表达猪瘟病毒E0-E2基因重组腺病毒疫苗在小鼠体内的存留时间分析
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摘要
研究E0-E2基因和表达蛋白及抗体水平在小鼠体内的最长存留时间,以确定表达猪瘟病毒E0-E2基因重组腺病毒疫苗(rAd-E0-E2)在小鼠体内的存留规律。本试验选用30只SPF级别的健康小鼠,随机分为A、B组,各免疫组以1×106 IFU/只剂量接种rAd-E0-E2,对照组注射生理盐水。A组20只于接种后不同特定时间(2只/次)采集血液和组织进行PCR和IFA检测E0-E2基因及表达蛋白残留情况;B组10只于接种后1、3、5、7、10、14、21、28d采集血清检测猪瘟抗体水平。PCR和IFA检测显示,在接种12、24、36h后肝、脾、肾组织和血清中E0-E2基因和蛋白检出率为100%,接种48h后小鼠肝、脾组织检出率为50%,肾组织和血清检出率为100%。接种72h以后,小鼠血液和组织检测均为阴性。接种后7d试验组体内抗体阳性率为40%,接种后10d小鼠体内抗体阳性率为100%,抗体阻断率在43%～51.2%,接种后28d,猪瘟抗体阳性率为100%,抗体阻断率在75.6%～87.5%。表明rAd-E0-E2在小鼠体内的存留时间最长72h,接种时间与E2抗体水平呈正相关。
In order to determine the safty of recombinant adenovirus vaccine expressing swine fever virus E0-E2 gene(rAd-E0-E2)in mice,the retention time of E0-E2 gene,E0-E2 protein and response antibody in mice were tested.30 SPF healthy mice were randomly divided into group A and group B.Each group was inoculated with 1×106 IFU/dose of rAd-E0-E2.The control group was given with saline.Group A had 20 mice,the blood and tissues were collected after inoculation at different times(10 mice/time)for PCR and IFA to detect the E0-E2 gene and E0-E2 protein residue.Group B had 10 mice,serum samples were collected on 1,3,5,7,10,14 days after inoculation to detect the antibody levels.The PCR and IFA detection results showed that the E0-E2 gene and protein were detected in the liver,spleen,kidney tissue and sera 12 hours and 24 hours after inoculation.After 48 hours of vaccination,the detection rate of liver and spleen tissue in mice was 50%,and the detection rate in renal tissues and sera were 100%.After 72 hours of inoculation,the mice were negative in blood and tissues.The antibody positive rate was 40%in the test group 7 days after the immunization.The antibody positive rate in mice 10 days after immunization was 100%,the antibody blocking rates were betwean 43%-51.2%.The positive rate of antibody was 100% 28 days after immunization,the antibody blocking rates were between 75.6%-87.5%.The above results indicated that rAd-E0-E2 retention time in mice can reach up to 72 hours,the time after immunization and E2 antibody levels are positive correlation.
In order to determine the safty of recombinant adenovirus vaccine expressing swine fever virus E0-E2 gene(rAd-E0-E2)in mice,the retention time of E0-E2 gene,E0-E2 protein and response antibody in mice were tested.30 SPF healthy mice were randomly divided into group A and group B.Each group was inoculated with 1×106 IFU/dose of rAd-E0-E2.The control group was given with saline.Group A had 20 mice,the blood and tissues were collected after inoculation at different times(10 mice/time)for PCR and IFA to detect the E0-E2 gene and E0-E2 protein residue.Group B had 10 mice,serum samples were collected on 1,3,5,7,10,14 days after inoculation to detect the antibody levels.The PCR and IFA detection results showed that the E0-E2 gene and protein were detected in the liver,spleen,kidney tissue and sera 12 hours and 24 hours after inoculation.After 48 hours of vaccination,the detection rate of liver and spleen tissue in mice was 50%,and the detection rate in renal tissues and sera were 100%.After 72 hours of inoculation,the mice were negative in blood and tissues.The antibody positive rate was 40%in the test group 7 days after the immunization.The antibody positive rate in mice 10 days after immunization was 100%,the antibody blocking rates were betwean 43%-51.2%.The positive rate of antibody was 100% 28 days after immunization,the antibody blocking rates were between 75.6%-87.5%.The above results indicated that rAd-E0-E2 retention time in mice can reach up to 72 hours,the time after immunization and E2 antibody levels are positive correlation.
引文
[1]任世斌,赵亮,郭抗抗,等.猪瘟综合防控研究进展[J].动物医学进展,2018,39(9):100-106.
[2]McOrist S,Khampee K,Guo A.Modern pig farming in the People's Republic of China:growth and veterinary challenges[J].Rev Sci Tech,2011,30(3):961-968.
[3]李亚菲.猪瘟病毒E2蛋白纳米疫苗的初步研究[D].陕西杨凌:西北农林科技大学,2018.
[4]孙永科,杨玉艾,王养会,等.表达猪瘟病毒石门株E2抗原的重组腺病毒构建及免疫性研究[J].病毒学报,2007,23(2):137-142.
[5]Sun S Q,Yin S H,Guo H C,et al.Genetic typing of classical swine fever virus isolates from China[J].Transb Emerg Dis,2013,60(4):370-375.
[6]Zhang H,Sun Y K,Zhang X M,et al.A rabbit model for evaluating the efficacy of recombinant adenovirus vaccine expressing E0-E2antigens of classical swine fever virus[J].Thai J Vet Med,2017,47(3):363-372.
[7]张恒,李传龙,崔治中,等.禽白血病A亚群病毒gp85的单因子血清制备及其特异性鉴定[J].微生物学报,2011,51(1):134-140.
[8]祖立闯,谢金文,沈志强,等.猪瘟疫苗质量的替代检验方法研究进展[J].动物医学进展,2015,36(2):76-79.
[9]程树军.实验动物替代方法原理与应用[M].北京:科学出版社,2010.
[10]中华人民共和国科学技术部.关于善待实验动物的指导性意见[Z].国科发字,2006,398号.
[11]杨洋,高博,宫婷,等.猪瘟病毒E0-E2蛋白基因重组人5型腺病毒的构建及鉴定[J].中国生物制品学杂志,2012(7):805-808.
[12]张恒,范根成,杜元钊,等.一种猪瘟病毒基因重组腺病毒载体疫苗病毒含量测定方法[P].中华人民共和国国家知识产权局.CN104535764A,2015-04-22.
[13]杨玉艾,初晓辉,毕保良,等.猪瘟病毒E0和E0-E2蛋白融合表达的重组腺病毒构建及免疫保护性研究[J].中国预防兽医学报,2010,32(6):419-423.
[14]Sun Y,Tian D Y,Li S,et al.Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2against classical swine fever[J].Vaccine,2013,31(3):538-544.
[15]Gao F,Jiang Y,Li G,et al.Porcine reproductive and respiratory syndrome virus expressing E2of classical swine fever virus protects pigs from a lethal challenge of highly-pathogenic PRRSV and CSFV[J].Vaccine,2018,36(23):3269-3277.
[2]McOrist S,Khampee K,Guo A.Modern pig farming in the People's Republic of China:growth and veterinary challenges[J].Rev Sci Tech,2011,30(3):961-968.
[3]李亚菲.猪瘟病毒E2蛋白纳米疫苗的初步研究[D].陕西杨凌:西北农林科技大学,2018.
[4]孙永科,杨玉艾,王养会,等.表达猪瘟病毒石门株E2抗原的重组腺病毒构建及免疫性研究[J].病毒学报,2007,23(2):137-142.
[5]Sun S Q,Yin S H,Guo H C,et al.Genetic typing of classical swine fever virus isolates from China[J].Transb Emerg Dis,2013,60(4):370-375.
[6]Zhang H,Sun Y K,Zhang X M,et al.A rabbit model for evaluating the efficacy of recombinant adenovirus vaccine expressing E0-E2antigens of classical swine fever virus[J].Thai J Vet Med,2017,47(3):363-372.
[7]张恒,李传龙,崔治中,等.禽白血病A亚群病毒gp85的单因子血清制备及其特异性鉴定[J].微生物学报,2011,51(1):134-140.
[8]祖立闯,谢金文,沈志强,等.猪瘟疫苗质量的替代检验方法研究进展[J].动物医学进展,2015,36(2):76-79.
[9]程树军.实验动物替代方法原理与应用[M].北京:科学出版社,2010.
[10]中华人民共和国科学技术部.关于善待实验动物的指导性意见[Z].国科发字,2006,398号.
[11]杨洋,高博,宫婷,等.猪瘟病毒E0-E2蛋白基因重组人5型腺病毒的构建及鉴定[J].中国生物制品学杂志,2012(7):805-808.
[12]张恒,范根成,杜元钊,等.一种猪瘟病毒基因重组腺病毒载体疫苗病毒含量测定方法[P].中华人民共和国国家知识产权局.CN104535764A,2015-04-22.
[13]杨玉艾,初晓辉,毕保良,等.猪瘟病毒E0和E0-E2蛋白融合表达的重组腺病毒构建及免疫保护性研究[J].中国预防兽医学报,2010,32(6):419-423.
[14]Sun Y,Tian D Y,Li S,et al.Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2against classical swine fever[J].Vaccine,2013,31(3):538-544.
[15]Gao F,Jiang Y,Li G,et al.Porcine reproductive and respiratory syndrome virus expressing E2of classical swine fever virus protects pigs from a lethal challenge of highly-pathogenic PRRSV and CSFV[J].Vaccine,2018,36(23):3269-3277.
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