enJSRV和Hyal-2在绵羊胎儿和初生羔羊免疫器官中的表达研究
摘要
本研究为了揭示感染JSRV病羊体内检测不到循环抗体的免疫学机理,参照GeneBank中已发表的内源性绵羊肺腺瘤病毒(enJSRV)及其受体Hyal-2的基因序列(EF6803000和NM-001009754),设计合成2对引物,选择无绵羊肺腺瘤病发病历史的羊场母羊10只,经同期受孕后,取经临床病理学检查处于健康状况的妊娠70d绵羊胎儿,妊娠130d绵羊胎儿,初生3日龄羔羊(各3只)的胸腺、脾脏、肠系膜淋巴结和肺脏并提取其总RNA,经RT-PCR扩增,用地高辛(DIG)标记制备enJSRV-env和Hyal-2的cDNA探针,原位杂交法检测妊娠70 d的绵羊胎儿(免疫器官初步形成期)、妊娠130 d胎儿(即将出生的)和出生后3日龄羔羊的胸腺、脾脏、肠系膜淋巴结和肺脏内enJSRV mRNA和Hyal-2 mRNA表达情况。结果表明,enJSRV和Hyal-2 mRNA特异性的表达在胸腺小叶中,特别是在胸腺的皮髓交界处和髓质中,在淋巴结的各种淋巴细胞,脾脏的白髓和肺脏的支气管上皮细胞和肺泡上皮细胞也均可检测到阳性信号,而阴性对照组均没有阳性信号,大鼠相应组织(胸腺,脾脏,肺脏)阴性对照组中亦无阳性信号出现。利用Real-Time PCR法,对enJSRV mRNA和Hyal-2mRNA在以上组织中的表达进行了定量分析。结果表明,不同组织间enJSRV mRNA的表达差异很大,以enJSRV在妊娠70d胎儿肺脏组织中的表达为对照,在妊娠130d脾脏组织的表达为对照的26.36倍,在出生3日龄羔羊胸腺的表达为对照的22.42倍,在妊娠130d胸腺表达约为对照的13.73倍,在妊娠130d肠系膜淋巴结的表达为对照的11.57倍,并均存在着显著差异或极显著差异。这表明enJSRV mRNA在免疫组织中有高水平的表达,而在肺脏中有低水平的表达。另外,在妊娠70d所有组织中enJSRV的表达水平都相对较低,而在妊娠130d和初生3日龄羔羊其表达量较高。然而,Hyal-2在以上各时期组织中的表达水平差异不显著。以上试验结果为机体对exJSRV的感染产生免疫耐受这一假说提供了有力的证据。
The aim of this study was to investigate the function of enJSRV and the immunology mechanism why can not detected circulating antibody in infected sheep. Primers were designed on the basis of published enJSRV and Hyal-2 sequences in GeneBank. Gestation day 70 fetus, gestation day 130 fetus and 3d newborn lambs were assigned randomly to be necropsied. The lung,thymus,spleen and mesenteric lymph node were collected and stored at -80°C for RNA extraction. Total RNA was isolated from these tissues .we amplificated enJSRV and Hyal-2 fragment using RT-PCR and Labelled the specific probes using DIG , then analysised the expression of enJSRV envelope (env) and Hyal-2 mRNAs in the collected tissues through In Situ Hybridization. The results revealed that enJSRV envelope (env) and Hyal-2 mRNAs expressed in thymus, spleen, mesenteric lymph node and lung of different periods, but there were no positive signals in negative control. At the same time, the expression level of each target mRNA was analyzed by Real-Time reverse transcription PCR, and the results showed that based on the fold changes relative to the lung of 70d fetus, the enJSRV mRNA was most abundant by 26-fold in the spleen of 130d fetus and 22-fold in the thymus of 3d newborn. The expression level of enJSRV mRNA in the thymus and mesenteric lymph node of 130d fetal lambs was approximately 10-fold. It is clear that the level of enJSRV mRNA was much higher in immune organs than that in lungs. In addition that, the level of enJSRV mRNA was lower in all the tissues of 70d fetus than in other periods. While There was no significant difference among the expression levels of Hyal-2 mRNA in all collected tissues. This study will unravel the pathogenesis that sheep are tolerized towards infection by the exJSRV.
The aim of this study was to investigate the function of enJSRV and the immunology mechanism why can not detected circulating antibody in infected sheep. Primers were designed on the basis of published enJSRV and Hyal-2 sequences in GeneBank. Gestation day 70 fetus, gestation day 130 fetus and 3d newborn lambs were assigned randomly to be necropsied. The lung,thymus,spleen and mesenteric lymph node were collected and stored at -80°C for RNA extraction. Total RNA was isolated from these tissues .we amplificated enJSRV and Hyal-2 fragment using RT-PCR and Labelled the specific probes using DIG , then analysised the expression of enJSRV envelope (env) and Hyal-2 mRNAs in the collected tissues through In Situ Hybridization. The results revealed that enJSRV envelope (env) and Hyal-2 mRNAs expressed in thymus, spleen, mesenteric lymph node and lung of different periods, but there were no positive signals in negative control. At the same time, the expression level of each target mRNA was analyzed by Real-Time reverse transcription PCR, and the results showed that based on the fold changes relative to the lung of 70d fetus, the enJSRV mRNA was most abundant by 26-fold in the spleen of 130d fetus and 22-fold in the thymus of 3d newborn. The expression level of enJSRV mRNA in the thymus and mesenteric lymph node of 130d fetal lambs was approximately 10-fold. It is clear that the level of enJSRV mRNA was much higher in immune organs than that in lungs. In addition that, the level of enJSRV mRNA was lower in all the tissues of 70d fetus than in other periods. While There was no significant difference among the expression levels of Hyal-2 mRNA in all collected tissues. This study will unravel the pathogenesis that sheep are tolerized towards infection by the exJSRV.
引文
1. Palmarini M, Fan H. Retrovirus-Induced Ovine Pulmonary Adenocarcinoma,an Animal Model for Lung Cancer[J]. Journal of the National Cancer Institute, 2001, 93: 1603-1614.
2. Palmarini M, Delasheras M, lnglis NF, et al. Epithelial tumour cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus[J]. J Gen Virol, 1995, 76: 2731-2737.
3. Palmarini M, Dalziel RG, Bai J, et al. The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep[J]. J Virol, 1996, 70: 1618-1623.
4.中华人民共和国农业部公告动物疫病病种名录[J].农业科技通讯,2000.10:35.
5.国际动物卫生组织(OIE)编,农业部畜牧兽医局译.国际动物卫生法典[M].兵器工业出版社,2000,285-289.
6 . Perk K,Hod I.Sheep lung carcinoma:an endemic analogue of a sporadic human neoplasm[J].JNCI,1982,69:747-749.
7.Riley LK.A method for biotinylating oligonucletide probes for use in molecular hybridizations[J].DNA,1986,5(4):333-336.
8. Dungal N. Experiments with jaagsiekte.Amer J of Path,1946,22:737-759.
9. Martin WB,Scott FMM,Sharp JM,et al.Experimental production of sheep pulmonary adenomatosis (Jaagsiekte)[J].Nature,1976,264:183-184.
10. Sharp JM, Angus KW,Gray EW,et al. Rapid transmission of sheep pulmonary adenomatosis (jaasiekte) in young lambs[J].Archives of Virology,1983,78:89-95.
11. Sharp JM,Herring AJ.Sheep pulmonary adenomatosis:demonstration of a protein which cross-reacts with the major core proteins of Mason-Pfizer monkey virus and mouse mammary tumour virus[J].J Gen Virol,1983,64:2323-2327.
12. York DF,Vigne R,Verwoerd DW,et al.Nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats[J].J Virol, 1992,66(8):4930-4939.
13. Palmarini M,Sharp JM,Lee C, et al. In vitro infection of ovine cell lines by jaagsiekt sheep retrovirus (JSRV)[J].Journal of Virology,1999b,73:10070-10078.
14. Demartini JC,Bishop JV,Allen TE,et al.Jaagsiekte sheep retrovirus proviral clone JSRV(JS7), derived from the JS7 lung tumor cell line,induces ovine pulmonary carcinoma and is integrated into the surfactant protein A gene[J].Journal of Virology,2001,75(9):239-246
15. Gifford R,Tristem M.The evolution,distribution diversity of endogenous retroviruses [J].Virus Genes,2003,26:291-315.
16.Christina , Leenadevi T, et al.Stuart L,Jaagsiekte sheep retrovirus is present at high concentration in lung fluid produced by ovine pulmonary adenocarcinoma-affected sheep and can survive for several weeks at ambient temperatures[J]. Research in Veterinary Science, 2009, 87: 154–156.
17. Caporale M,Centorame P,Giovannini A,at al.Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of naturally occurring JSRV infection during the commercial lifespan of sheep[J].Virology,2005, 10:1-10.
18. Ortin A,Minguijqn E,Dewar P,et al.Lack of specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis[J]. Veterinary Immunology and Immunopothology,1998,61:229-237.
19. Bai J, Zhu RY, Stedman K, et al. Unique long terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome[J].J Virol,1996,70(5):3159-3168.
20. Gonzalez L,Garcia-Goti M,Cousens C,et al.Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre-clinical period of sheep pulmonary adenomatosis[J].J Gen Virol,2001,82:1355-1358.
21. Maeda N,Palmarini M,Murgia C.Direct transformation of rodent fibroblasts by jaagsikte sheep retrovirus DNA[J].Proc Natl Acad Sci USA,2001,98:4449-4454.
22. Palmarini M, Maeda N, Murgia C. A phosphatidylinositol-3-kinase (PI23K) docking site in the cytoplasmic tail of the jaagsikte sheep retrovirus transmembrane protein is essential for envelope induced transformation of NIH-3T3[J].J Virol,2001,25:3056- 3062.
23. Nishigaki K, Hanson C,Ohashi T. Erythroid cells rendered erythropoietin independent by infection with Friend spleen focusforming virus show constitutive activation of phosphatidylino sitol3-kinase and Ake kinase:involvement of insulin recptor substrate-related adapter proteins[J].J Virol,2000,74:3037-3045.
24. White MK,Straver DS. Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol3-kinase in typeⅡalveolar cells Exp[J]. Cell Res,2000,255:67-76.
25. Margana RK,Boggaram V.Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3,TTF-1,and HNF-3alpha transcription factors are necessary for lung cell-specific activation of SP-B gene transcription[J].J Biol Chem,1997,272(5): 3083-3090.
26. Palmarini M,Mura M,Spencer T.Endogenous betaretroviruses of sheep: teaching new lessons in retroviral interference and adaptation[J].J Gen Virol,2004,85:1-13.
27. Thomas Spencer, Proceedings of the National Academy of Sciences [J]. Virology , 2006,258:201-213.
28. Palmarini M, Holland MJ, Cousens C, et al. Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[J]. J Gen Virol, 1996, 77: 2991-2998.
29. Spencer T E, Mura M, Gray CA, et al. Receptor Usage and Fetal Expression of Ovine Endogenous Betaretroviruses: Implications for Coevolution of Endogenous and Exogenous Retroviruses[J]. J Virol , 2003, 77: 749-753.
30. Palmarini M,Hallwirth C,York D,et al.Molecular cloning and functional analysis of three type D endogenous retrovirus of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus[J].J Virol, 2000,74(17):8065-8076.
31. Bai J,Bishop JV,Carlson JO,et al.Sequence comparison of JSRV with endogenous proviruses:Envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor[J].Virology,1999,258:333-343.
32. Nakagawa K,Harrison LC.The potential roles of endogenous retroviruses in autoimmunity[J].Immunol Rev,1996,152:193-236.
33. Rai SK,Demartini JC,Miller AD.Retrovirus vector bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3[J].J Virol,2000,74:4698-4704.
34.HollandPM,AbramsonRD,矶厄tsonR,etal.Detectionofspeeifiepol娜eraseehai reactionProduetbyutilizingthes’一3’exonueleaseactivityofthermusaquaticu DNA Polymerase.ProcNatlAeadSeiUSA,1991,88(16),7276一7280.
35.陈凤花,王琳,胡丽华.实时荧光定量RT-PCR内参基因的选择[J].临床检验杂志,2005,23(5):393-395.
36. Nakagawa K, Harrison LC. The potential roles of endogenous retroviruses in autoimmunity [J]. Immunol Rev, 1996, 152: 193-236.
37. Palmarini M, Holland MJ, Cousens C, et al. Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[J]. J Gen Virol, 1996, 77: 2991-2998.
38. de Parseval N, Heidmann T (2005). Human endogenous retroviruses: from infectious elements to human genes.Cytogenet Genome Res 110: 318-332.
39. Ruprecht,K., Gronen, F., Sauter, M., Best, B., Rieckmann, P.,2008. Lack of immuneresponses against multiple sclerosis-associated retrovirus/human endogenous retrovirus W in patients with multiple sclerosis. Journal of NeuroVirology, 14: 143–-151.
40. Griebel, P. J., 1998. Sheep immunology. In Handbook of Vertebrate Immunology, pp. 485-554.
41.丛蓉,杜冰,孔德升等等.免疫生物学[M].第五版。北京科学出版社:2008.
42.冯永淼,蒙古绵羊胚胎肝-脾的组织学发生[M]。内蒙古农业大学,2004,40-43.
43. Rai SK, Demartini JC, Miller AD. Retrovirus vector bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3 [J].J Virol, 2000, 74: 4698-4704.
44. Golovkina TV, Chervonsky AV, Dudley JP, et al. Transgenic mouse mammary tumor virus superantigen expression prevents viral infection[J] , Cell , 1992, 69: 637-645.
45. Held H, Shakhov AN, Izui S, et al. Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus[J]. J Exp Med, 1993, 177: 359-366.
2. Palmarini M, Delasheras M, lnglis NF, et al. Epithelial tumour cells in the lungs of sheep with pulmonary adenomatosis are major sites of replication for Jaagsiekte retrovirus[J]. J Gen Virol, 1995, 76: 2731-2737.
3. Palmarini M, Dalziel RG, Bai J, et al. The exogenous form of Jaagsiekte retrovirus is specifically associated with a contagious lung cancer of sheep[J]. J Virol, 1996, 70: 1618-1623.
4.中华人民共和国农业部公告动物疫病病种名录[J].农业科技通讯,2000.10:35.
5.国际动物卫生组织(OIE)编,农业部畜牧兽医局译.国际动物卫生法典[M].兵器工业出版社,2000,285-289.
6 . Perk K,Hod I.Sheep lung carcinoma:an endemic analogue of a sporadic human neoplasm[J].JNCI,1982,69:747-749.
7.Riley LK.A method for biotinylating oligonucletide probes for use in molecular hybridizations[J].DNA,1986,5(4):333-336.
8. Dungal N. Experiments with jaagsiekte.Amer J of Path,1946,22:737-759.
9. Martin WB,Scott FMM,Sharp JM,et al.Experimental production of sheep pulmonary adenomatosis (Jaagsiekte)[J].Nature,1976,264:183-184.
10. Sharp JM, Angus KW,Gray EW,et al. Rapid transmission of sheep pulmonary adenomatosis (jaasiekte) in young lambs[J].Archives of Virology,1983,78:89-95.
11. Sharp JM,Herring AJ.Sheep pulmonary adenomatosis:demonstration of a protein which cross-reacts with the major core proteins of Mason-Pfizer monkey virus and mouse mammary tumour virus[J].J Gen Virol,1983,64:2323-2327.
12. York DF,Vigne R,Verwoerd DW,et al.Nucleotide sequence of the jaagsiekte retrovirus, an exogenous and endogenous type D and B retrovirus of sheep and goats[J].J Virol, 1992,66(8):4930-4939.
13. Palmarini M,Sharp JM,Lee C, et al. In vitro infection of ovine cell lines by jaagsiekt sheep retrovirus (JSRV)[J].Journal of Virology,1999b,73:10070-10078.
14. Demartini JC,Bishop JV,Allen TE,et al.Jaagsiekte sheep retrovirus proviral clone JSRV(JS7), derived from the JS7 lung tumor cell line,induces ovine pulmonary carcinoma and is integrated into the surfactant protein A gene[J].Journal of Virology,2001,75(9):239-246
15. Gifford R,Tristem M.The evolution,distribution diversity of endogenous retroviruses [J].Virus Genes,2003,26:291-315.
16.Christina , Leenadevi T, et al.Stuart L,Jaagsiekte sheep retrovirus is present at high concentration in lung fluid produced by ovine pulmonary adenocarcinoma-affected sheep and can survive for several weeks at ambient temperatures[J]. Research in Veterinary Science, 2009, 87: 154–156.
17. Caporale M,Centorame P,Giovannini A,at al.Infection of lung epithelial cells and induction of pulmonary adenocarcinoma is not the most common outcome of naturally occurring JSRV infection during the commercial lifespan of sheep[J].Virology,2005, 10:1-10.
18. Ortin A,Minguijqn E,Dewar P,et al.Lack of specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis[J]. Veterinary Immunology and Immunopothology,1998,61:229-237.
19. Bai J, Zhu RY, Stedman K, et al. Unique long terminal repeat U3 sequences distinguish exogenous jaagsiekte sheep retroviruses associated with ovine pulmonary carcinoma from endogenous loci in the sheep genome[J].J Virol,1996,70(5):3159-3168.
20. Gonzalez L,Garcia-Goti M,Cousens C,et al.Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre-clinical period of sheep pulmonary adenomatosis[J].J Gen Virol,2001,82:1355-1358.
21. Maeda N,Palmarini M,Murgia C.Direct transformation of rodent fibroblasts by jaagsikte sheep retrovirus DNA[J].Proc Natl Acad Sci USA,2001,98:4449-4454.
22. Palmarini M, Maeda N, Murgia C. A phosphatidylinositol-3-kinase (PI23K) docking site in the cytoplasmic tail of the jaagsikte sheep retrovirus transmembrane protein is essential for envelope induced transformation of NIH-3T3[J].J Virol,2001,25:3056- 3062.
23. Nishigaki K, Hanson C,Ohashi T. Erythroid cells rendered erythropoietin independent by infection with Friend spleen focusforming virus show constitutive activation of phosphatidylino sitol3-kinase and Ake kinase:involvement of insulin recptor substrate-related adapter proteins[J].J Virol,2000,74:3037-3045.
24. White MK,Straver DS. Surfactant protein A regulates pulmonary surfactant secretion via activation of phosphatidylinositol3-kinase in typeⅡalveolar cells Exp[J]. Cell Res,2000,255:67-76.
25. Margana RK,Boggaram V.Functional analysis of surfactant protein B (SP-B) promoter. Sp1, Sp3,TTF-1,and HNF-3alpha transcription factors are necessary for lung cell-specific activation of SP-B gene transcription[J].J Biol Chem,1997,272(5): 3083-3090.
26. Palmarini M,Mura M,Spencer T.Endogenous betaretroviruses of sheep: teaching new lessons in retroviral interference and adaptation[J].J Gen Virol,2004,85:1-13.
27. Thomas Spencer, Proceedings of the National Academy of Sciences [J]. Virology , 2006,258:201-213.
28. Palmarini M, Holland MJ, Cousens C, et al. Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[J]. J Gen Virol, 1996, 77: 2991-2998.
29. Spencer T E, Mura M, Gray CA, et al. Receptor Usage and Fetal Expression of Ovine Endogenous Betaretroviruses: Implications for Coevolution of Endogenous and Exogenous Retroviruses[J]. J Virol , 2003, 77: 749-753.
30. Palmarini M,Hallwirth C,York D,et al.Molecular cloning and functional analysis of three type D endogenous retrovirus of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus[J].J Virol, 2000,74(17):8065-8076.
31. Bai J,Bishop JV,Carlson JO,et al.Sequence comparison of JSRV with endogenous proviruses:Envelope genotypes and a novel ORF with similarity to a G-protein-coupled receptor[J].Virology,1999,258:333-343.
32. Nakagawa K,Harrison LC.The potential roles of endogenous retroviruses in autoimmunity[J].Immunol Rev,1996,152:193-236.
33. Rai SK,Demartini JC,Miller AD.Retrovirus vector bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3[J].J Virol,2000,74:4698-4704.
34.HollandPM,AbramsonRD,矶厄tsonR,etal.Detectionofspeeifiepol娜eraseehai reactionProduetbyutilizingthes’一3’exonueleaseactivityofthermusaquaticu DNA Polymerase.ProcNatlAeadSeiUSA,1991,88(16),7276一7280.
35.陈凤花,王琳,胡丽华.实时荧光定量RT-PCR内参基因的选择[J].临床检验杂志,2005,23(5):393-395.
36. Nakagawa K, Harrison LC. The potential roles of endogenous retroviruses in autoimmunity [J]. Immunol Rev, 1996, 152: 193-236.
37. Palmarini M, Holland MJ, Cousens C, et al. Jaagsiekte retrovirus establishes a disseminated infection of the lymphoid tissues of sheep affected by pulmonary adenomatosis[J]. J Gen Virol, 1996, 77: 2991-2998.
38. de Parseval N, Heidmann T (2005). Human endogenous retroviruses: from infectious elements to human genes.Cytogenet Genome Res 110: 318-332.
39. Ruprecht,K., Gronen, F., Sauter, M., Best, B., Rieckmann, P.,2008. Lack of immuneresponses against multiple sclerosis-associated retrovirus/human endogenous retrovirus W in patients with multiple sclerosis. Journal of NeuroVirology, 14: 143–-151.
40. Griebel, P. J., 1998. Sheep immunology. In Handbook of Vertebrate Immunology, pp. 485-554.
41.丛蓉,杜冰,孔德升等等.免疫生物学[M].第五版。北京科学出版社:2008.
42.冯永淼,蒙古绵羊胚胎肝-脾的组织学发生[M]。内蒙古农业大学,2004,40-43.
43. Rai SK, Demartini JC, Miller AD. Retrovirus vector bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3 [J].J Virol, 2000, 74: 4698-4704.
44. Golovkina TV, Chervonsky AV, Dudley JP, et al. Transgenic mouse mammary tumor virus superantigen expression prevents viral infection[J] , Cell , 1992, 69: 637-645.
45. Held H, Shakhov AN, Izui S, et al. Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus[J]. J Exp Med, 1993, 177: 359-366.