TGEV S基因B和C抗原位点片段的原核表达及5’核酸探针制备
|
摘要
猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)是冠状病毒科、冠状病毒属成员，引起猪的传染性胃肠炎(Transmissible gastroenteritis TGE)，死亡率很高，呈世界范围分布，严重危害养猪业的发展。TGEV与猪流行性腹泻病毒(PEDV)、猪呼吸道冠状病毒(PRCV)在临床症状上很相似，基因组结构上与TGEV具有较高的同源性，传统的方法很难鉴别。
为了鉴别诊断TGEV与PRCV及对TGEV进行流行病学调查，本研究采用原核表达系统(GST融合表达系统)表达TGEV纤突蛋白(S蛋白)中含有B和C抗原位点的多肽，并且制备了非放射性地高辛标记的核酸探针，通过斑点杂交(Dot-Blot)检测TGEV核酸RNA。
TGEV S蛋白可诱导产生中和抗体，是主要的保护性抗原，其N端抗原位点B和C在PRCV中缺失。根据GenBank TH-98株序列设计第1对引物，应用RT-PCR方法体外扩增TGEV TH-98株S基因5’端含有B、C抗原位点的片段(ts)，大小为357bp，并将ts连接到pMD18-T载体上。序列分析结果为，该片段的核苷酸序列与TGEV其它分离株FS772／70、Miller、Purdue15、Pur46-Mad、133的同源性分别为96.92％、97.76％、98.88％、99.72％、99.44％；氨基酸同源性分别为94.92％、95.76％、97.46％、99.07％、98.31％，表明该片段在TGEV不同分离株中较为保守。将ts片段亚克隆到原核表达载体pGEX-6P-1中，将鉴定为阳性的重组质粒转化到感受态细胞BL21(DE_3)pLysS中。挑选含重组质粒pGEX-6P-ts的菌落，经IPTG(终浓度为0.2mmol／L)诱导后，SDS-PAGE分析目的蛋白与谷胱苷肽硫转移酶(GST大小约为26ku)融合后大小约为40ku，目的蛋白分子量约为13.2ku，与理论值相符。优化表达条件后表达量达37.9％。
TGEV S基因5’端285bp片段在TGEV分离株中较为保守，与其它冠状病毒同源性较低。根据GenBank TH-98株序列设计第2对引物，应用RT-PCR方法体外扩增该片段(命名为tp)，琼脂糖凝胶纯化后测定其浓度和纯度，进行非放射性地高辛标记。对标记效率测定后，以带正电的尼龙膜为介质进行特异性和灵敏性检测，经非放射性自显影来显示靶核酸。本研究中探针tp检测到TGEV cDNA最低量为120pg。
本研究高效表达了TGEV含有B和C抗原位点的多肽片段(TS)。制备了TGEV非放射性标记的核酸探针，并初步应用于检测TGEV。二者为TGEV鉴别诊断、血清学调查奠定了基础。
Transmissible gastroenteritis ( TGE ) was an enteric disease of pigs caused by transmissible gastroenteritis virus (TGEV), a member of coronaviridae family. TGEV was characterized with high mortality and had spread all over the world. The genome structure of TGEV, PEDV and PRCV had high homology and it was difficult to diagnose TGEV by traditional means.
Two studies were carried out to differentiate the TGEV from PRCV and investigate the epidemiology of TGE. One was cloning, sequence analysis and expression of the fragment containing the B and C antigenic sites locating at the 5' terminus in spike gene of TGEV in prokaryotic expression system (fused with GST), the other was preparation of non-radioactive probe labeled by digoxigenin for detecting the RNA extracted from TGEV by assay of Dot-Blot.
Spike protein was the main protective antigen inducing the neutralization antibody. The fragment containing antigenic sites B and C locating at the 5' terminus in spike gene of TGEV was deleted in PRCV. The 1st pair of primer was designed according to strain TH-98.The fragment (named ts) was amplified by RT-PCR and inserted into pMD18-T vector. Sequence analysis showed that homology of nucleotide and amino acid between ts and other TGEV strains Fs772/70, Miller, Purdue15, Pur46-Mad, 133 were 96.92%, 97.76%, 98.88%, 99.72%, 99.44% and 94.92%, 95.76%, 97.46%, 99.07%, 98.31% respectively. Fragment ts was subcloned into expression plasmid pGEX-6P-1, then was transformed into competent cell of BL21 (DE3) pLysS. After screening the positive colony containing recombinant plasmid pGEX-6P-ts, the E.coli was induced by 0.2mmol/L IPTG and was analyzed by SDS-PAGE. The overexpressed protein (TS) was 13.2 ku and showed 40ku in gel fused with GST (26ku) . The amount of TS expressed was 37.9% under optimized condition.
The 285 bp fragment (named tp) , locating at 5'terminus of spike gene, had high conservation in TGEV isolations. The 2nd pair of primers was designed according to the sequence of strain TH-98 collected in GenBank. Concentration of tp was analyzed after amplification invitro by RT-PCR, purified by low melt agarose and labeled by digoxigenin. After the estimation of labeling efficiency tp was applicated to hybridize the RNA of TGEV, then was subjected to self-development of X-ray exposed by CSPD to show the target nucleic acid. The minimal amount of TGEV cDNA can be detected was about 120 pg in this research.
In the research TS containing antigenic sites B and C was expressed with high efficiency. Nonradioactive nucleic probe to TGEV was prepared and was applicated to detect TGEV at
first stage, which established foundational work for differentiating TGEV from PRCV and investigation on epidemiology of TGEV.
Candidate: Hu Sen Major: Preventive Veterinary Science
Supervisor: Prof. Wang Junwei
为了鉴别诊断TGEV与PRCV及对TGEV进行流行病学调查，本研究采用原核表达系统(GST融合表达系统)表达TGEV纤突蛋白(S蛋白)中含有B和C抗原位点的多肽，并且制备了非放射性地高辛标记的核酸探针，通过斑点杂交(Dot-Blot)检测TGEV核酸RNA。
TGEV S蛋白可诱导产生中和抗体，是主要的保护性抗原，其N端抗原位点B和C在PRCV中缺失。根据GenBank TH-98株序列设计第1对引物，应用RT-PCR方法体外扩增TGEV TH-98株S基因5’端含有B、C抗原位点的片段(ts)，大小为357bp，并将ts连接到pMD18-T载体上。序列分析结果为，该片段的核苷酸序列与TGEV其它分离株FS772／70、Miller、Purdue15、Pur46-Mad、133的同源性分别为96.92％、97.76％、98.88％、99.72％、99.44％；氨基酸同源性分别为94.92％、95.76％、97.46％、99.07％、98.31％，表明该片段在TGEV不同分离株中较为保守。将ts片段亚克隆到原核表达载体pGEX-6P-1中，将鉴定为阳性的重组质粒转化到感受态细胞BL21(DE_3)pLysS中。挑选含重组质粒pGEX-6P-ts的菌落，经IPTG(终浓度为0.2mmol／L)诱导后，SDS-PAGE分析目的蛋白与谷胱苷肽硫转移酶(GST大小约为26ku)融合后大小约为40ku，目的蛋白分子量约为13.2ku，与理论值相符。优化表达条件后表达量达37.9％。
TGEV S基因5’端285bp片段在TGEV分离株中较为保守，与其它冠状病毒同源性较低。根据GenBank TH-98株序列设计第2对引物，应用RT-PCR方法体外扩增该片段(命名为tp)，琼脂糖凝胶纯化后测定其浓度和纯度，进行非放射性地高辛标记。对标记效率测定后，以带正电的尼龙膜为介质进行特异性和灵敏性检测，经非放射性自显影来显示靶核酸。本研究中探针tp检测到TGEV cDNA最低量为120pg。
本研究高效表达了TGEV含有B和C抗原位点的多肽片段(TS)。制备了TGEV非放射性标记的核酸探针，并初步应用于检测TGEV。二者为TGEV鉴别诊断、血清学调查奠定了基础。
Transmissible gastroenteritis ( TGE ) was an enteric disease of pigs caused by transmissible gastroenteritis virus (TGEV), a member of coronaviridae family. TGEV was characterized with high mortality and had spread all over the world. The genome structure of TGEV, PEDV and PRCV had high homology and it was difficult to diagnose TGEV by traditional means.
Two studies were carried out to differentiate the TGEV from PRCV and investigate the epidemiology of TGE. One was cloning, sequence analysis and expression of the fragment containing the B and C antigenic sites locating at the 5' terminus in spike gene of TGEV in prokaryotic expression system (fused with GST), the other was preparation of non-radioactive probe labeled by digoxigenin for detecting the RNA extracted from TGEV by assay of Dot-Blot.
Spike protein was the main protective antigen inducing the neutralization antibody. The fragment containing antigenic sites B and C locating at the 5' terminus in spike gene of TGEV was deleted in PRCV. The 1st pair of primer was designed according to strain TH-98.The fragment (named ts) was amplified by RT-PCR and inserted into pMD18-T vector. Sequence analysis showed that homology of nucleotide and amino acid between ts and other TGEV strains Fs772/70, Miller, Purdue15, Pur46-Mad, 133 were 96.92%, 97.76%, 98.88%, 99.72%, 99.44% and 94.92%, 95.76%, 97.46%, 99.07%, 98.31% respectively. Fragment ts was subcloned into expression plasmid pGEX-6P-1, then was transformed into competent cell of BL21 (DE3) pLysS. After screening the positive colony containing recombinant plasmid pGEX-6P-ts, the E.coli was induced by 0.2mmol/L IPTG and was analyzed by SDS-PAGE. The overexpressed protein (TS) was 13.2 ku and showed 40ku in gel fused with GST (26ku) . The amount of TS expressed was 37.9% under optimized condition.
The 285 bp fragment (named tp) , locating at 5'terminus of spike gene, had high conservation in TGEV isolations. The 2nd pair of primers was designed according to the sequence of strain TH-98 collected in GenBank. Concentration of tp was analyzed after amplification invitro by RT-PCR, purified by low melt agarose and labeled by digoxigenin. After the estimation of labeling efficiency tp was applicated to hybridize the RNA of TGEV, then was subjected to self-development of X-ray exposed by CSPD to show the target nucleic acid. The minimal amount of TGEV cDNA can be detected was about 120 pg in this research.
In the research TS containing antigenic sites B and C was expressed with high efficiency. Nonradioactive nucleic probe to TGEV was prepared and was applicated to detect TGEV at
first stage, which established foundational work for differentiating TGEV from PRCV and investigation on epidemiology of TGEV.
Candidate: Hu Sen Major: Preventive Veterinary Science
Supervisor: Prof. Wang Junwei
引文
1 程焕春．刘操．金梅林 等．猪传染性胃肠炎病毒的分离鉴定．华中农业大学学报．1992，11(1)：82-86
2 鄂征 主编．组织培养和分子细胞学技术．科学出版社，1994
3 方定一 等主编．兽医微生物学．农业出版社，1979
4 冯立．佟有恩．李伟杰 等．猪传染性胃肠炎华毒弱毒株纤突蛋白B.C抗原位点片段基因的克隆及序列分析．中国预防兽医学报 2000，22(5)．361-365
5 何琚．严家新．李承平．朱家鸿．狂犬病毒aG株糖蛋白在PET原核系统中的表达及纯化微生物免疫学进展．1999，27(3)：1-6
6 秦翠丽 金宁一 佟明华 等 狂犬病病毒糖蛋白在原核细胞中的表达 中国兽医学报 1996，(16)1：34-37
7 冉智光 等．猪传染性胃肠炎病毒的血凝作用．中国兽医科技．1997，27(9)：3-4
8 苏忠兰．金宁一．段正芳．HIV-2 gag 蛋白在大肠杆菌中的表达纯化中华皮肤科杂志 1999，32(5)：328-332
9 王继科．刘常明．马思奇等．猪传染性胃肠炎和流行性腹泻免疫电镜的诊断研究．中国畜禽传染病．1991，(2)：22-25
10 王明俊等．兽医生物制品．中国农业出版社，1996
11 吴冠芸．潘华珍．生物化学与分子生物学实验常用数据手册．第一版．科学出版社．1999．北京．346—349
12 吴乃虎 基因工程原理(下)．第二版．科学出版社．2001
13 殷震．刘景华．动物病毒学．第二版，北京．科学出版社．1997：681-688
14 Bae Ⅰ.Jackwood DJ.Benfield DA. Deffereantiation of transmissible gastroenteritis virus from porcine respiratory eoronavirus an antigenically related coronavirus by using probes specific for the 5'region or-the S glycoprotein gene. J Clin Microbiol. 1991, 29. (1): 215-218
15 Ballesteros L.Sanches C.Enjuanes L. Two amino acid changes at the N-terminal of TGEV spike protein result in the loss of enteric tropism. Virology. 1997,227 (2): 378-388
16 Benfield DA.Jackwood DJ.Bae Ⅰ.Saif LJ.Wesley RD. Detection of transmissible gastroenteritis virus using eDNA probes. Arch Virol. 1991, 116: 91-106
17 Bernard S.Laude H. Site-specific alteration of transmissible gastroenteritis virus spike protein results in markedly reduced pathogenity. Gen Virol. 1995,76:2235-2241
18 Bohl EH. Diagnosis of diarrhea in pigs due to transmissible gastroenteritis or rotavirus. In: Viral Enteritis in Humans and Animals. Bricout F. & Seherrer R. eds. INSERM. Paris.France. 1979,90. 341-343.
19 Britton P. Page KW. Sequence of the S gene from a virulent British field isolate of transmissible gastroenteritis virus. Virus Res. 1990,18: 71-80.
20 Callbaut P. Pensaert M. Jimenezg. Antigenic differrntiation between TGEV and a related
PRCV. J Gn Virol.1988.69:1725-1730
21 Callebaut P. Pensaert MB.Hooyberghs J. A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus. Vet.Microbiol. 1989,20: 9-19
22 Cavanagh D. Brian DA. Bruton MA. Enjuanes L. Holmes KV. Coronavidae In Virus Taxonomy. The Classification and Nomenclature of Viruses,6th Rep of ICTV.F.A.Murphy et al. Vienna and NewYork 1995,pp.407-411
23 Chomczynski P. Sacchi N..Single-step methods of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987,162:156-159
24 Christine K.Graham D.Yolken R.et al. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of TGEV [J]. Journal of Virology.1997,71(4): 3285-3287
25 Correa Ⅰ, Jimenze G, Sune C, Bullido M J, Enjuanes L. Antigenic structure of E2-glycoprotein of transmissible gastroenteritis coronavirus Virus Res. 1988,10:77-94.
26 Correa Ⅰ.Gebauer F. Buillido M J. Sune C et al. Location of antigenic site of the E2 glycoprotein of transmissible gastroenteritis virus. J Gert Virol. 1990,71: 271-279.
27 Delmas B, Gelfi J, Laude H. Antigenic structure of transmissible gastroenteritis coronavirus Ⅱ.Domains in the peplomer protein. J. Gen: Virol. 1986,67:1405-1418
28 Doyle LP. Hutching LM. A transmissible gastroenteritis virus in pigs. J Am Vet Assoc.1946,108:257-259
29 Dulac GC.Ruckerbauer GM.Boulanger P. Transmissible gastroenteritis: demonstration of the virus from field specimens by means of cell culture and pig inoculation. Can. J. Comp.Med. 1997,41.357-363
30 Eleouet J.Bett A.Koolen M.et al. Complete sequence of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus.[J] .Journal of Virology. 1995,206: 817-822.
31 Enjuanes L. Vanderzeijst BA. Molecular basis ofTGEV epidemiplogy in the coroviridae Ed.S.G Siddell.New York: Pleum Press,pp1995.337-376
32 Enjuanes L.Van der Zeijst BAM..Molecular basis of transmissible gastroenteritis epidemiology. In.S.G.Siddle (ed).The coronaviridae Plenum Press.New York..1995,337-376
33 Escors D., Camafeita E., Ortego J. et al. Organization of two transmissible gastroenteritis coronavirus membrane protein tepologies within the virion and core. J. Vriol.2001,75(24): 12228-12240
34 Fatima G.Willewp A.Correa C.e.t al. Residues involved in the antigenic sites of transmissible gastroenteritis virus S glycoprotein. Virology. 1991,183: 225-238
35 Godet M.Stewart F. Higgins D.Processing and antigenicity of entire and anchor-free S protein of TGEV expressed by recombinant Baculovirus. Journal of Virol. 1991,185:
732-740
36 Gomez N.Carrillo J.Parra F. et al. Expression of immunogenic glycoprotein S polypeptides from TGEV in transgenic plants [J]. Virology. 1998,249(2): 352-358
37 Haelterman EO. Epidemiological studies of transmissible gastroenteritis of swine. Proc US Livest Sunit Assoc. 1962,66:305-315
38 Haelterman EO. Hutchings LM. Epidemic diarrheal diseases of viral origin in newborn swine .Ann NY Acad Sci 195666:186190
39 Harada K. Kaji J. Kumagai T. Sasahara J. Studies on TGE in pigs Physicochemical and biological properties of TGE virus. Natl Inst Anim Health Q. 1968, (Tokyo) 8:140-147
40 Hill H. Biwer J. Wood R. PRCV isolated from two U.S swine herds.In.Proc. Am. Assoc. Swine. Pract. Desmoines. Iowa. 1990,333
41 Hiscox JA.Characterization of the transmissible gastroenteritis virus (TGEV) transcription initiation sequence.Adv Exp Med Biol. 1995,380:529-535
42 Horinek MC. Lutzh H. Pedersen NC. Antigenic relationships among homologous structural polypeptides of porcine. Feline and canine eoronavirus. Infect Immun 1982,37:1148-1155
43 Jackwood DJ.Bae Ⅰ.Jackwood RJ. Saif LJ. Transmissible gastroenteritis virus and porcine respiratory coronavirus: molecular characterization of the S gene using cDNA probes and nucleotide sequence analysis. Adv. Exp. Med. Biol. 1993,3. 42: 43-48
44 Jackwood DJ.won HM.Saif LJ.Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains correlation with antigenicity and pathogenicity.. Adv Exp Med Biol. 1995,380:35-41
45 Jones T. Shenk T. Transmissible gastroenteritis coronavirus of pigs. [J] Journal of Veterinary Record. 1997,41(16): 427-428
46 Julian A.Hiscox JA.David C.et al. Quantification of individual subgenomic mRNA species during replication of the transmissible gastroenteritis virus. Virus Research. 1995,36:119-130
47 Kemeny LJ. Antibody response in pigs in pigs inoculated with TGEV and cross reaction among ten isolates. Can. J. Comp. Med. 40:209-214
48 Kim B.Chae C. In situ hybridization technique for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods. Can J Vet Res. 2001,65(1): 33-37
49 Kim L.Chang KO.Sestak K.Parwani A. Development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs.Vet Diagn Invest. 2000,124): 385-388
50 Kim L.Hayes J.Lewis P. Parwani AV. Chang KO.Salf LJ. Molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus and porcine respiratory
coronavirus fields isolates co-circulating in a swine herd. Arch Virol.2000,145: 1133-1147
51 Kim O.Choi C.Kim B.Chae CDetection and differentiation of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in clinical samples by multiplex RT-PCR.Vet Rec May.2000,146 (22): 637-640
52 Klein RC.and Ristic M. The effect of propagation of transmissible gastroenteritis (TGE) virus in pups and the lung of baby pigs on the immunologic properties of the virus. Proc 4th Int Conger Pig. Vet. Soc.1976,Iowa. State Uniu.p.k 11.
53 Krempl C. Schultze B. Laude H. Herrler G. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.J Virol. 1997,71:3285-3287.
54 Kwon HM.Saif LJ.Jackwood DJ.Field isolates of transmissible gastroenteritis virus differ at the molecular level from the Miller and Purdue virulent and attenuated strains and from porcine respiratory coronavirus. J Vet Med Sci. 1998, 60(5): 589-597
55 Laude H.Godet M.Bernard S.Gelfi.Duarte M.Delmas B.Functions domains in the spike protein of transmissible gastroenteritis virus.Adv. Exp.Med. Biol. 1995, 380: 299-304.
56 Laude H.Rasscgaert D.Delmas B.Godet M.Gelfi J.Charley B. Molecular biology of transmissible gastroenteritis. Vet. Microbiol. 1990. (4) 23:147-154.
57 Laude H.Reeh KV. Pensaert M. Porcine respiratory coronavirus molecular feature and virus-host interactions. Vet Res. 1993,24: 125-150.
58 Lowings P. Laude H.Charley B. Discrimination between transmissible gastroenteritis and isolaties. Archives of Virol. 1997,142(8): 1703-1722.
59 Mc Clurkin AW. Stark S.Norman JO.Transmissible gastroenteritis (TGE) of swine:the possible role of dog in the epizootiology of TGE. Can. J. Comp.Med. 1970, 34:3457-3459
60 McGoldrick A.Lowings JP. Pato DJ. Characterisaion of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a. Arch Virol. 1999,144(4) :763-770.
61 Meny L.J. Isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter.Am J Vet Res. 1978, 39:703-705.
62 Motokawa K. Comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid protein of feline, canine and porcine coronavirus. Microbiology and Immunology. 1996,40 (6): 425-433.
63 Nelson L.D. Kehling CL.Enzyme-linked immunosorbent assay for detection of transmissible gastroenteritis virus antibody in swine sera. Am. J. Vet. Res. 1984, 45. 1645-1657.
64 Noda M.Yamashita H. Koide K. Hemagglutination with TGEV. Arch. Virol 1987,96:109-115.
65 Paton D.Ibata G.Sands J.McGoldrick A. Detection transmissible gastroenteritis virus by
RT-PCR and differentiation from porcine respiratory coronavirus. Virol Methods. 1997,66(2): 303-309.
66 Paul B.Kevin W. The cloning and sequencing of the virion protein genes from a British isolate of PRCV:Comparison with TGEV. Virus Research. 1991,21: 181-198.
67 Paul PS. Halbur PG. Vaugh EM. Significance of PRCV infection Compendium. Cont EDUC Pract. Vet 1994, (16):1223-1234.
68 Pederden MC.Warde J.Mengeling WL. Antigenic relationship of feline infectious peritonitis virus to coronavirus of other species. Arch Viol.1978,58: 45-53.
69 Pensaert D.Callebaut RVergote J. Isolation of a porcine respiratory.non-enteric coronavirus related to transmissible gastroenteritis coronavirus.Vet.Q.1986,8: 257-261
70 Rasschaert D.Laude H. The predicted primary structure of the peplomer protein E2 of the porcine transmissible gastroenteritis virus.J Gen Virol. 1987,68: 1883-1890.
71 Reynolds DJ.Garwes DJ.Lucey S. Differentiation of canine coronavirus and porcine transmissible gastroenteritis virus by neutralization with canine.porcine.and feline sera.Vet Microbiol. 1980, 5283-290.
72 Riffault S.Baric R.Levy L. In vivo induction of inferno-alpha in pig by non-infectious coronacirus: tissue localization and in situ phenotypic characterization of inferno-alpha producing cell. [J].Journal of General Virology. 1997,78(10): 2483-2487.
73 Saif LJ.Wesley RD. Transmissible gastroenteritis and porcine respiratory coronavirus in Straw BE.D'Allaire S.Mengeling WL.Taylor DJ. (eds).Diseases of swine 8th ed. Iowa State University Press.Ames.pp. 1999, 295-325.
74 Sail LJ.Wesley RD. Transmissible gastroenteritis, pp: In: Diseases of Swine (Leman AD.Straw BE and Mengeling WL.et al) Iowa state university Press.Ames. IA. 1992, 362-386.
75 Sambrook J. Fritsch EF. Maniatis T. Molecular Cloning [M].2nd.ed.Cold Spring Harbor Laboratory Press 1998, 19-21.
76 Schltze B. Krempl C. Ballesteros M. TGE coronavirus, but not the related PRCV has a sialic acid binding activity. J Virol 1996,70:5636-5637
77 Shockley LJ. Kapke PA.Lapps W. Brian DA.Potgeier. J Clin Microbiol.1987,Sep. 25: 1591-1596.
78 Shoup DI..Active and passive immune responses to TGEV in swine inoculated with recombinant baculovirus-expressed TGEV spike glycoprotein vaccines.American Journal of Veterinary Reseach. 1997,58(3): 242-250.
79 Siddle SG. The coronaviridae: An introdution.in the coronaviridae. Ed.S.G Siddell. New York: plenum Press. 1995, PP, 1-10.
80 Simkins RA.Weilnau PA.Bias J.Saif LJ.Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV.Am J Vet Res. 1992,53: 1253-1258.
81 Sirinarumitr T, Paul PS, Kluge JP, Halbur PG. In situ hybridization technique for the detection of swine enteric and respiratory coronavirus, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embeddedtissues. Virol Methods. 1996,56(2): 149-160.
82 Sirinarumitr T, Siddell S, Grahare F. Porcine transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures. Archives of Virology. 1998,143(12): 2471-2485.
83 Sirinarumitr T. Paul PS.Halbur PG.Kluge JP. An overview of immunological and genetic methods for detecting swine coronavirus.transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues.Adv Exp Med Biol. 1997,412: 37-46.
84 Sirinarumitr T. Paul PS.Halbur PG.Kluge JP. Rapid in situ hybridization technique for the detection of ribonucleic acids in tissues using radiolabelled and fluorescein-labelled riboprobes. Mol Cell Probes. 1997,11(4): 273-280.
85 Torres JM.Alonso C.Mittal S.et al. Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against TGEV. Journal of Virology. 1996, 70:3770-3780
86 Torres JM.Sanchz C.Mittal S. et al. Induction of antibodies protecting against of TGEV by recombinant adenovirus expressing TGEV spike protein Virology. 1995,213:503-516
87 Tuboly T. Nagy E. Dennis JR, Derbyshine JB. Immunogenicity of the S protein of transmissible gastroenteritis coronavirus expressed in baclovirus. Arch Virol. 1994,137: 55-67.
88 Vaughn EM.Halbur PG.Paul PS. Use of nonradioactive cDNA probes to differentiate porcine respiratory coronavirus and transmissible gastroenteritis virus isolates. Vet Diagn Invest. 1996,8(2): 241-244.
89 Weslay R.Woods R.Chuny A.Nucleotide basis of the pathogenesis of transmissible gastroenteritis coronavirus. Journal of Virol. 1990,64(10): 4761-4766.
90 Wesley R.Woods R.Cheuny A. Genetic basis of the pathogenesis of transmissible gastroenteritis virus. Journal of Virol. 1990,64(10): 4761-4766.
91 Wesley R.Woods R.Cheuny A.Nucleotide sequence of the E2-peplomer protein gene and partial nucleotide sequence of upstream polymerase of transmissible gastroenteritis virus (Miller strain).Adv Exp Med Biol. 1990,276:301-306.
92 Woods RD.Studies of enteric coronavirus in a feline cell line. Vet Microbiol.1982,(7): 427-435.
2 鄂征 主编．组织培养和分子细胞学技术．科学出版社，1994
3 方定一 等主编．兽医微生物学．农业出版社，1979
4 冯立．佟有恩．李伟杰 等．猪传染性胃肠炎华毒弱毒株纤突蛋白B.C抗原位点片段基因的克隆及序列分析．中国预防兽医学报 2000，22(5)．361-365
5 何琚．严家新．李承平．朱家鸿．狂犬病毒aG株糖蛋白在PET原核系统中的表达及纯化微生物免疫学进展．1999，27(3)：1-6
6 秦翠丽 金宁一 佟明华 等 狂犬病病毒糖蛋白在原核细胞中的表达 中国兽医学报 1996，(16)1：34-37
7 冉智光 等．猪传染性胃肠炎病毒的血凝作用．中国兽医科技．1997，27(9)：3-4
8 苏忠兰．金宁一．段正芳．HIV-2 gag 蛋白在大肠杆菌中的表达纯化中华皮肤科杂志 1999，32(5)：328-332
9 王继科．刘常明．马思奇等．猪传染性胃肠炎和流行性腹泻免疫电镜的诊断研究．中国畜禽传染病．1991，(2)：22-25
10 王明俊等．兽医生物制品．中国农业出版社，1996
11 吴冠芸．潘华珍．生物化学与分子生物学实验常用数据手册．第一版．科学出版社．1999．北京．346—349
12 吴乃虎 基因工程原理(下)．第二版．科学出版社．2001
13 殷震．刘景华．动物病毒学．第二版，北京．科学出版社．1997：681-688
14 Bae Ⅰ.Jackwood DJ.Benfield DA. Deffereantiation of transmissible gastroenteritis virus from porcine respiratory eoronavirus an antigenically related coronavirus by using probes specific for the 5'region or-the S glycoprotein gene. J Clin Microbiol. 1991, 29. (1): 215-218
15 Ballesteros L.Sanches C.Enjuanes L. Two amino acid changes at the N-terminal of TGEV spike protein result in the loss of enteric tropism. Virology. 1997,227 (2): 378-388
16 Benfield DA.Jackwood DJ.Bae Ⅰ.Saif LJ.Wesley RD. Detection of transmissible gastroenteritis virus using eDNA probes. Arch Virol. 1991, 116: 91-106
17 Bernard S.Laude H. Site-specific alteration of transmissible gastroenteritis virus spike protein results in markedly reduced pathogenity. Gen Virol. 1995,76:2235-2241
18 Bohl EH. Diagnosis of diarrhea in pigs due to transmissible gastroenteritis or rotavirus. In: Viral Enteritis in Humans and Animals. Bricout F. & Seherrer R. eds. INSERM. Paris.France. 1979,90. 341-343.
19 Britton P. Page KW. Sequence of the S gene from a virulent British field isolate of transmissible gastroenteritis virus. Virus Res. 1990,18: 71-80.
20 Callbaut P. Pensaert M. Jimenezg. Antigenic differrntiation between TGEV and a related
PRCV. J Gn Virol.1988.69:1725-1730
21 Callebaut P. Pensaert MB.Hooyberghs J. A competitive inhibition ELISA for the differentiation of serum antibodies from pigs infected with transmissible gastroenteritis virus (TGEV) or with the TGEV-related porcine respiratory coronavirus. Vet.Microbiol. 1989,20: 9-19
22 Cavanagh D. Brian DA. Bruton MA. Enjuanes L. Holmes KV. Coronavidae In Virus Taxonomy. The Classification and Nomenclature of Viruses,6th Rep of ICTV.F.A.Murphy et al. Vienna and NewYork 1995,pp.407-411
23 Chomczynski P. Sacchi N..Single-step methods of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987,162:156-159
24 Christine K.Graham D.Yolken R.et al. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of TGEV [J]. Journal of Virology.1997,71(4): 3285-3287
25 Correa Ⅰ, Jimenze G, Sune C, Bullido M J, Enjuanes L. Antigenic structure of E2-glycoprotein of transmissible gastroenteritis coronavirus Virus Res. 1988,10:77-94.
26 Correa Ⅰ.Gebauer F. Buillido M J. Sune C et al. Location of antigenic site of the E2 glycoprotein of transmissible gastroenteritis virus. J Gert Virol. 1990,71: 271-279.
27 Delmas B, Gelfi J, Laude H. Antigenic structure of transmissible gastroenteritis coronavirus Ⅱ.Domains in the peplomer protein. J. Gen: Virol. 1986,67:1405-1418
28 Doyle LP. Hutching LM. A transmissible gastroenteritis virus in pigs. J Am Vet Assoc.1946,108:257-259
29 Dulac GC.Ruckerbauer GM.Boulanger P. Transmissible gastroenteritis: demonstration of the virus from field specimens by means of cell culture and pig inoculation. Can. J. Comp.Med. 1997,41.357-363
30 Eleouet J.Bett A.Koolen M.et al. Complete sequence of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus.[J] .Journal of Virology. 1995,206: 817-822.
31 Enjuanes L. Vanderzeijst BA. Molecular basis ofTGEV epidemiplogy in the coroviridae Ed.S.G Siddell.New York: Pleum Press,pp1995.337-376
32 Enjuanes L.Van der Zeijst BAM..Molecular basis of transmissible gastroenteritis epidemiology. In.S.G.Siddle (ed).The coronaviridae Plenum Press.New York..1995,337-376
33 Escors D., Camafeita E., Ortego J. et al. Organization of two transmissible gastroenteritis coronavirus membrane protein tepologies within the virion and core. J. Vriol.2001,75(24): 12228-12240
34 Fatima G.Willewp A.Correa C.e.t al. Residues involved in the antigenic sites of transmissible gastroenteritis virus S glycoprotein. Virology. 1991,183: 225-238
35 Godet M.Stewart F. Higgins D.Processing and antigenicity of entire and anchor-free S protein of TGEV expressed by recombinant Baculovirus. Journal of Virol. 1991,185:
732-740
36 Gomez N.Carrillo J.Parra F. et al. Expression of immunogenic glycoprotein S polypeptides from TGEV in transgenic plants [J]. Virology. 1998,249(2): 352-358
37 Haelterman EO. Epidemiological studies of transmissible gastroenteritis of swine. Proc US Livest Sunit Assoc. 1962,66:305-315
38 Haelterman EO. Hutchings LM. Epidemic diarrheal diseases of viral origin in newborn swine .Ann NY Acad Sci 195666:186190
39 Harada K. Kaji J. Kumagai T. Sasahara J. Studies on TGE in pigs Physicochemical and biological properties of TGE virus. Natl Inst Anim Health Q. 1968, (Tokyo) 8:140-147
40 Hill H. Biwer J. Wood R. PRCV isolated from two U.S swine herds.In.Proc. Am. Assoc. Swine. Pract. Desmoines. Iowa. 1990,333
41 Hiscox JA.Characterization of the transmissible gastroenteritis virus (TGEV) transcription initiation sequence.Adv Exp Med Biol. 1995,380:529-535
42 Horinek MC. Lutzh H. Pedersen NC. Antigenic relationships among homologous structural polypeptides of porcine. Feline and canine eoronavirus. Infect Immun 1982,37:1148-1155
43 Jackwood DJ.Bae Ⅰ.Jackwood RJ. Saif LJ. Transmissible gastroenteritis virus and porcine respiratory coronavirus: molecular characterization of the S gene using cDNA probes and nucleotide sequence analysis. Adv. Exp. Med. Biol. 1993,3. 42: 43-48
44 Jackwood DJ.won HM.Saif LJ.Molecular differentiation of transmissible gastroenteritis virus and porcine respiratory coronavirus strains correlation with antigenicity and pathogenicity.. Adv Exp Med Biol. 1995,380:35-41
45 Jones T. Shenk T. Transmissible gastroenteritis coronavirus of pigs. [J] Journal of Veterinary Record. 1997,41(16): 427-428
46 Julian A.Hiscox JA.David C.et al. Quantification of individual subgenomic mRNA species during replication of the transmissible gastroenteritis virus. Virus Research. 1995,36:119-130
47 Kemeny LJ. Antibody response in pigs in pigs inoculated with TGEV and cross reaction among ten isolates. Can. J. Comp. Med. 40:209-214
48 Kim B.Chae C. In situ hybridization technique for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods. Can J Vet Res. 2001,65(1): 33-37
49 Kim L.Chang KO.Sestak K.Parwani A. Development of a reverse transcription-nested polymerase chain reaction assay for differential diagnosis of transmissible gastroenteritis virus and porcine respiratory coronavirus from feces and nasal swabs of infected pigs.Vet Diagn Invest. 2000,124): 385-388
50 Kim L.Hayes J.Lewis P. Parwani AV. Chang KO.Salf LJ. Molecular characterization and pathogenesis of transmissible gastroenteritis coronavirus and porcine respiratory
coronavirus fields isolates co-circulating in a swine herd. Arch Virol.2000,145: 1133-1147
51 Kim O.Choi C.Kim B.Chae CDetection and differentiation of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in clinical samples by multiplex RT-PCR.Vet Rec May.2000,146 (22): 637-640
52 Klein RC.and Ristic M. The effect of propagation of transmissible gastroenteritis (TGE) virus in pups and the lung of baby pigs on the immunologic properties of the virus. Proc 4th Int Conger Pig. Vet. Soc.1976,Iowa. State Uniu.p.k 11.
53 Krempl C. Schultze B. Laude H. Herrler G. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.J Virol. 1997,71:3285-3287.
54 Kwon HM.Saif LJ.Jackwood DJ.Field isolates of transmissible gastroenteritis virus differ at the molecular level from the Miller and Purdue virulent and attenuated strains and from porcine respiratory coronavirus. J Vet Med Sci. 1998, 60(5): 589-597
55 Laude H.Godet M.Bernard S.Gelfi.Duarte M.Delmas B.Functions domains in the spike protein of transmissible gastroenteritis virus.Adv. Exp.Med. Biol. 1995, 380: 299-304.
56 Laude H.Rasscgaert D.Delmas B.Godet M.Gelfi J.Charley B. Molecular biology of transmissible gastroenteritis. Vet. Microbiol. 1990. (4) 23:147-154.
57 Laude H.Reeh KV. Pensaert M. Porcine respiratory coronavirus molecular feature and virus-host interactions. Vet Res. 1993,24: 125-150.
58 Lowings P. Laude H.Charley B. Discrimination between transmissible gastroenteritis and isolaties. Archives of Virol. 1997,142(8): 1703-1722.
59 Mc Clurkin AW. Stark S.Norman JO.Transmissible gastroenteritis (TGE) of swine:the possible role of dog in the epizootiology of TGE. Can. J. Comp.Med. 1970, 34:3457-3459
60 McGoldrick A.Lowings JP. Pato DJ. Characterisaion of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a. Arch Virol. 1999,144(4) :763-770.
61 Meny L.J. Isolation of transmissible gastroenteritis virus from pharyngeal swabs obtained from sows at slaughter.Am J Vet Res. 1978, 39:703-705.
62 Motokawa K. Comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid protein of feline, canine and porcine coronavirus. Microbiology and Immunology. 1996,40 (6): 425-433.
63 Nelson L.D. Kehling CL.Enzyme-linked immunosorbent assay for detection of transmissible gastroenteritis virus antibody in swine sera. Am. J. Vet. Res. 1984, 45. 1645-1657.
64 Noda M.Yamashita H. Koide K. Hemagglutination with TGEV. Arch. Virol 1987,96:109-115.
65 Paton D.Ibata G.Sands J.McGoldrick A. Detection transmissible gastroenteritis virus by
RT-PCR and differentiation from porcine respiratory coronavirus. Virol Methods. 1997,66(2): 303-309.
66 Paul B.Kevin W. The cloning and sequencing of the virion protein genes from a British isolate of PRCV:Comparison with TGEV. Virus Research. 1991,21: 181-198.
67 Paul PS. Halbur PG. Vaugh EM. Significance of PRCV infection Compendium. Cont EDUC Pract. Vet 1994, (16):1223-1234.
68 Pederden MC.Warde J.Mengeling WL. Antigenic relationship of feline infectious peritonitis virus to coronavirus of other species. Arch Viol.1978,58: 45-53.
69 Pensaert D.Callebaut RVergote J. Isolation of a porcine respiratory.non-enteric coronavirus related to transmissible gastroenteritis coronavirus.Vet.Q.1986,8: 257-261
70 Rasschaert D.Laude H. The predicted primary structure of the peplomer protein E2 of the porcine transmissible gastroenteritis virus.J Gen Virol. 1987,68: 1883-1890.
71 Reynolds DJ.Garwes DJ.Lucey S. Differentiation of canine coronavirus and porcine transmissible gastroenteritis virus by neutralization with canine.porcine.and feline sera.Vet Microbiol. 1980, 5283-290.
72 Riffault S.Baric R.Levy L. In vivo induction of inferno-alpha in pig by non-infectious coronacirus: tissue localization and in situ phenotypic characterization of inferno-alpha producing cell. [J].Journal of General Virology. 1997,78(10): 2483-2487.
73 Saif LJ.Wesley RD. Transmissible gastroenteritis and porcine respiratory coronavirus in Straw BE.D'Allaire S.Mengeling WL.Taylor DJ. (eds).Diseases of swine 8th ed. Iowa State University Press.Ames.pp. 1999, 295-325.
74 Sail LJ.Wesley RD. Transmissible gastroenteritis, pp: In: Diseases of Swine (Leman AD.Straw BE and Mengeling WL.et al) Iowa state university Press.Ames. IA. 1992, 362-386.
75 Sambrook J. Fritsch EF. Maniatis T. Molecular Cloning [M].2nd.ed.Cold Spring Harbor Laboratory Press 1998, 19-21.
76 Schltze B. Krempl C. Ballesteros M. TGE coronavirus, but not the related PRCV has a sialic acid binding activity. J Virol 1996,70:5636-5637
77 Shockley LJ. Kapke PA.Lapps W. Brian DA.Potgeier. J Clin Microbiol.1987,Sep. 25: 1591-1596.
78 Shoup DI..Active and passive immune responses to TGEV in swine inoculated with recombinant baculovirus-expressed TGEV spike glycoprotein vaccines.American Journal of Veterinary Reseach. 1997,58(3): 242-250.
79 Siddle SG. The coronaviridae: An introdution.in the coronaviridae. Ed.S.G Siddell. New York: plenum Press. 1995, PP, 1-10.
80 Simkins RA.Weilnau PA.Bias J.Saif LJ.Antigenic variation among transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus strains detected with monoclonal antibodies to the S protein of TGEV.Am J Vet Res. 1992,53: 1253-1258.
81 Sirinarumitr T, Paul PS, Kluge JP, Halbur PG. In situ hybridization technique for the detection of swine enteric and respiratory coronavirus, transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), in formalin-fixed paraffin-embeddedtissues. Virol Methods. 1996,56(2): 149-160.
82 Sirinarumitr T, Siddell S, Grahare F. Porcine transmissible gastroenteritis virus induced apoptosis in swine testes cell cultures. Archives of Virology. 1998,143(12): 2471-2485.
83 Sirinarumitr T. Paul PS.Halbur PG.Kluge JP. An overview of immunological and genetic methods for detecting swine coronavirus.transmissible gastroenteritis virus, and porcine respiratory coronavirus in tissues.Adv Exp Med Biol. 1997,412: 37-46.
84 Sirinarumitr T. Paul PS.Halbur PG.Kluge JP. Rapid in situ hybridization technique for the detection of ribonucleic acids in tissues using radiolabelled and fluorescein-labelled riboprobes. Mol Cell Probes. 1997,11(4): 273-280.
85 Torres JM.Alonso C.Mittal S.et al. Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against TGEV. Journal of Virology. 1996, 70:3770-3780
86 Torres JM.Sanchz C.Mittal S. et al. Induction of antibodies protecting against of TGEV by recombinant adenovirus expressing TGEV spike protein Virology. 1995,213:503-516
87 Tuboly T. Nagy E. Dennis JR, Derbyshine JB. Immunogenicity of the S protein of transmissible gastroenteritis coronavirus expressed in baclovirus. Arch Virol. 1994,137: 55-67.
88 Vaughn EM.Halbur PG.Paul PS. Use of nonradioactive cDNA probes to differentiate porcine respiratory coronavirus and transmissible gastroenteritis virus isolates. Vet Diagn Invest. 1996,8(2): 241-244.
89 Weslay R.Woods R.Chuny A.Nucleotide basis of the pathogenesis of transmissible gastroenteritis coronavirus. Journal of Virol. 1990,64(10): 4761-4766.
90 Wesley R.Woods R.Cheuny A. Genetic basis of the pathogenesis of transmissible gastroenteritis virus. Journal of Virol. 1990,64(10): 4761-4766.
91 Wesley R.Woods R.Cheuny A.Nucleotide sequence of the E2-peplomer protein gene and partial nucleotide sequence of upstream polymerase of transmissible gastroenteritis virus (Miller strain).Adv Exp Med Biol. 1990,276:301-306.
92 Woods RD.Studies of enteric coronavirus in a feline cell line. Vet Microbiol.1982,(7): 427-435.