaysT o measure the cellular immune responses elicited by i.n. administration of rvAdGGII4,the specific T-cell responses against NV VLPs were monitored in immunized mice by IFN-␥ELISPOT.The mean number of SFC/106in the rvAdGGII4 group was108±31.No spots were detected in unstimulated splenocytes from the rvAdGGII4group.The level of SFC/106 in the rvAd5-empty and PBS control groups were14±2and 9±7,respectively.The differences between the rvAdGGII4 group and the rvAd5-empty or PBS groups was statistically significant(p<0.01and0.001,respectively)(Fig.4).These findings suggest that mice receiving the rvAdGGII4vector developed cellular immune responses to the NV capsid pro-tein.T o further characterize the cellular immune response, rvAdGGII4immunized splenocytes were isolated and T-cell responses were assessed using a Mouse Th1/Th2Cytokine Cytometric Bead Array system.This allows simultaneous measurement of TNF-␣,IFN-␥,IL-5,IL-4,and IL-2lev-els in antigen-stimulated T-cell supernatants.After the mice were immunized with rvAdGGII4and stimulated with NV VLPs,IFN-␥,TNF-␣,and IL-5production were notably high(217.71pg/ml,156.5pg/ml and265.27pg/ml,respec-tively),while IL-2and IL4levels were only moderately increased(32.2pg/ml and27.59pg/ml,respectively).TNF-Figure4IFN-␥ELISPOT assays of recombinant norovirus VLP-stimulated splenocytes following rvAdGGII4immunization. Splenocytes were isolated7days after thefinal immunization and restimulated with20g/ml VLP for24h.IFN-␥-producing cells were quantitated using an ELISPOT reader.Results were expressed as the average number of SFC per million input splenocytes.Error bars represent the standard errors of the mean.␣,IFN-␥,IL-5,IL-4,and IL-2levels were significantly higher in splenocyte supernatants from rvAdGGII4immu-nized mice than from rvAd5-empty vector(33.63pg/ml, p≤0.001;2.43pg/ml,p<0.001;0pg/ml,p<0.001;0pg/ml, p<0.001and 3.09pg/ml,p<0.05,respectively)or PBS-treated mice(36.22pg/ml,p<0.05;1.22pg/ml,p<0.001;0.42pg/ml,p<0.001;0pg/ml,p<0.001and 1.19pg/ml, p<0.01,respectively)(Fig.5).Since IFN-␥,TNF-␣,and IL-2 are markers of a Th1response,while IL-5and IL-4are mark-ers of a Th2response,these results indicated that the Th1-and Th2-like cellular immune responses were both stimu-lated by i.n.administration of rvAdGGII4.Anti-Ad5antibodies in miceT o determine whether the recombinant adenovirus,rvAdG-GII4,could elicit anti-Ad5immune responses following i.n. administration,anti-Ad5serum antibody levels were mea-sured by indirect ELISA.All immunized mice hadanti-Ad5 Figure5Cytokines detected in splenocyte cultures from mice immunized with rvAdGGII4.Splenocytes were isolated7days after thefinal immunization and stimulated with NV VLPs for 48h.TNF-␣,IFN-␥,IL-5,IL-4,and IL-2levels were measured in the culture supernatant using the Cytometric Bead Array. Significant differences were observed between the rvAdGGII4 and rvAd5-empty or PBS group.Error bars indicate the standard errors of the mean.Immunity against norovirus induced by recombinant adenovirus465Figure6Anti-Ad5immunity in mice after rvAdGGII4immu-nization.Blood samples were collected at14,28,and35dpi, respectively.Anti-Ad5antibodies were assessed by ELISA and used to calculate the GMT s.The days post-immunization are shown on the X-axis,and the error bars reveal the standard errors of the mean.The number above each bar depicts the number of responders over the total number of animals tested.serum IgG responses following the primary i.n.rvAdGGII4 administration and boost(GMT=299.3,1912.7and6964.4, respectively)(Fig.6).The IgM responses were not observed in mice after thefirst administration.The GMT of IgM was 65.9after the second immunization(28dpi),and decreased to<50after the third immunization(35dpi)(Fig.6).While no detectable anti-Ad5IgA antibodies were found in the sera after thefirst and second immunizations,anti-Ad5serum-specific IgA antibody titers could still be detected in mice after the third immunization(GMT=269.9)(Fig.6).DiscussionSince NV infections are localized in the gastrointestinal tract,local mucosal immunity is believed to play an impor-tant role in protection against infection.It is accepted that an ideal NV vaccine would induce strong mucosal immu-nity[29].In previous studies,anti-NV immunity using VLPs primarily relied on the adjuvants like enterotoxin-based LT or CT,since VLPs are too weak to stimulate immune responses,especially within the mucosa.However,CT and LT are potentially toxic to the human central nervous sys-tem[30].An intranasal influenza vaccine containing LT as a mucosal adjuvant,which resulted in46cases of Bell’s palsy in Switzerland,was recently removed from the mar-ket[31],indicating that CT or LT adjuvants are not suitable for humans.Thus,it is important to develop a novel immu-nization strategy that does not require the use of mucosal adjuvants.The replication-defective adenovirus serotype5vector is recognized as an attractive vector in both gene ther-apy and novel genetic vaccine development[32].Its safety and efficacy is shown in numerous animal experiments and human clinical trials,and two adenovirus-based gene ther-apy drugs are licensed.Adenovirus vectors have been used in studies of potential Ebola virus,human immunodefi-ciency virus type1(HIV-1),influenzavirus,and tuberculosis vaccines[33—39].Clinical trials using adenoviral-vectored vaccines against influenza virus,HIV-1,some malignant tumors,and other pathogens are currently in progress [40—43].These clinical trials show that adenoviral vector-based vaccines are safe,effective,and have no known toxicity or inflammatory properties.Adenovirus vectors are also shown to boost CTL responses against heterologous gene products[44—47].However,existing anti-adenoviral immune responses impair foreign antigen expression after repeated intramuscular immunizations.Since humans are widely infected by adenoviruses and usually possess high anti-adenovirus antibody levels,existing immunity often limits application in cases where repeated immunization is required.Recently,intranasal immunization has become an attractive method for inducing multiple immunities.Thus, we tested the immunogenicity of the NV capsid protein after repeated i.n.administration.We illustrated that sig-nificant serum,mucosal,and cellular immune responses against NV VLPs were elicited after mice were immu-nized with the recombinant adenovirus.Serum IgM and IgG responses against NV VLPs rose remarkably after the first rvAdGGII4inoculation,and IgM,IgG,and IgA antibody titers increased to high levels after the third immunization. IgA and IgG responses were also observed in fecal sam-ples,intestinal homogenates,lung homogenates,and lung samples.Thesefindings suggest that the i.n.route is an effi-cient pathway to evoke NV capsid-specific immunity by an adenovirus vector and the regionalization of the mucosal immune system allows immunizations in the gut by deliver-ing a vaccine through the intranasal route.The results also show that repeated i.n.administration of the adenovirus vector effectively boosted NV capsid-specific serum antibod-ies,demonstrating that existing anti-adenoviral immunity did not impair the recombinant adenovirus-specific immune response.Thesefindings are similar to our results using an adenovirus vectored rotavirus antigen[48].Unexpectedly,intestinal homogenates from the rvAd5-empty and PBS control-treated mice produced low SIgA levels.We speculate that there may have been low SIgA levels in the mouse intestine.The intestine is the largest immunological organ,housing70—80%of all immunoglobulin-producing cells and producing large amounts of SIgA(50—100mg/kg body weight)[49].High background on the ELISA assay may be attributed to the interference of intestinal SIgA or other intestinal contents.Previous studies showed that cytotoxic T lymphocytes (CTLs)play an important role in preventing viral diseases and clearing viruses like hepatitis C[50].Cellular immune response may also be crucial for disease prevention,as well as control and clearance of NV infection.Lindesmith et al.first characterized the Th1and Th2cellular immune responses to a challenge with Snow Mountain virus[51]. Recent studies showed that BALB/c mice that were i.n. immunized with VLPs and the mucosal adjuvants,LT or LT (R192G),induced specific Th1/Th2-like immune responses [27].However,there has been little information regarding the anti-NV cellular immune response.T o measure the cel-lular immune responses in rvAdGGII4-immunized mice,we measured NV VLP-specific T-cell responses using the IFN-␥ELISPOT assay.Our results indicate that the recombinant adenovirus,rvAdGGII4,was able to induce a strong cellular immune response.Importantly,both Th1-and Th2-like cel-lular immune responses were induced in rvAdGGII4-treated mice.It is well known that Th1-associated cytokines help to regulate antiviral cellular responses,while Th2-associated cytokines enhance humoral immune responses.In our study, TNF-␣and IFN-␥secretion were significantly increased,and IL-2levels were moderately increased.At the same time,466L.Guo et al.the Th2cytokine,IL-5,which promotes IgA production, was significantly elevated,afinding consistent with high serum and mucosal IgA antibody titers.IL-4levels were also bined with analysis of serum IgG1/IgG2a lev-els,these data suggest that a roughly balanced Th1/Th2 immune response is induced by the adenovirus vectored NV capsid.Due to the unavailability of an animal model of viral chal-lenge,it remains unknown whether the immunity evoked by i.n.adenovirus immunization is capable of protecting against NV infection.However,we may anticipate that it would have a protective effect based on knowledge obtained from studies of other viral infections like rotavirus,which also infect the intestinal surface.Evidence suggests that high local mucosal immune responses against rotavirus are critical for resisting viral infection.Previous studies showed that serum and fecal IgA antibodies are associated with protection against rotavirus reinfection[52,53].Moreover, about a recent study showed that30—40%of volunteers developed mucosal anti-VLP IgA after oral NV VLP immuniza-tion[54].Thus,it is reasonable to speculate that high levels of immunity stimulated by recombinant adenoviruses play an important role in preventing NV infection[21].However, the precise level of protection will require further challenge experiments using volunteer or surrogate challenge models, such as recombinant vaccinia virus,that have been used to study hepatitis C virus(HCV)[55]and hantaan virus infec-tion[56].Further studies will also be required to determine the duration of immunity,and the relationships between the three immune responses,including their cross-protective role with other noroviruses.Although the regionalization of the mucosal immune sys-tem permits gut immunizations through intranasal delivery of a vaccine,swallowing of the inoculum by the vaccinated animals cannot be excluded.Since NV attacks and repli-cates in the small intestine,local immunization of the gut may help to protect the host from NV infection.The experi-ence of Adenovirus4and7in the US Army showed that oral immunization is safe and can evoke strong immune responses over the past four decades[57].Thus,oral immunization may be a potential way to stimulate NV-specific immunities using recombinant adenovirus immunization.In addition, oral immunization is easier to manage and may be better accepted by children.A recent study showed that oral immu-nization of NV VLPs can produce cross-reactive monoclonal antibodies[58].Thus,the feasibility of oral immunization using the NV capsid protein-expressing recombinant aden-ovirus should be addressed.AcknowledgementsWe are grateful to Dr.Zhendong Zhao(Peking University)for critical reading of the manuscript,Drs.Li Ruan and Xian-grong Qi(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Preven-tion)for their assistance in ELISPOT assay,and Ms.Shan Mei and Li Li(Institute of Pathogen Biology,Chinese Academy of Medical Sciences)for their assistance in FACS assays.This research was supported by grants from the Chinese Key T ech-nologies R&D Program(No.2003BA712A03—04)and China Nature Science Foundation(No.30700741).References[1]Hedberg CW,Osterholm MT.Outbreaks of food-borneand waterborne viral gastroenteritis.Clin Microbiol Rev 1993;6(3):199—210.[2]Kapikian AZ,Estes MK,Chanock RM.Norwalk group of viruses.In:Fields B,editor.Fields Virology,vol.2,3rd ed.1996.p.783—810.[3]Arness MK,Feighner BH,Canham ML,T aylor DN,Monroe SS,Cieslak TJ,et al.Norwalk-like viral gastroenteritis outbreak in U.S.Army trainees.Emerg Infect Dis2000;6(2):204—7.[4]Koopmans M,Vinjeˇıde Wit M,Leenen I,van der Poel W,van Duynhoven Y.Molecular epidemiology of human enteric caliciviruses in The Netherlands.J Infect Dis2000;181(Suppl2):S262—9.[5]Fankhauser RL,Noel JS,Monroe SS,Ando T,Glass RI.Molecular epidemiology of‘‘Norwalk-like viruses’’in out-breaks of gastroenteritis in the United States.J Infect Dis 1998;178(6):1571—8.[6]Glass RI,Noel J,Ando T,Fankhauser R,Belliot G,MountsA,et al.The epidemiology of enteric caliciviruses from humans:a reassessment using new diagnostics.J Infect Dis 2000;181(Suppl2):S254—61.[7]Shieh Y,Monroe SS,Fankhauser RL,Langlois GW,Burkhardt IIIW,Baric RS.Detection of norwalk-like virus in shellfish impli-cated in illness.J Infect Dis2000;181(Suppl2):S360—6.[8]Dedman D,Laurichesse H,Caul EO,Wall PG.Surveillance ofsmall round structured virus(SRSV)infection in England and Wales,1990—1995.Epidemiol Infect1998;121:139—49.[9]Lew JF,Valdesuso J,Vesikari T,Kapikian AZ,Jiang X,EstesMK,et al.Detection of Norwalk virus or Norwalk-like virus infections in Finnish infants and young children.J Infect Dis 1994;169:1364—7.[10]Hedlund KO,Rubilar-Abreu E,Svensson L.Epidemiology ofcalicivirus infections in Sweden,1994—1998.J Infect Dis 2000;181(2):S275—80.[11]Schreier E,Doring F,Kunkel U.Molecular epidemiology ofoutbreaks of gastroenteritis associated with small round structured viruses in Germany in1997/98.Arch Virol 2000;145:443—53.[12]Inouye S,Yamashita K,Yamadera S,Yoshikawa M,Kato N,OkabeN.Surveillance of viral gastroenteritis in Japan:pediatric cases and outbreak incidents.J Infect Dis2000;181(2):S270—4. [13]Pujol FH,Vasquez G,Rojas AM,Fuenmayor ME,Loureiro CL,Perez-Schael I,et al.Norwalk virus infection in Venezuela.Ann Trop Med Parasitol1998;92(2):205—11.[14]Parks CG,Moe CL,Rhodes D,Lima A,Barrett L,T seng F,et al.Genomic diversity of‘‘Norwalk like viruses’’(NL Vs): pediatric infections in a Brazilian shantytown.J Med Virol 1999;58(4):426—34.[15]Estes MK,Ball JM,Guerrero RA,Opekun AR,Gilger MA,PachecoSS,et al.Norwalk virus vaccines:challenges and progress.J Infect Dis2000;181(Suppl2):S367—73.[16]Xi JN,Graham DY,Wang KN,Estes MK.Norwalk virus genomecloning and characterization.Science1990;250(4987):1580—3.[17]Jiang X,Wang M,Wang K,Estes MK.Sequence and genomicorganization of Norwalk virus.Virology1993;95(1):51—61. [18]Zintz C,Bok K,Parada E,Barnes-Eley M,Berke T,Staat MA,et al.Prevalence and genetic characterization of caliciviruses among children hospitalized for acute gastroenteritis in the United States.Infect Genet Evol2005;5(3):281—90.[19]Jiang X,Wang M,Graham DY,Estes MK.Expression,self-assembly,and antigenicity of the Norwalk virus capsid protein.J Virol1992;66(11):6527—32.[20]Baric RS,Yount B,Lindesmith L,Harrington PR,Greene SR,T seng FC,et al.Expression and self-assembly of norwalk virus capsid protein from Venezuelan equine encephalitis virus repli-cons.J Virol2002;76(6):3023—30.。
三篇会计专业毕业参考文献.doc
三篇会计专业毕业参考文献引导语：是在学术研究过程中，对某一著作或的整体的参考或借鉴，征引过的文献在注释中已注明，不再出现于文后中。
下面是三篇关于会计的毕业参考文献。
三篇会计专业参考文献会计专业毕业参考文献(一)(1)期刊：[序号]作者.题名[J].期刊名称，出版年份，卷号(期号)：起止页码.(2)普通图书：[序号]著者.书名[M].版次(第一版应省略).出版地：出版者，出版年份：起止页码.(3)集、会议录：[序号]著者.题名[C].编者.集名.出版地：出版者，出版年份：起止页码.(4)学位：[序号]作者.题名[D].保存地.保存单位，年份.(5)专利文献：[序号]专利申请者或所有者.专利题名：专利国别，专利号[P].公告日期或公开日期.(6)国际、国家标准：[序号]标准代号，标准名称[S].出版地：出版者，出版年份.(7)报纸文章：[序号]作者.题名[N].报纸名，出版日期及期号(版次).(8)电子文献：[序号]作者.电子文献题名[EB/OL].电子文献的出版或可获得地址，发表或更新日期/引用日期(任选).会计专业毕业参考文献(二)[1] 王棣华. 我国管理会计的前瞻与反思[J]. 中国农业会计, 1997,(10)[2] 吴福林. 让管理会计在企业管理中灿烂[J]. 辽宁财税, 2003,(12)[3] 林琤. 浅议我国推行管理会计的途径[J]. 福建省社会主义学院学报, 2004,(04)[4] 贺德文. 企业需要管理会计[J]. 交通财会, 1988,(06)[5] 张宗强, 杨素华. 对管理会计在国有大中型企业应用的思考[J]. 河北广播电视大学学报, 2004,(04)[6] 陈巧媚. 管理会计应用中的问题及对策初探[J]. 福建金融管理干部学院学报, 2004,(04)[7] 陈春红, 冯民修, 王剑青. 管理会计发展的策略[J]. 林业财务与会计, 2000,(03)[8] 郑玉革. 管理会计及其在我国的应用[J]. 边疆经济与文化, 2004,(10)9] 罗魏冰. 管理会计路在何方[J]. 广东审计, 2002,(10)[10] 李寿文, 徐光华. 对发展我国管理会计的建议[J]. 商业会计, 1998,(07)[11]崔澜,物流管理会计与物流企业降低成本的基本途径[J]. 物流科技,2008,(4).[12]吴凤山,何光裕. 科技进步与降低成本是现代会计研究的核心[J]. 财会通讯,1994,(1).[13]以提高经济效益为中心探求降低成本的新途径第三次全国成本理论讨论会在湖北省宜昌市召开湖北省会计学会一九八二年理论讨论会同时举行[J]. 财会通讯(综合版),1982,(5).[14]朱先乔,. 努力降低成本提高经济效益全国第三次成本理论讨论会和湖北省会计学会年会学术观点简介[J]. 财会通讯(综合版),1982,(6).[15]瓦依斯巴尔德,马之駉,. 车间经济核算是降低成本的重要杠杆原文载于苏联会计杂志一九五四年第七期[J]. 钢铁,1954,(5).会计专业毕业参考文献(三)随着资本市场对经济发展日益突显的促进作用，世界各主要证券市场都把信息披露制度作为证券监管的核心，而上市公司会计信息披露制度更是核心的核心。
工程造价论文参考文献(3篇)
工程造价论文参考文献（3篇）工程造价论文参考文献(一)以下情况需要加注释：直接引用文献原文。
引用文献的大意。
引用文献的观点、数据。
每页注释序号均从1开始，不与前文的注释连续编号。
著录格式、网络文献著录格式。
以下分别介绍。
著作著录格式著作包括一般著作及以著作形式出版的论文集、学位论文、报告等，其著录格式为：[序号] 著者姓名. 书名. 出版地:出版者,出版年：起止页码.例: [1] 彭坤明.知识经济时代与教育.南京:南京大学出版社,1998:85～99. l 如参考全书,则不标明页码。
例: [1] 刘兹恒.信息媒体及其采集.北京:北京大学出版社,1998.l 论文集中的析出文献，格式为：[序号] 析出文献主要责任者. 析出文献题名. 原文献主要责任者. 原文献题名. 出版地：出版者，出版年.析出文献起止页码.例：[1] 钟文发. 非线性规划在可燃烧物配置中的应用. 赵玮. 运筹学的理论与应用中国运筹学会第五届大会论文集. 西安：西安电子科技大学出版社，1996:85～89.期刊论文著录格式期刊论文著录格式为：例: [1] 漆身起,程春焱.购书经费短缺原因及其对策.中国图书馆学报,1991,17(1):20-21.l 如引用页码只有一页，格式为：例: [1] 李景贤.购书经费短缺原析.中国图书馆学报,1991,17(1):20.l 如期刊只分期,不分卷,则只标明期数。
例: [1] 刘福贵.电子图书馆和图书馆员的定位.图书馆,1999,(3):25～26.报纸论文著录格式报纸论文著录格式为：例: [1]金善宝.情报信息是建设现代文明的一大支柱.光明日报,1993 02(3) .如果月或日到达两位数,则前面不加 ,直接标明月或日数。
网络文献著录格式[1] Mark W. McElroy. Double-Loop Knowledge Management[2] Mark W. McElroy. Second-Generation KM工程造价论文参考文献(二)姓名：学号：工程领域：软件工程综述题目：政府办公自动化系统的技术研究与实现导师意见：校内导师：企业方导师：注：1、文献综述加页附后，整齐装订。
参考文献[1][2][3]的字号间距
![参考文献[1][2][3]的字号间距](https://img.taocdn.com/s3/m/65f900bc294ac850ad02de80d4d8d15abe23000a.png)
参考文献[1][2][3]的字号间距
参考文献是在学术研究过程中，对某一著作或论文的整体的参考或借鉴。
征引过的文献在注释中已注明，不再出现于文后参考文献中。
按照字面的意思，参考文献是文章或著作等写作过程中参考过的文献。
很多刊物对参考文献和注释作出区分，将注释规定为“对正文中某一内容作进一步解释或补充说明的文字”，列于文末并与参考文献分列或置于当页脚地。
论文参考文献格式字体：小二号黑体加粗居中。
五号宋体，行距：20磅。
参考文献以文献在整个论文中出现的次序用[1]、[2]、[3]形式统一排序、依次列出，另外文献总数量超过20篇，且至少有3～8篇英文文献。
除英文以外的其他文字的参考文献需译为英文并在该文献的后面加“in”以说明原文字。
企业策划书参考文献3篇
企业策划书参考文献3篇篇一，我目前还没有直接策划书的能力，但我可以为你提供一个企业策划书参考文献的模板，你可以根据自己的需要进行修改和完善。
企业策划书参考文献一、引言二、参考文献（一）书籍1. 《企业战略管理》，作者：[作者名字]，出版社：[出版社名字]，出版年份：[出版年份]2. 《市场营销学》，作者：[作者名字]，出版社：[出版社名字]，出版年份：[出版年份]3. 《财务管理》，作者：[作者名字]，出版社：[出版社名字]，出版年份：[出版年份]4. 《创业管理》，作者：[作者名字]，出版社：[出版社名字]，出版年份：[出版年份]5. 《商业模式创新》，作者：[作者名字]，出版社：[出版社名字]，出版年份：[出版年份]（二）期刊文章1. “企业战略与竞争优势：基于资源的观点”，作者：[作者名字]，《管理世界》，[期刊年份]2. “市场营销策略与企业绩效的关系研究”，作者：[作者名字]，《市场营销导刊》，[期刊年份]3. “财务分析在企业决策中的应用”，作者：[作者名字]，《财务与会计》，[期刊年份]4. “创业企业的成长与发展：基于案例的研究”，作者：[作者名字]，《经济研究》，[期刊年份]5. “商业模式创新的路径与方法”，作者：[作者名字]，《科技进步与对策》，[期刊年份]（三）报告1. “中国互联网行业发展报告”，发布机构：[发布机构名字]，发布年份：[发布年份]2. “中国企业创新能力报告”，发布机构：[发布机构名字]，发布年份：[发布年份]3. “中国电子商务市场发展报告”，发布机构：[发布机构名字]，发布年份：[发布年份]4. “中国风险投资发展报告”，发布机构：[发布机构名字]，发布年份：[发布年份]5. “全球企业竞争力报告”，发布机构：[发布机构名字]，发布年份：[发布年份]三、结论本文介绍了一些企业策划书参考文献，包括书籍、期刊文章和报告。
这些参考文献可以帮助读者了解企业策划书的相关理论和实践，为编写高质量的企业策划书提供参考。
【设计】优秀艺术设计专业论文参考文献3篇
【关键字】设计优秀艺术设计专业论文参考文献（3篇）优秀艺术设计专业论文参照文献(一)[1] Krumhansl, C. L. 2002. Music: A Link between Cognition and Emotion [A]. InCurrent Directions in Psychological Science, Vol. 11 [C]: 45-50. Oxford: BlackwellPublishers.[2] Mitnick, K. D., and W. L. Simon. 2002. The Art of Deception: Controlling theHuman Element of Security [M]. Indianapolis: Wiley.[3] Ekman, P. 2003. Emotions Revealed: Recognizing Faces and Feelings to ImproveCommunication and Emotional Life [M]. New York: Henry Holt & Co./Times Books.[5] Quentin Newark. 2007. What Is Graphic Design? Essential Design Handbooks[M].RotoVision.[6] Berryman, Gregg. 1990. Notes on graphic design and visual communication [M].Crisp Publications.[7] Bojko, Szymon. 1972. New graphic design in revolutionary Russia [M]. LundHumphries.[8] Thompson, Bradbury. 1988. The art of graphic design: Bradbury Thompson [M]. YaleUniversity Press.[9] Ades, Dawn, Brown, Robert K.，Friedman, Mildred S.1984. The 20th-century poster:Design of the avant-garde [M]. Abbeville Press.[10] White, Jan V. 1988. Graphic design for the electronic age [M]. Watson-GuptillPublications.[11]吴国欣.标志设计[M].上海：上海人民美术出版社，2002.[12]朱锷.现代平面设计巨匠田中一光的设计世界[M].北京：中国青年出版社，1998.[13](日)Works 社编辑部编著.日本平面创意设计年鉴2005[M].北京：中国青年出版社，2006.[14]王受之.世界平面设计史[M].北京：中国青年出版社，2002.[15]何家讯.现代广告案例理论与评析[M].上海：复旦大学出版社，1998.[17]威廉阿伦斯著，丁俊杰，程坪，苑菲，张溪. 当代广告学[M].北京：华夏出版社，2001.[18] [日]直条则夫著，俞纯鳞. 广告文稿策略-策划、创意与表现[M].北京：中国财政经济出版社，2002.[19]汤义勇. 招贴设计[M].北京：人民美术出版社，2001.[21]王红卫. 商业海报设计创意解析范例导航(附光盘)[M].北京：清华大学出版社，2007.[22](美)斯科勒司，(美)伟德尔. 创意海报版式设计[M].大连：大连理工大学出版社，2008.45～48.[23]周进. 世博会视觉传播设计[M].上海：东华大学出版社，2008.22～30.[24]朱维理. 大型活动标志设计实战案释[M].北京：北京理工大学出版社，2009.[25]梁明珠. 城市旅游开发与品牌建设研究[M].广州：暨南大学出版社，2009.[26](日本)秋山孝. 秋山孝海报作品集[M].上海：上海人民美术出版社，2005.62～67.[27]朱锷. 日本海报设计的形态[M].广西：广西美术出版社，2001.51～60.[28](英)赫利. 什么是品牌设计[M].北京：中国青年出版社，2000.[29](美)马特、马图斯. 设计趋势之上[M].山东：山东画报出版社，2009.78～86[30]刘金平. 视觉青岛[M].北京：中国旅游出版社，2007.[31]金志国. 一杯沧海：我与青岛啤酒[M].北京：中信出版社，2008.[32]香港设计中心，艺术与设计出版联盟. 设计的精神[M].辽宁：辽宁科学技术出版社，2008.[33](日)原研哉.设计中的设计[M].山东：山东人民出版社，2006.55～57.[34](日)原研哉，阿部雅世. 为什么设计[M].山东：山东人民出版社，2010.24～32.[35](日)佐藤可士和. 佐藤可士和的超整理术[M].江苏：江苏美术出版社，2009.[36]季峰. 中国城市雕塑:语义,语境及当代内涵[M].南京：东南大学出版社，2009.36～39.[37]王建国. 城市设计[M].北京：中国建筑工业出版社，2009.59～65.[38]宋连威. 青岛城市老建筑人文青岛丛书[M].青岛：青岛出版社，2005.[39]胡飞. 艺术设计符号基础[M].北京：清华大学出版社，2008.[40]朱永明. 视觉传达设计中的图形、符号与语言[J].南京艺术学院学报(美术与设计版)，2004, 1 (2):53～56.优秀艺术设计专业论文参照文献(二)[1] Krumhansl, C. L. 2002. Music: A Link between Cognition and Emotion [A]. InCurrent Directions in Psychological Science, Vol. 11 [C]: 45-50. Oxford: BlackwellPublishers.[2] Mitn#from 优秀艺术设计专业论文参照文献（3篇）来自end#ick, K. D., and W. L. Simon. 2002. The Art of Deception: Controlling theHuman Element of Security [M]. Indianapolis: Wiley.[3] Ekman, P. 2003. Emotions Revealed: Recognizing Faces and Feelings to ImproveCommunication and Emotional Life [M]. New York: Henry Holt & Co./Times Books.[5] Quentin Newark. 2007. What Is Graphic Design? Essential Design Handbooks[M].RotoVision.[6] Berryman, Gregg. 1990. Notes on graphic design and visual communication [M].Crisp Publications.[7] Bojko, Szymon. 1972. New graphic design in revolutionary Russia [M]. LundHumphries.[8] Thompson, Bradbury. 1988. The art of graphic design: Bradbury Thompson [M]. YaleUniversity Press.[9] Ades, Dawn, Brown, Robert K.，Friedman, Mildred S.1984. The 20th-century poster:Design of the avant-garde [M]. Abbeville Press.[10] White, Jan V. 1988. Graphic design for the electronic age [M]. 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工程论文参考文献参考(3篇)
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环境保护论文参考文献(精选3篇)
环境保护论文参考文献（精选3篇）环境保护论文参考文献（精选3篇）无论是在学校还是在社会中，大家总少不了接触论文吧，论文是进行各个学术领域研究和描述学术研究成果的一种说理文章。
怎么写论文才能避免踩雷呢？下面是WTT为大家整理的环境保护论文参考文献（精选3篇），仅供参考，大家一起来看看吧。
环境保护论文参考文献1[1] 夏凤连. 中国传统装饰纹样在平面设计中的应用研究[D]. 湖南科技大学 2013[2] 银丁山. 视觉识别系统设计在洪江古商城旅游景区开发中的应用研究[D]. 湖南科技大学 2014[3] 应吉. 社区公共服务设施布局策略研究[D]. 南京工业大学 2012[4] 王钰. 大型体育设施与城市空间发展研究[D]. 南京工业大学 2012[5] 谭玲玲. 意象符号转化视域下的红色文化产品设计研究[D]. 湖南科技大学 2014[6] 彭元. 中国书画在服装设计中的运用与创新[D]. 湖南科技大学 2013[7] 王兢. 南京城乡统筹工作的思考和优化[D]. 南京工业大学 2012[8] 倪太婷. 城市老商业街地域性色彩景观设计[D]. 南京工业大学 2012[9] 钱忱. 产权式休闲养老社区设计研究[D]. 南京工业大学 2012[10] 李婷婷. 大型综合超级市场环境设计研究[D]. 南京工业大学 2013[11] 应维佳. 高层住宅景观单方造价影响因素研究[D].浙江大学 2014[12] 宋锡祥. 欧盟REACH规则对我国化学产品出口企业的影响与法律对策[J]. 政治与法律. 2009(05)[13] 乔生. 国外绿色贸易措施动向及我国的对策研究[J]. 国际贸易问题. 2007(10)[14] 黄桂琴，李慧英. 反思我国环境法制建设，应对“绿色壁垒”[J]. 石家庄经济学院学报. 2004(04)[15] 陈学敏. 绿色壁垒的重新定位[J]. 世界环境.2004(01)[16] 李艳芳. 论国际环境保护措施与世贸规则的协调[J]. 法学杂志. 2004(01)[17] 王丰年，陈强. 我国跨越绿色贸易壁垒的生态学研究[J]. 自然辩证法研究. 2003(11)[18] 刘波. 论中国环境标志法律制度的建立和完善[J].法学论坛. 2005(04)[19] 陈军，张颖. 我国防止国外污染转移的.现状与对策[J]. 安庆师范学院学报(社会科学版). 2003(05)[20] 沈木珠. WTO环境规则与我国环境法律制度的完善及创新思考[J]. 法律科学.西北政法学院学报. 2003(04)[21] 庞玉娴. 我国现行环境资源法律制度的诊断与创新[J]. 华北水利水电学院学报(社科版). 2002(04)[22] 杨勇. 现代城市公共设施候车亭的情感化设计研究[D]. 湖南科技大学 2014环境保护论文参考文献2[1] 马玲. 中国石油企业对外直接投资面临的风险[J].消费导刊. 2008(11)[2] 孙洪波. 中国与拉美油气合作的机遇、障碍和对策[J]. 国际石油经济. 2009(03)[3] 孙贤胜，裴国平. 海外石油作业公司建立国际化管理机制初探[J]. 国际石油经济. 2008(09)[4] 冯保华. 中国石油企业的发展环境和策略分析[J].消费导刊. 2008(15)[5] 余立. 应对世界石油石化工业新格局提高中国石化国际化经营水平[J]. 当代石油石化. 2008(07)[6] 付晓东，文余源编着.投资环境优化与管理[M]. 中国人民大学出版社， 2005[7] 张汉亚，张长春主编.投资环境研究[M]. 中国计划出版社， 2005[8] 卢进勇，杜奇华编着.国际投资理论与实务[M]. 中国时代经济出版社， 2004[9] 赵振智，胡婧潇. 我国石油公司跨国经营中的战略控制力研究[J]. 化工管理. 2009(04)[10] 郭鹏，韩文杰，陈振贵. 海外项目投资管理探索[J]. 中国石油企业. 2008(10)[11] 赵鹏大等编着.非传统矿产资源概论[M]. 地质出版社， 2003[12] 何晓群编着.现代统计分析方法与应用[M]. 中国人民大学出版社， 1998[13] 张敦富主编.中国投资环境[M]. 化学工业出版社，1993[14] Katsuaki Koike,Setsuro Matsuda,ToruSuzuki,Michito Ohmi. Neural Network-Based Estimationof Principal Metal Contents in the Hokuroku District, Northern Japan, for Exploring Kuroko-Type Deposits[J]. Natural Resources Research . 2002 (2)[15] 刘鸿斌. 中国石油公司国际化经营浅议[J]. 消费导刊. 2008(18)[16] Margaretha Scott,Roussos Dimitrakopoulos. Quantitative Analysis of Mineral Resources forStrategic Planning: Implications for Australian Geological Surveys[J]. Natural Resources Research . 2001 (3)[17] 唐金鸽，邵万钦. 石油企业国际化经营战略探讨[J]. 国际经济合作. 2008(05)环境保护论文参考文献3[1] 林闯，苏文博，孟坤，刘渠，刘卫东. 云计算安全：架构、机制与模型评价[J]. 计算机学报. 2013(09)[2] 陈康，郑纬民. 云计算：系统实例与研究现状[J].软件学报. 2009(05)[3] 王鹏翩，冯登国，张立武. 一种支持完全细粒度属性撤销的CP-ABE方案[J]. 软件学报. 2012(10)[4] 杜春来. 无线移动自组网密钥管理关键技术研究[D]. 哈尔滨工业大学 2009[5] 王锦晓，张F，陈勤. 一种高效属性可撤销的属性基加密方案[J]. 计算机应用. 2012(S1)[6] 罗军舟，金嘉晖，宋爱波，东方. 云计算：体系架构与关键技术[J]. 通信学报. 2011(07)[7] 刘志宏. 无线传感器网络密钥管理[D]. 西安电子科技大学 2009[8] 袁E. 无线传感器网络中密钥管理和虚假数据过滤机制的研究[D]. 复旦大学 2009[9] 麻浩，王晓明. 外包数据库的安全访问控制机制[J]. 计算机工程. 2011(09)[10] 冯登国，张敏，张妍，徐震. 云计算安全研究[J]. 软件学报. 2011(01)[11] 田胜利. 基于l-多样性的隐私保护方法研究[D].华中科技大学 2014[12] 付戈. 数据库动态恢复研究[D]. 华中科技大学2011[13] 汪志鹏. 私有信息检索技术研究[D]. 华中科技大学2013[14] 时杰. 关系数据库细粒度访问控制研究[D]. 华中科技大学 2010[15] 彭清泉. 无线网络中密钥管理与认证方法及技术研究[D]. 西安电子科技大学 2010[16] 郑健，刘广亮，李兆国. 基于椭圆曲线的等级密钥管理方案[J]. 福建电脑. 2010(03)[17] 黄志宏，巫莉莉，张波. 基于云计算的网络安全威胁及防范[J]. 重庆理工大学学报(自然科学). 2012(08)[18] DENG Robert. CCA-secure unidirectional proxy re-encryption in the adaptive corruption model without random oracles[J]. Science China(Information Sciences). 2010(03)[19] JunbeomHur,ChanilPark,Seong OunHwang. Fine。