减蛋综合征病毒在不同品系小鼠体内的组织分布
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摘要
目的通过人工感染减蛋综合征病毒(egg drop syndrome virus,EDSV),观察病毒在不同品系小鼠体内增殖情况以及动态变化规律,为EDSV构建载体提供理论依据与数据支持。方法选取免疫系统正常的BALB/c小鼠、T细胞免疫缺陷裸鼠(Nu)以及高度免疫缺陷小鼠(NSG)为研究对象,每品系32只,雌性,5～6周龄,经腹腔注射人工感染EDSV,分别于攻毒后1、3、5、7、14、21、28、35 d采集血清,应用间接ELISA方法进行抗体监测;选择攻毒后1、7、14、21、28 d小鼠,采集心脏、肺、肝、脾、肾、小肠、子宫、气管、食管、脑10种组织,应用荧光定量PCR相对定量比较Ct法(△△CT)进行各组织内病毒载量的检测。结果 BALB/c小鼠于攻毒后3 d即可在血清内检测到抗体的表达,14 d抗体水平达到最高,并一直维持至监测期内35 d;Nu小鼠也可于攻毒后3 d检测到抗体,表达水平较BALB/c小鼠有所降低,攻毒14 d后,Nu小鼠血清中抗体水平出现下降,至35 d抗体一直维持在较低的水平;NSG小鼠在整个监测过程中,抗体水平一直处于阴性状态。核酸相对定量结果显示,BALB/c小鼠感染后1 d,肝组织中的病毒表达量最高,达到5.45个数量级,其次由高到低依次是脾、食管、子宫、小肠、肺、气管、肾、心脏,脑组织中病毒含量最低,随感染时间的延长,各组织内病毒表达量较感染1 d均有所下降,至攻毒后28 d,肝、脾病毒表达量依然维持着较高的水平;Nu小鼠和NSG小鼠感染1 d表现为脾中病毒表达量最高,分别为3.95和4.05个数量级,其次为肝,攻毒28 d,两种小鼠体内各器官内仍可以检出阳性信号,肝、脾病毒表达量较高。结论 EDSV可刺激小鼠产生免疫应答,在免疫缺陷小鼠体内抗体水平表达量较低。该病毒在小鼠体内有肝、脾等组织嗜性,为EDSV开发成为载体以及在实验动物模型上的进一步研究与应用提供了参考数据。
Objective Through artificial infection with egg drop syndrome virus(EDSV), to observe the proliferation and dynamic changes of the distribution of EDSV in vivo in different mouse strains, and to provide a theoretical basis and data support for construction of the virus vectors. Methods Three mouse strains with different immune status, BALB/c(normal immunity), Nu(T-cell immunodeficiency) and NSG(high immunodeficiency) mice were used. Thirty-two 5-6 week old female mice per strain were grouped. The mice were infected by intraperitonealy injection of EDSV and serum samples were collected 1, 3, 5, 7, 14, 21, 28 and 35 days later. Antibodies were monitored by an indirect ELISA. Samples of the heart, lung, liver, spleen, kidney, small intestine, uterus, trachea, esophagus and brain were collected from the mice at 1, 7, 14,21 and 28 days after infection. The viral load in each tissue was detected by quantitative fluorescence PCR and comparative Ct(2~(-ΔΔCT)) method. Results Antibodies were detected 3 days after infection in the sera of BALB/c mice, reached the highest level at 14 days, and this level was maintained until 35 days. Antibodies were detected in the Nu mice 3 days post-infection,(but at a lower level than that in BALB/c mice), the levels were reduced after 14 days of infection and maintained at a low level until 35 days post-infection. The antibody of NSG mice was negative during the whole process of infection monitoring. Relative quantification of nucleic acids showed that EDSV expression in the liver tissue of BALB/c mice reached 5.45 orders of magnitude at 1 day after infection, followed by the spleen, esophagus, uterus, small intestine, lung, trachea, kidney and heart. EDSV content in the brain tissue was the lowest. With the extension of infection period, EDSV expression in each tissue was lower than that on the first day of infection, and EDSV expression in the liver and spleen remained high 28 days after infection. Nu mice and NSG mice showed the highest EDSV expression in the spleen 1 day after infection(3.95 and 4.05 orders of magnitude, respectively), followed by that in the liver. Positive signals could be detected in the organs of Nu mice and NSG mice 28 days after infection. EDSV expression in the liver and spleen remained high. Conclusions EDSV can stimulate immune response in mice, and the level of antibody expression in immunodeficient mice is low. EDSV exhibites hepatic and splenic tropism in mice in vivo. The present study provides reference data for development of EDSV vectors, as well as for their further application in laboratory animals.
Objective Through artificial infection with egg drop syndrome virus(EDSV), to observe the proliferation and dynamic changes of the distribution of EDSV in vivo in different mouse strains, and to provide a theoretical basis and data support for construction of the virus vectors. Methods Three mouse strains with different immune status, BALB/c(normal immunity), Nu(T-cell immunodeficiency) and NSG(high immunodeficiency) mice were used. Thirty-two 5-6 week old female mice per strain were grouped. The mice were infected by intraperitonealy injection of EDSV and serum samples were collected 1, 3, 5, 7, 14, 21, 28 and 35 days later. Antibodies were monitored by an indirect ELISA. Samples of the heart, lung, liver, spleen, kidney, small intestine, uterus, trachea, esophagus and brain were collected from the mice at 1, 7, 14,21 and 28 days after infection. The viral load in each tissue was detected by quantitative fluorescence PCR and comparative Ct(2~(-ΔΔCT)) method. Results Antibodies were detected 3 days after infection in the sera of BALB/c mice, reached the highest level at 14 days, and this level was maintained until 35 days. Antibodies were detected in the Nu mice 3 days post-infection,(but at a lower level than that in BALB/c mice), the levels were reduced after 14 days of infection and maintained at a low level until 35 days post-infection. The antibody of NSG mice was negative during the whole process of infection monitoring. Relative quantification of nucleic acids showed that EDSV expression in the liver tissue of BALB/c mice reached 5.45 orders of magnitude at 1 day after infection, followed by the spleen, esophagus, uterus, small intestine, lung, trachea, kidney and heart. EDSV content in the brain tissue was the lowest. With the extension of infection period, EDSV expression in each tissue was lower than that on the first day of infection, and EDSV expression in the liver and spleen remained high 28 days after infection. Nu mice and NSG mice showed the highest EDSV expression in the spleen 1 day after infection(3.95 and 4.05 orders of magnitude, respectively), followed by that in the liver. Positive signals could be detected in the organs of Nu mice and NSG mice 28 days after infection. EDSV expression in the liver and spleen remained high. Conclusions EDSV can stimulate immune response in mice, and the level of antibody expression in immunodeficient mice is low. EDSV exhibites hepatic and splenic tropism in mice in vivo. The present study provides reference data for development of EDSV vectors, as well as for their further application in laboratory animals.
引文
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[10] 郭建军,范汉东,杨一兵.减蛋综合征病毒纤维蛋白主要抗原结构域原核表达及抗原性鉴定[J].畜牧与兽医,2012,44(2):22-25.Guo JJ,Fan HD,Yang YB.Prokaryotic expression of structural domains of fiber protein of egg drop syndrome virus and antigenicity identification[J].Anim Husbandry Vet Med,2012,44(2):22-25.
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[13] 王翠娥,陈立超,周倩,等.实验大鼠小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31(2):20-24.Wang CE,Chen LC,Zhou Q,et al.Testing of multiple viral antibodies in laboratory rats and mice [J].Lab Anim Sci,2014,31(2):20-24.
[14] 罗银珠,张钰,潘金春,等.广东小鼠腺病毒血清学调查及病毒和抗体在人工感染小鼠体内消长规律研究[J].中国比较医学杂志,2017,27(1):43-48.Luo YZ,Zhang Y,Pan JC,et al.Serological survey of Mad infection among mice in Guangdong and distribution and serological studies in artificial infection[J].Chin J Comp Med,2017,27(1):43-48.
[15] The Jackson Laboratory,NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ.http://www.jax.org/strain/005557
[16] Hess M,Bl?cker H,Brandt P.The complete nucleotide sequence of the egg drop syndrome virus:an intermediate between mastadenoviruses and aviadenoviruses [J].Virology,1997,238(1):145-156.
[2] 程安春,汪铭书,钟妮娜,等.鸭腺病毒感染的研究—病毒分离鉴定及病原特性[J].中国兽医科技,1996,26(10):3-5.Cheng AC,Wang MS,Zhong NN,et al.Stadies of duck adenovirus infection—isolation,identification and pathogenic characteristics of virus [J].Chin J Vet Sci Technol,1996,26(10):3-5.
[3] 王玉泉,马立宾,于秋莲.动物腺病毒载体的研究进展[J].内蒙古民族大学学报(自然科学版),2009,24(2):180-183.Wang YQ,Ma LB,Yu QL.Construction of animal adenovirus vectors — A review [J].J Inner Mongolia Univ for Natl,2009,24(2):180-183.
[4] 柳云帆,吴小兵,阮力.腺病毒载体在疫苗研究中的应用[J].生物技术通讯,2011,22(4):552-558.Liu YF,Wu XB,Ruan L.Application of adenovirus as vaccine vectors [J].Lett Biotech,2011,22(4):552-558.
[5] 赵莉,周洁,高诚.禽腺病毒载体研究进展[J].动物医学进展,2012,33(11):99-103.Zhao L,Zhou J,Gao C.Progress on fowl adenovirus vector [J].Prog Vet Med,2012,33(11):99-103.
[6] 马震原,李刚,李文超,等.TaqMan荧光定量PCR检测鸡产蛋下降综合征病毒方法的建立及应用[J].畜牧兽医学报,2012,43(5):767-772.Ma ZY,Li G,Li WC,et al.Development and application of TaqMan fluorescent real-time quantitative PCR for the detection of egg drop syndrome virus [J].Acta Vet Et Zootech Sin,2012,43(5):767-772.
[7] Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT[J].Methods,2001,25(4):402-408.
[8] 肖妙,于志丹,马波,等.减蛋综合征病毒五邻体重组蛋白的原核表达及抗原性鉴定[J].东北农业大学学报,2009,40(2):83-87.Xiao M,Yu ZD,Ma B,et al.Cloning and expression of recombinent of protein penton gene from egg drop syndrome virus and identification of expressed protein antigenicity [J].J Northeast Agric Univ,2009,40(2):83-87.
[9] 杜冬华.鸡减蛋综合征病毒河北分离株六邻体蛋白基因的克隆表达及免疫原性研究[D].河北农业大学,2007.Du DH.Cloning and expression of hexon gene from egg drop syndrome virus detected in Hebei and analysis of immunogenicity[D].Agricultural University of Hebei,2007.
[10] 郭建军,范汉东,杨一兵.减蛋综合征病毒纤维蛋白主要抗原结构域原核表达及抗原性鉴定[J].畜牧与兽医,2012,44(2):22-25.Guo JJ,Fan HD,Yang YB.Prokaryotic expression of structural domains of fiber protein of egg drop syndrome virus and antigenicity identification[J].Anim Husbandry Vet Med,2012,44(2):22-25.
[11] 冯柳柳,程冰花,刁有祥,等.鸡源减蛋综合征病毒SD01株对种鸭的致病性的研究[J].中国预防兽医学报,2015,37(11):821-824.Feng LL,Cheng BH,Diao YX,et al.Pathogenicity of chicken-origin egg drop syndrome virus SD01 strain in laying ducks [J].Chin J Prev Vet Med,2015,37(11):821-824.
[12] 王晓东.EDSV京911株五邻体优势抗原表位的筛选及鉴定[D].东北农业大学,2012.Wang XD.Screeing and identification the dominant epitopes on penton of EDSV [D].Northeast Agricultural University,2012.
[13] 王翠娥,陈立超,周倩,等.实验大鼠小鼠多种病毒的血清学检测结果分析[J].实验动物科学,2014,31(2):20-24.Wang CE,Chen LC,Zhou Q,et al.Testing of multiple viral antibodies in laboratory rats and mice [J].Lab Anim Sci,2014,31(2):20-24.
[14] 罗银珠,张钰,潘金春,等.广东小鼠腺病毒血清学调查及病毒和抗体在人工感染小鼠体内消长规律研究[J].中国比较医学杂志,2017,27(1):43-48.Luo YZ,Zhang Y,Pan JC,et al.Serological survey of Mad infection among mice in Guangdong and distribution and serological studies in artificial infection[J].Chin J Comp Med,2017,27(1):43-48.
[15] The Jackson Laboratory,NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ.http://www.jax.org/strain/005557
[16] Hess M,Bl?cker H,Brandt P.The complete nucleotide sequence of the egg drop syndrome virus:an intermediate between mastadenoviruses and aviadenoviruses [J].Virology,1997,238(1):145-156.