屠宰猪猪肺炎支原体P36、P46、P97 R1、P146氨基酸序列的差异分析
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摘要
为了解猪肺炎支原体流行菌株遗传变异情况,选取2014-2017年不同时间、地点收集的17头猪肺炎支原体阳性屠宰猪肺脏灌洗液DNA为模板,进行猪肺炎支原体P36(编码具有强免疫原性的胞浆蛋白)、P46(编码具有强免疫原性的表面蛋白)、P97 R1区、P146高变区(猪肺炎支原体黏附气管纤毛细胞的重要功能区域)基因的PCR扩增、测序。结合GenBank登录的猪肺炎支原体相关序列进行氨基酸序列的比对分析。结果表明,比对的序列之间,P36、P46蛋白氨基酸序列同源性均在98.9%以上,但P97功能区R1区、P146多变区在菌株间存在较大差异。11个屠宰猪样本和5个GenBank登录菌株P97 R1区重复氨基酸序列数量达到14个以上,而GenBank登录的3个疫苗相关菌株(168株、168-L株、J株)及另外4个菌株、4个屠宰猪样本重复序列为9～11个不等;P146高变区氨基酸序列的差异主要集中在2个重复氨基酸序列区(R1、R2),GenBank登录的168株、168-L株、J株以及另外2个菌株,1个屠宰猪样本在R1区出现较多氨基酸缺失。168株、168-L株,6个屠宰猪样本及另外3个GenBank登录菌株在R2区出现了3～9个不等的氨基酸缺失。利用DNAStar进行抗原表位预测发现,菌株间P97 R1、P146 R1、P146 R2区氨基酸序列的差异,导致了抗原表位出现明显的变化。提示开展P97、P146等黏附因子的筛选和研究或许能进一步提升疫苗阻止猪肺炎支原体黏附气管纤毛细胞的作用,从而进一步改善疫苗的免疫效果。
To understand the genetic variation of Mycoplasma hyopneumoniae(M.hyo) epidemic strain, a conventional PCR was employed to amplify the fragments of M. hyo P36, P46, P97 R1 and P146 gene from the DNA mix of bronchoalveolar lavage fluid of 17 slaugher pigs, which had been tested positive to M.hyo, and the PCR products were sequenced directly and analyzed. The deduced amino acid sequences of P36 and P46 were conservative between M.hyo of 17 slagughter pig lung samples and those strains registered in GenBank, with a homology of more than 98.9%. Among the amino acid sequences, there were large variations in P97 R1 area and 2 repetitive sequence regions(R1, R2) of P146. P97 amino acid sequences R1 area of M.hyo from 11 slaughter pig samples and 5 strains registered in GenBank were composed of 14 or more tandem copies. It was composed of 9-11 tandem copies in three vaccine-related strains(168 strain, 168-L strain, J strain), four other strains registered in GenBank, and 4 strains from slaughter pigs. A few amino acid deficiency was observed in R1 area of P146 from 5 strains registered in GenBank(168 strain, 168-L strain, J strain, and two other strains) and 1 slaughter pig samples, and 3-9 amino acid deficiency was observed in R2 area of P146 from 11 strains(168-L strain, 168 strain, three other strains registered in GenBank, and 6 strains detected from slaughter pig). Consequently, antigen epitope prediction showed significant difference in P97 R1 area and two repetitive sequence regions(R1, R2) of P146. To the best of our knowledge, this study was the first preliminary investigation to M.hyo strain in slaughter pigs in Hunan province, and it could provide some significant data for understanding the prevalence of M.hyo strain in different areas and screening of vaccine candidate strains.
To understand the genetic variation of Mycoplasma hyopneumoniae(M.hyo) epidemic strain, a conventional PCR was employed to amplify the fragments of M. hyo P36, P46, P97 R1 and P146 gene from the DNA mix of bronchoalveolar lavage fluid of 17 slaugher pigs, which had been tested positive to M.hyo, and the PCR products were sequenced directly and analyzed. The deduced amino acid sequences of P36 and P46 were conservative between M.hyo of 17 slagughter pig lung samples and those strains registered in GenBank, with a homology of more than 98.9%. Among the amino acid sequences, there were large variations in P97 R1 area and 2 repetitive sequence regions(R1, R2) of P146. P97 amino acid sequences R1 area of M.hyo from 11 slaughter pig samples and 5 strains registered in GenBank were composed of 14 or more tandem copies. It was composed of 9-11 tandem copies in three vaccine-related strains(168 strain, 168-L strain, J strain), four other strains registered in GenBank, and 4 strains from slaughter pigs. A few amino acid deficiency was observed in R1 area of P146 from 5 strains registered in GenBank(168 strain, 168-L strain, J strain, and two other strains) and 1 slaughter pig samples, and 3-9 amino acid deficiency was observed in R2 area of P146 from 11 strains(168-L strain, 168 strain, three other strains registered in GenBank, and 6 strains detected from slaughter pig). Consequently, antigen epitope prediction showed significant difference in P97 R1 area and two repetitive sequence regions(R1, R2) of P146. To the best of our knowledge, this study was the first preliminary investigation to M.hyo strain in slaughter pigs in Hunan province, and it could provide some significant data for understanding the prevalence of M.hyo strain in different areas and screening of vaccine candidate strains.
引文
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[2] Simionatto S,Marchioro S B,Maes D,Dellagostin O A.Mycoplasma hyopneumoniae:From disease to vaccine development[J].Veterinary Microbiology,2013,165(3/4):234-242.doi:10.1016/j.vetmic.2013.04.019.
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[4] Villarreal I,Meyns T,Dewulf J,Vranckx K,Calus D,Pasmans F,Haesebrouck F,Maes D.The effect of vaccination on the transmission of Mycoplasma hyopneumoniae in pigs under field conditions[J].Veterinary Journal,2011,188(1):48-52.doi:10.1016/j.tvjl.2010.04.024.
[5] 沈青春,吴思杰,范学政,张广川,高和义,宁宜宝.猪支原体肺炎活疫苗(RM48株)的最小免疫剂量测定及活菌滴度与免疫保护率平行关系试验[J].中国兽药杂志,2014,48(12):1-4.Shen Q C,Wu S J,Fan X Z,Zhang G C,Gao H Y,Ning Y B.The minimum immune dose measure of Mycoplasma hyopneumoniae live vaccine(RM48 strain)and the parallel assay of vaccine CCU titer and potency[J].Chinese Journal of Veterinary Drug,2014,48(12):1-4.
[6] 田瑞雨,熊祺琰,刘茂军,韦艳娜,武昱孜,冯志新,姜平,邵国青.猪肺炎支原体抗原定量ELISA检测方法的建立[J].中国兽医科学,2017,47(4):471-475.doi:10.16656/j.issn.1673-4696.2017.04.011.Tian R Y,Xiong Q Y,Liu M J,Wei Y N,Wu Y Z,Feng Z X,Jiang P,Shao G Q.Development of a quantitative enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae[J].Chinese Veterinary Science,2017,47(4):471-475.
[7] 沈青春,王芳,韩明远,覃青松,范学政,杨汉春,宁宜宝.猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立[J].畜牧兽医学报,2012,43(3):431-437.Shen Q C,Wang F,Han M Y,Qin Q S,Fan X Z,Yang H C,Ning Y B.Expression of P46 protein of Mycoplasma hyopneumoniae in Escherichia coli and used for indirect ELISA[J].Acta Veterinaria et Zootechnica Sinica,2012,43(3):431-437.
[8] Okamba F R,Arella M,Music N,Jia J J,Gottschalk M,Gagnon C A.Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine[J].Vaccine,2010,28(30):4802-4809.doi:10.1016/j.vaccine.2010.04.089.
[9] Okamba F R,Moreau E,Bouh K,Gagnon C A,Massie B,Arella M.Immune responses induced by replication-defective adenovirus expressing the C-terminal portion of the Mycolasma hyopneumoniae p97 adhesin[J].Clinical and Vaccine Immunology,2007,14(6):767-774.doi:10.1128/CVI.00415-06.
[10] 杜改梅,刘茂军,韦艳娜,甘源,马庆红,邵国青.猪肺炎支原体不同蛋白抗原检测疫苗诱导IgG产生规律的研究[J].畜牧兽医学报,2013,44(6):925-930.doi:10.11843/j.issn.0366-6964.2013.06.014.Du G M,Liu M J,Wei Y N,Gan Y,Ma Q H,Shao G Q.Detection of IgG antibodies regularity induced by vaccine using different Mycoplasma hyopneumoniae antigen proteins[J].Acta Veterinaria et Zootechnica Sinica,2013,44(6):925-930.
[11] Bogema D R,Deutscher A T,Woolley L K,Raymond B B,Tacchi J L,Dixon N E,Minion F,Walker M J,Djordjevic S P.Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography[J].mBio,2012,3(2):1-10.doi:10.1128/mBio.00282-11.
[12] Wilton J L,Scarman A L,Walker M J,Djordjevic S P.Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae[J].Microbiology,1998,144(7):1931-1943.doi:10.1099/00221287-144-7-1931.
[13] Minion F C,Adams C,Hsu T.R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia[J].Infection and Immunity,2000,68(5):3056-3060.doi:10.1128/IAI.68.5.3056-3060.2000.
[14] Caron J,Sawyer N,Moumen B B,Bouh K C,Dea S.Species-specific monoclonal antibodies to Escherichia coli-expressed p36 cytosolic protein of Mycoplasma hyopneumoniae[J].Clinical and Diagnostic Laboratory Immunology,2000,7(4):528-535.doi:10.1128/CDLI.7.4.528-535.2000.
[15] Bouh K S,Shareck F,Dea S.Monoclonal antibodies to Escherichia coli-expressed P46 and P65 membranous proteins for specific immunodetection of Mycoplasma hyopneumoniae in lungs of infected pigs[J].Clinical and Diagnostic Laboratory Immunology,2003,10(3):459-468.doi:10.1128/CDLI.10.3.459-468.2003.
[16] 彭丽萍,刘思远,朱晓萍,陈珍,姚昌茹,赵宝华.猪肺炎支原体168弱毒株P46-P65融合蛋白的免疫原性[J].中国兽医学报,2017,37(4):1005-4545.doi:10.16303/j.cnki.1005-4545.2017.04.08.Peng L P,Liu S Y,Zhu X P,Chen Z,Yao C R,Zhao B H.Immunogenicity of P46-P65 fusion protein of avirulent Mycoplasma hyopneumoniae strain 168 in mice[J].Chinese Journal of Veterinary Science,2017,37(4):1005-4545.
[17] Feng Z X,Bai Y,Yao J T,Pharr G,Xiao S B,Chi L Z,Gan Y A,Wang H Y,Wei Y N,Liu M J,Xiong Q Y,Bai F F,Li B,Wu X S,Shao G Q.Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1,P46 and P36 in the diagnosis of infection[J].Veterinary Journal,2014,202(1):128-133.doi:10.1016/j.tvjl.2014.06.019.
[18] Mebus C A,Underdahl N R.Scanning electron microscopy of trachea and bronchi from gnotobiotic pigs inoculated with Mycoplasma hyopneumoniae[J].American Journal of Veterinary Research,1977,38(8):1249-1254.doi:10.2754/avb197847030203.
[19] Ferreira H B,De Castro L A.A preliminary survey of M.hyopneumoniae virulence factors based on comparative genomic analysis[J].Genetics and Molecular Biology,2007,30(1,S):245-255.doi:10.1590/S1415-47572007000200012.
[20] 王凯,杨青春,陈绍红,刘艳芬,刘铀.猪肺炎支原体P97基因重组腺病毒的构建及其免疫原性[J].中国兽医科学,2015,45(11):1114-1121.Wang K,Yang Q C,Chen S H,Liu Y F,Liu Y.Construction and immunogenicity of the recombinant adenovirus containing P97 gene of Mycoplasma hyopeumoniae[J].Chinese Veterinary Science,2015,45(11):1114-1121.
[21] Zhang Q J,Young T F,Ross R F.Identification and Characterization of a Mycoplasma hyopneumoniae Adhesin[J].Infection and Immunity,1995,63(3):1013-1019.doi:10.1016/0162-3109(94)00058-N.
[22] Zimmerman J J,Karriker L A,Ramirez A,Schwartz A,Stevenson G W.猪病学[M].10版.北京:中国农业大学出版社,2014:826.Zimmerman J J,Karriker L A,Ramirez A,Schwartz A,Stevenson G W.Diseases of swine[M].10th ed.Beijing:China Agricultural University Press,2014:826.
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