禽传染性支气管炎病毒S1基因RT-PCR、RT-套式PCR扩增及其序列分析
摘要
本研究采用RT-PCR（reverse translation-polymerase chain reaction）和
RT-套式PCR（reverse translation-nest polymerase chain reaction）对7株禽传
染性支气管炎病毒（IBV：M_(41)、Ma_5、SAIB_4、SAIB_(14)、SAIB_(WJ)、SAIB_6、
SAIB_9）进行了S1基因的扩增，首次对四川地区分离的3株IBV的S1
基因进行了序列分析。本研究利用SDS-蛋白酶K法提取7株IBV的基
因组RNA。根据国际基因库Genbank注册的IBV呼吸型标准毒株（M_(41)
株）的S糖蛋白基因全序列设计了内、外两对引物。用外引物对7株IBV
的S1基因进行了RT-PCR扩增，其中M_(41)、Ma_5、SAIB_4、SAIB_(14)、SAIB
_(WJ)5株获得了长约1.72kb特异条带，SAIB_6、SAIB_9 2株未扩增出特异
条带。7株IBV的RT-PCR扩增产物经稀释后用内引物进行RT-套式PCR
扩增，7株都得到长约1.62kb的特异扩增条带，结果表明RT-套式PCR
扩增较RT-PCR具有更高的敏感性。将SAIB_4、SAIB_(14)、SAIB_(WJ)三株IBV
S1基因的RT-PCR特异扩增条带插入到pUC18质粒多克隆位点，转化
DH_(5α)载体菌，筛选获得阳性克隆菌株。对克隆质粒中外源片段序列测定
表明，所测序列内均含有长为1611bp的IBV S1结构基因，编码537个
氨基酸，N-端有一编码18个氨基酸的信号肽序列。本研究首次将3株四
川分离株SAIB_4、SAIB_(14)、SAIB_(WJ)的S1基因在国际基因库Genbank注
册（注册号：SAIB_4为bankit404421、SAIB_(14)为bankit404417、SAIB_(WJ)
为bankit404422）。与国际基因库Genbank注册的IBV M_(41)标准株S1基
因序列比较，SAIB_(14)、SAIB_4、SAIB_(WJ)的S1基因与其核苷酸序列同源
率分别为95.10％、79.24％、80.45％，相应的氨基酸序列同源率分别为
91.44％、75.61％、76.16％。本研究丰富了IBV分子流行病学资料，为进
一步研制IB DNA疫苗提供了基础材料。
RT-PCR and RT-NPCR was employed to amplify 7 strain IBV (M41,
Ma5, SAIB4, SAIB ~ SAIB w~, SAIB6, SAIB 9) , including two standard
stains(M4 1, Ma5) and 5 strain isolates (SAIB4, SAIB 14, SAIB wj, SAIB6,
SAIB9) . And for the first time, the sequence analysis of three IBV isolates
from Sichuan Province in China had been done. Two pairs of primer were
designed referring to the S glycoprotein (including Si and S2 glycoprotein)
gene sequence of IBV M4, strain published in Genbank (Accession number:
M21883). A specific fragment about 1.72kb was produced by RT-PCR from
five stains (M41, Ma5, SAIB4, SAIB,4 and SAIBwj) respectively, while no
band from the rest two strains (SAIB6 and SAIB9). Then a band about 1.62kb,
was amplified by RT-NPCR with the inside primer from all of the 7 strains. It
is shown that RT-NPCR is more sensitive than RT-PCR. The RI-PeR
product of three isolates (SAIB4, SAIB ~ and SAIBw1) was cloned and
sequenced. The result shows that, the coding region is 1611 bp in size, coding
a 1 8-amino signal-peptide and a 519-amino mat-peptide. All of the three gene
sequences have been accessed by Genbank (Accession number: SAIB4 is
bankit 404421, 5MB ,~ bankit 404417, and SAIB wj bankit 404422).
Comparison of the Si gene sequences of the three IBV isolates with IBV M~
strain, the nucleotide homology of SAIB ,4,SAIB4 and SAIBw~ is respectively
95.10%, 79.24% and 80.45%, and the correspondingly amino-acid homology
equals to 91.44%, 75.6i%and 76.16%.
RT-套式PCR（reverse translation-nest polymerase chain reaction）对7株禽传
染性支气管炎病毒（IBV：M_(41)、Ma_5、SAIB_4、SAIB_(14)、SAIB_(WJ)、SAIB_6、
SAIB_9）进行了S1基因的扩增，首次对四川地区分离的3株IBV的S1
基因进行了序列分析。本研究利用SDS-蛋白酶K法提取7株IBV的基
因组RNA。根据国际基因库Genbank注册的IBV呼吸型标准毒株（M_(41)
株）的S糖蛋白基因全序列设计了内、外两对引物。用外引物对7株IBV
的S1基因进行了RT-PCR扩增，其中M_(41)、Ma_5、SAIB_4、SAIB_(14)、SAIB
_(WJ)5株获得了长约1.72kb特异条带，SAIB_6、SAIB_9 2株未扩增出特异
条带。7株IBV的RT-PCR扩增产物经稀释后用内引物进行RT-套式PCR
扩增，7株都得到长约1.62kb的特异扩增条带，结果表明RT-套式PCR
扩增较RT-PCR具有更高的敏感性。将SAIB_4、SAIB_(14)、SAIB_(WJ)三株IBV
S1基因的RT-PCR特异扩增条带插入到pUC18质粒多克隆位点，转化
DH_(5α)载体菌，筛选获得阳性克隆菌株。对克隆质粒中外源片段序列测定
表明，所测序列内均含有长为1611bp的IBV S1结构基因，编码537个
氨基酸，N-端有一编码18个氨基酸的信号肽序列。本研究首次将3株四
川分离株SAIB_4、SAIB_(14)、SAIB_(WJ)的S1基因在国际基因库Genbank注
册（注册号：SAIB_4为bankit404421、SAIB_(14)为bankit404417、SAIB_(WJ)
为bankit404422）。与国际基因库Genbank注册的IBV M_(41)标准株S1基
因序列比较，SAIB_(14)、SAIB_4、SAIB_(WJ)的S1基因与其核苷酸序列同源
率分别为95.10％、79.24％、80.45％，相应的氨基酸序列同源率分别为
91.44％、75.61％、76.16％。本研究丰富了IBV分子流行病学资料，为进
一步研制IB DNA疫苗提供了基础材料。
RT-PCR and RT-NPCR was employed to amplify 7 strain IBV (M41,
Ma5, SAIB4, SAIB ~ SAIB w~, SAIB6, SAIB 9) , including two standard
stains(M4 1, Ma5) and 5 strain isolates (SAIB4, SAIB 14, SAIB wj, SAIB6,
SAIB9) . And for the first time, the sequence analysis of three IBV isolates
from Sichuan Province in China had been done. Two pairs of primer were
designed referring to the S glycoprotein (including Si and S2 glycoprotein)
gene sequence of IBV M4, strain published in Genbank (Accession number:
M21883). A specific fragment about 1.72kb was produced by RT-PCR from
five stains (M41, Ma5, SAIB4, SAIB,4 and SAIBwj) respectively, while no
band from the rest two strains (SAIB6 and SAIB9). Then a band about 1.62kb,
was amplified by RT-NPCR with the inside primer from all of the 7 strains. It
is shown that RT-NPCR is more sensitive than RT-PCR. The RI-PeR
product of three isolates (SAIB4, SAIB ~ and SAIBw1) was cloned and
sequenced. The result shows that, the coding region is 1611 bp in size, coding
a 1 8-amino signal-peptide and a 519-amino mat-peptide. All of the three gene
sequences have been accessed by Genbank (Accession number: SAIB4 is
bankit 404421, 5MB ,~ bankit 404417, and SAIB wj bankit 404422).
Comparison of the Si gene sequences of the three IBV isolates with IBV M~
strain, the nucleotide homology of SAIB ,4,SAIB4 and SAIBw~ is respectively
95.10%, 79.24% and 80.45%, and the correspondingly amino-acid homology
equals to 91.44%, 75.6i%and 76.16%.
引文
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2、B．W．卡尔尼克主编，禽病学，第九版，北京：北京农业大学出版社，1991，407-418。
3、Kusters..J.G., et al., Niesters, H. G. M., et al. 1989. Phylogeny of antigenic variants of avian coronavirus IBV. Virology. 169:217-221.
4、Boursnell M. E. C., Brow T. D. K., Foulds I. J., et al. 1987. Completion of the sequence of the genome of the Coronavirus infectious bronchitis virus. J. Gen. Virol. 68:57～77.
5、LB ENDO-MUNOZ and JT FARAGHER. 1989. Avian infectious bronchitis: crossprotection studies using different Australian subtypes. 66:345～348.
6、King, D.J., Cavangh, D. Infectious bronchitis. Disease of Poulty, 1991,471-484.
7、郭文和，鸡传染性支气管炎病毒的免疫研究进展，中国兽医杂志，1992，10：38～39。
8、王红宁，鸡肾病变型传染性支气管炎诊断治，中国兽医杂志，1994，20(3)：18～19。
9、Mcca E，传染性支气管炎现状：有效的防治措施，国外兽医学——畜禽传染病，1987，9(4)：39～40。
10、Davelaar. F. G., et al., Kouwenhoven B, et al. 1984. Occurrence and significance of infectious bronchitis vires variant strain in egg and broiler production in the Netherlands. Vet. Q, 1984, 6: 114-120.
11、Finney P. M, et al., Studies with a bivalent infectious bronchitis killed virus vaccine. Avian Pathology. 1990, 19: 435-450.
12、Avellaneda G. E, Villeges P, Jackwood M. W, et al. 1994. In vivo evaluation of the pathogenecity of isolates of infectious bronchitis virus. Avian Dis. 38: 589～597.
13、Albassam M. A, Winterfield R.W, Thacker H. L. 1986. Comparision of the nephropathogenecity of four strains of infectious bronchitis virus. Avian Dis. 30: 468～476.
14、Schalk A. K. and Hawn M. C. 1931. An apparently new respiratory disease of baby chicks. J .Am. Vet. Med. Assoc. 78:413～422.
15、Beach, J. R., and O. W. Schalm. 1936. A filterable vires, distinct from that of laryngotrachitis, the cause of a respiratory disease of chicks. Poult Sci 15:199～206.
16、Beaudette, F. R., and C. B. Hudson. 1937,. Cultivation of the vires of infectious bronchitis. J Am Vet Med Assoc 90:51～60.
17、Jungherr, E. L., T. W. Chomiak, and R. E. Luginbuhl. 1956. Immunologic differences in strains of infectious bronchitis. Proc 60th Annu Meet US Livestock Sanit Assoc, pp. 203～209.
18、Ambal A．G．鸡传染性支气管炎病毒嗜肠型毒株的近期研究，国外兽医学——畜禽传染病，1993，3：30～32。
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