猪繁殖与呼吸综合征新疆株ORF2-7基因的克隆及序列分析
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摘要
【目的】研究新疆PRRS流行毒株的ORF2–7基因的变异及进化情况,为揭示PRRSV在新疆的流行特点提供参考,为新疆PRRS的防控提供理论依据。【方法】将新疆某规模化和散养户猪场疑似猪繁殖与呼吸综合征(PRRS)的猪肺脏病料进行处理,提取病毒RNA,应用PT-PCR技术扩增出4株猪繁殖与呼吸综合征病毒(PRRSV)ORF2–7基因序列,并进行序列测定。【结果】PRRSV ORF2–7基因核苷酸长度为3 206bp。序列分析表明,新疆分离株XJTK-02株、XJERG-03株、XJ-04株和XJFCH-05株之间核苷酸的同源率较高,为90.3%~98.6%;XJTK-02株与经典PRRSV CH-1a株核苷酸同源率最高为93%;而其它3株新疆分离株与高致病性PRRSV JXA1、HuN4、JXwn06、pJX143和HPBEDV苷酸同源率较高,为95.1%~98.9%;4株新疆分离株ORF2–7与美洲型代表株VR-2332核苷酸同源性为88.9%~90.5%;而与欧洲型代表株LV4.2.1的核苷酸同源性仅为54.2%~54.6%。【结论】新疆分离株为PRRSV美洲型。
【Objective】Research To research genetic evolution of Xinjiang PRRS strains ORF2-7 amino acid and provide a theoretical basis for the effective perventionprevention and control of the virus. 【Method】Four strains were treated from pig lung of clinical tissue samples originated from the suspected cases were treated with porcine reproductive and respiratory syndrome virus( PRRSV) in Xinjiang Province,RNA was extracted,and genes of PRRSV ORF2-7 were amplificated by RT-PCR and sequenced. 【Result】Nucleotide of PRRSV ORF2-7 was 3206 bp. Sequence analysis of PRRSV ORF2-7 indicated that XJTK-02 strain,XJERG-03 strain,XJ-04 strain and XJFCH-05 strain shared 90. 3% ~ 98. 6% nucleotide; XJTK-02 strain shared 93% nucleotide identities to with CH-1a; XJERG-03 strain,XJ-04 strain and XJFCH-05 strain shared 95. 1% ~ 98. 9% nucleotide identities to with JXA1,HuN4,JXwn06,pJX143 and HPBEDV;Tthe four strains shared 88. 9% ~ 90. 5% nucleotide identities to with North American genotype VR-2332,54. 2% ~ 54. 6% nucleotide identities with the Dutch Lelystad strain LV4. 2. 1. The results showed that the four strains PRRSV of Xinjiang belongs to North American genotype。.
【Objective】Research To research genetic evolution of Xinjiang PRRS strains ORF2-7 amino acid and provide a theoretical basis for the effective perventionprevention and control of the virus. 【Method】Four strains were treated from pig lung of clinical tissue samples originated from the suspected cases were treated with porcine reproductive and respiratory syndrome virus( PRRSV) in Xinjiang Province,RNA was extracted,and genes of PRRSV ORF2-7 were amplificated by RT-PCR and sequenced. 【Result】Nucleotide of PRRSV ORF2-7 was 3206 bp. Sequence analysis of PRRSV ORF2-7 indicated that XJTK-02 strain,XJERG-03 strain,XJ-04 strain and XJFCH-05 strain shared 90. 3% ~ 98. 6% nucleotide; XJTK-02 strain shared 93% nucleotide identities to with CH-1a; XJERG-03 strain,XJ-04 strain and XJFCH-05 strain shared 95. 1% ~ 98. 9% nucleotide identities to with JXA1,HuN4,JXwn06,pJX143 and HPBEDV;Tthe four strains shared 88. 9% ~ 90. 5% nucleotide identities to with North American genotype VR-2332,54. 2% ~ 54. 6% nucleotide identities with the Dutch Lelystad strain LV4. 2. 1. The results showed that the four strains PRRSV of Xinjiang belongs to North American genotype。.
引文
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[9]Mardassi,H.,Massie,B.,&Dea,S.(1996).Intracellular synthesis,processing,and transport of proteins encoded by ORFs5 to7 of porcine reproductive and respiratory syndrome virus[J].Virology,221(1):98-112.
[10]Dea,S.,Gagnon,C.A.,Mardassi,H.,&Milane,G.(1996).Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein[J].Journal of clinical microbiology,34(6):1,488-1,493.
[11]Rodriguez,M.J.,Sarraseca,J.,Garcia,J.,Sanz,A.,Plana-Dur,J.,&Casal,J.I.(1997).Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus[J].Journal of general virology,78(9):2,269-2,278.
[2]蔡宝祥.我国高致病性猪繁殖与呼吸综合征研究进展[J].畜牧与兽医,2010,42(10):1-4.CAI Bao-xiang.(2010).Advance on highly pathogenic porcine reproductive and respiratory syndrome in China[J].Animal Husbandry&Veterinary Medicine,42(10):1-4.(in Chinese)
[3]杨汉春.我国新发猪病的流行现状与防控的科学问题[J].兽医导刊,2012,(2):26-27.YANG Han-chun.(2012).Scientific problems of epidemic situation and prevention and control of new swine diseases in China[J].Veiterinary Orientation,(2):26-27.(in Chinese)
[4]Hao-tai,C.H.E.N.,Jie,Z.H.A.N.G.,Li-na,M.A.,&Yong-sheng,L.I.U.(2008).Characterization of Porcine Reproductive and Respiratory Syndrome Virus Deletion Mutant[J].Agricultural Sciences in China,7(11):1,379-1,386.
[5]童光志,周艳君,都晓芳,等.高致病性猪繁殖与呼吸综合征病毒的分离鉴定及其分子流行病学分析[J].中国预防兽医学报,2007,29(5):323-327.TONG Guang-zhi,ZHOU Yan-jun,DOU Xiao-fang,et al.(2007).ldentifieation Identification and moleeularmolecular epidemiology of the very virulent porcine reproductive and respiratory syndrome viruses emerged in China[J].Chinese Journal of Preventive Veterinary Medicine,29(5):323-327.(in Chinese)
[6]周伦江,王隆柏,魏宏,等.猪繁殖与呼吸综合征病毒FJ04A株的分离及其结构蛋白基因特异分析[J].福建农林大学学报(自然科学版),2006,35(5):527-531.ZHOU Lun-jiang,WANG Long-bai,WEI Hong,et al.(2006).Isolation and characteristics analyses of structure protein genes of porcine reproductive and respiratory syndrome virus strain FJ04A[J].Journal of Fujian Agricultural and Forestry University(Natural Science EditionEd.),35(5):527-531.(in Chinese)
[7]Saito,A.,Kanno,T.,Murakami,Y.,Muramatsu,M.,&Yamaguchi,S.(1996).Characteristics of major structural protein coding gene and leader-body sequence in subgenomic mRNA of porcine reproductive and respiratory syndrome virus isolated in Japan[J].The Journal of veterinary medical science/the Japanese Society of Veterinary Science,58(4):377-380.
[8]Urniza,A.,Climent,I.,Duran,J.P.,Corte,E.,Sarraseca,J.,Casal,J.I.,&Vela,C.(1997).Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91.Involvement of ORF3 and ORF5 proteins in protection[J].Virus genes,14(1):19-29.
[9]Mardassi,H.,Massie,B.,&Dea,S.(1996).Intracellular synthesis,processing,and transport of proteins encoded by ORFs5 to7 of porcine reproductive and respiratory syndrome virus[J].Virology,221(1):98-112.
[10]Dea,S.,Gagnon,C.A.,Mardassi,H.,&Milane,G.(1996).Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to the matrix protein[J].Journal of clinical microbiology,34(6):1,488-1,493.
[11]Rodriguez,M.J.,Sarraseca,J.,Garcia,J.,Sanz,A.,Plana-Dur,J.,&Casal,J.I.(1997).Epitope mapping of the nucleocapsid protein of European and North American isolates of porcine reproductive and respiratory syndrome virus[J].Journal of general virology,78(9):2,269-2,278.