CN103243112A - 预防大肠杆菌病的基因tsh-Ser及其制备以及利用其编码的蛋白 - Google Patents

预防大肠杆菌病的基因tsh-Ser及其制备以及利用其编码的蛋白 Download PDF

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CN103243112A
CN103243112A CN2013101811324A CN201310181132A CN103243112A CN 103243112 A CN103243112 A CN 103243112A CN 2013101811324 A CN2013101811324 A CN 2013101811324A CN 201310181132 A CN201310181132 A CN 201310181132A CN 103243112 A CN103243112 A CN 103243112A
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张鑫
马兴树
牛志勇
陈淑芳
李德斌
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HEBEI KEXING DRUG INDUSTRY Co Ltd
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Abstract

本发明属于新基因的制备，特别是指一种预防大肠杆菌病的基因tsh-Ser及其制备以及利用其编码的蛋白。包括以5’-atgaacagaatttatt-3’为上游引物zx1，以5’-gaatgaataacgaa-3’为下游引物zx2，以O78血清型大肠杆菌的质粒DNA为模板，采用PCR扩增方法获得的预防大肠杆菌病的基因tsh-Ser以及利用其编码的蛋白。本发明解决了现有技术存在的菌苗广谱性差等技术问题。具有广谱性好，免疫效果好、环境友好、工业化开发前景广阔等优点。

Description

预防大肠杆菌病的基因tsh-Ser及其制备以及利用其编码的蛋白
技术领域
本发明属于新基因的制备，特别是指一种预防大肠杆菌病的基因tsh-Ser及其制备以及利用其编码的蛋白。 
背景技术
大肠杆菌病是由大肠杆菌的某些致病性血清型菌株引起的疾病总称。禽大肠杆菌病是指部分或全部由禽致病性大肠杆菌(APEC)所引起的局部或全身性感染的疾病，包括大肠杆菌性败血症，大肠杆菌性肉芽肿(Hjarre病)、气囊病(慢性呼吸道疾病即CRD)、大肠杆菌性蜂窝织炎(即炎性过程)、肿头综合症、大肠杆菌性腹膜炎、大肠杆菌性输卵管炎、大肠杆菌性骨髓炎/滑膜炎(即火鸡骨髓炎综合征)、大肠杆菌性全眼球炎及大肠杆菌性脐炎/卵黄囊感染。大肠杆菌在哺乳动物主要引起肠道疾病，而当禽类受到高致病性大肠杆菌感染而使其防御能力不足以抵抗或完全丧失时，常会引起典型的继发性局部或全身性感染。大肠杆菌病给养禽业造成了严重的损失，是禽病调查中最常报道的疾病及酮体废弃的主要原因。有资料显示：禽肉加工中43％肉鸡酮体的报废与大肠杆菌性败血症有关。 
现有技术主要是通过利用抗生素来进行致病性大肠杆菌的治疗，但是由于抗生素的滥用，随着细菌耐药性的增强，大肠杆菌病也越来越难以防治。从食品安全角度考虑，导致直接食用这些禽畜类产品的人群感染大肠杆菌病的几率增加。抗生素的用量越来越大，致使农产品药残量增大，导致人间接性的食用大量的养殖用药，这都给人类健康造成隐患。另外传统疫苗都是通过改变培养条件，或在不同寄主动物上传代的办法使致病微生物的毒性减弱；或是通过物理化学的方法将它们灭活。但这样的疫苗都不理想。因为弱化的菌株可能发生回复突变而变成有毒的，或者失去免疫原性，灭活不适当的疫苗还会引起疾病的流行。因此，迫切需要发展一种性能更佳的新型预防大肠杆菌病的广谱菌苗。 
有研究表明，禽大肠杆菌的外膜蛋白在致病性和免疫方面都起着重要的作用。赵香汝和杨汉春的研究(赵香汝，杨汉春.不同血清型禽大肠杆菌外膜蛋白免疫原性研究[J].张家口农专学报.1999，第15卷第4期：4-6.)表明不 同血清型禽大肠杆菌混合的外膜蛋白免疫，无论对同型或异型菌株的攻击，都有一定的保护作用。李晓霞和邱玉玉的研究(李晓霞，邱玉玉，王海蓉.肠致病性大肠杆菌外膜蛋白免疫保护性研究[J].中国免疫学杂志.2007，第23卷第4期：394-397.)表明肠致病性大肠杆菌外膜蛋白可以诱导兔对大肠杆菌的特异性体液免疫和细胞免疫应答。 
禽致病性大肠杆菌中的tsh基因在应用中主要是其翻译表达的蛋白将大肠杆菌粘附于正常的体细胞表面，在文献(陈祥，刘静，高嵩，潘志明，焦新安，刘秀梵.禽病原性大肠杆菌iro和tsh基因缺失的构建[J].生物工程学报.2008，24(3)：401-408.)报道中出现过将tsh基因敲除后，大肠杆菌病的发病率将会降低的说明，但未见用单一细分的tsh基因及其翻译的蛋白对于对大肠杆菌病预防的报道。 
发明内容
本发明的目的在于提供一种预防大肠杆菌病的基因tsh-Ser。 
本发明的目的之二在于提供一种预防大肠杆菌病的基因tsh-Ser的制备方法。 
本发明的目的之三在于提供一种预防大肠杆菌病的基因tsh-Ser编码的蛋白。 
本发明的整体技术构思是： 
预防大肠杆菌病的基因tsh-Ser，其DNA序列为： 
atgaacagaatttattctcttcgctacagcgctgtggcccggggctttattgccatatctgagtttgctaggaaatgtgttcataagtctgtcagacgtctgtgtttcccggttttattactgatcccggtactattctctgcaggaagtcttgcgggaacggtcaataatgaactcgggtatcagttatttcgtgattttgctgaaaataaggggatgttccgcccgggggcaacgaatatcgctatttataataagcagggagaatttgtcggtacgctggataaggcagctatgcctgatttcagtgctgtggattcggaaatcggtgtggcgacactgataaacccgcagtatatcgccagcgtgaaacataacgggggatatacaaacgttagctttggtgatggtgaaaaccgttacaatatcgtggaccggaataatgcgccgtcactggattttcatgccccccggctggataaactggtgacagaggttgcccctactgcggtgacggcgcagggggcagtggctggcgcatatctggataaggagcgctatcctgttttttatcgtctggggtctggtactcagtatattaaggacagtaacggacagctgacaaaaatgggaggtgcatattcctggctgaccggcgggactgtcggtagcctgtcatcctatcagaatggagaaatgattagcaccagttcaggtctggttttt gattacaaacttaatggtgcaatgcccatttatggcgaggccggtgacagcggttcgcctttatttgcttttgatactgttcagaataaatgggtgctggtcggtgttcttactgcggggaatggcgcggggggcaggggaaataactgggctgttattccactggattttatcgggcagaaatttaatgaagacaatgatgccccggtcacgttcagaacatcggaaggtggtgcactggagtggagctttaacagcagtaccggagctggtgcgctgacacagggaaccaccacatatgccatgcacgggcagcagggaaatgacctgaatgctggtaagaacctgatatttcaggggcagaatggtcagattaaccttaaggattcggtttctcagggggcgggttccctgacgttccgtgataattacacagtaacaacctctaacggaagtacctggaccggtgccggtattgttgtggacaacggggtgtccgtaaactggcaggttaatggtgttaagggcgataacctgcataaaattggtgaaggtacgctgacggtacagggtacaggtattaatgaaggtggcctgaaggtcggggacggaaaggttgtactgaaccagcaggcggacaataaaggacaggtgcaggcgttcagcagtgttaatattgccagtggccggccgaccgtggtactgactgatgagcggcaggtaaatccggataccgtctcatggggatatcgtgggggcacactggatgttaatggtaacagtctgacgtttcatcagttgaaggcggcagattatggtgccgtgctggcgaataacgttgataaacgggccactatcacgctggactatgccctgcgggctgacaaagtagcactgaatggctggtcggaatcaggtaaaggaactgccggaaatttatataaatacaataacccgtacacaaatacgacggattacttcatcctgaagcagagcacctatggttatttccccacggaccagagcagcaacgccacctgggagtttgtggggcacagtcagggggatgcgcagaaactggtagctgaccgtttcaatactgcagggtatctgtttcacggacaactgaaaggcaatctgaatgtggacaatcgcctgcctgaaggcgttaccagtgctctggtgatggacggagctgcggatatctccggtacattcacccaggaaaacgggcgtctgacgctgcaggggcatccggttatccatgcatacaatactcagcctgtggctgacaaactggctgccagtggagaccattcggttctgactcagcctacgtcattcagtcaggaggactgggagaaccgcagttttacctttgacaggctgtcactgaagaacactgattttggtcttggtcgcaatgccacactgaacacaaccatccaggcagataactccagcgtcacgctgggcgacagccgggtatttatcgacaaaaacgatggccagggaacagcctttacccttgaagaaggcacatctgttgcaactaaagatgcagataaaagtgtcttcaacggcaccgtcaacctggataatcagtcagtgctgaatatcaatgatatattcaatggcggaatacaggcgaacaacagtaccgtgaatatctcctcagacagtgccgttctggggaactcaacactgaccagtaccgccctgaatctgaacaagggagcaaatgctctggccagtcagagttttgtttctgacggtccagtgaatatttctgatgccaccctgagtctgaacagccgtcctgatgaggtatctcacacacttttacctgtatacgattatgccggttcatggaacctgaagggagacgatgcccgcctgaacgtggggccgtacagtatgttgtcaggtaatatcaatgttcaggataaagggactgtcaccctcggaggggaaggggaa ctgagtcctgacctgactcttcagaatcagatgttgtacagcctgtttaacgggtaccgcaatatctggagcgggagcctgaatgcaccggatgccaccgtcagcatgacagacacccagtggtcgatgaacggaaactccacggcaggaaatatgaaacttaaccggacaatagtcggttttaacgggggaacatcaccgttcacgacactgacaacagataatctggacgcggttcagtcagcatttgtcatgcgtacagaccttaacaaggcagacaaactggtgataaacaagtcggcaacaggtcatgacaacagcatctgggttaacttcctgaaaaaaccttctaacaaggacacgcttgatattccactggtcagcgcacctgaagcgacagctgataatctgttcagggcatcaacacgggttgtgggattcagtgatgtcacccccatccttagtgtcagaaaagaggacgggaaaaaagagtgggtcctcgatggttaccaggttgcacgtaacgacggccagggtaaggctgccgccacattcatgcacatcagctataacaacttcatcactgaagttaacaacctgaacaaacgcatgggcgatttgagggatattaatggcgaagccggtacgtgggtgcgtctgctgaacggttccggctctgctgatggcggtttcactgaccactataccctgctgcagatgggggctgaccgtaagcacgaactgggaagtatggacctgtttaccggcgtgatggccacctacactgacacagatgcgtcagcagacctgtacagcggtaaaacaaaatcatggggtggtggtttctatgccagtggtctgttccggtccggcgcttactttgatgtgattgccaaatatattcacaatgaaaacaaatatgacctgaactttgccggagctggtaaacagaacttccgcagccattcactgtatgcaggtgcagaagtcggataccgttatcatctgacagatacgacgtttgttgaacctcaggcggaactggtctggggaagactgcagggccaaacatttaactggaacgacagtggaatggatgtctcaatgcgtcgtaacagcgttaatcctctggtaggcagaaccggcgttgtttccggtaaaaccctcagtggtaaggactggagtctgacagcccgtgccggcctgcattatgagttcgatctgacggacagtgctgacgttcatctgaaggatgcagcgggagaacatcagattaatggcagaaaagacagtcgtatgctttacggtgtggggttaaatgcccggtttggcgacaatacgcgtctggggctggaagttgaacgctctgcatttggtaaatacaacacagatgatgcgataaacgctaatattcgttattcattctga。 
预防大肠杆菌病的基因tsh-Ser的制备方法，以5’-atgaacagaatttatt-3’为上游引物zx1，以5’-gaatgaataacgaa-3’为下游引物zx2，以O78血清型大肠杆菌的质粒DNA为模板，采用PCR扩增方法获得。 
利用预防大肠杆菌病的基因tsh-Ser编码的蛋白，其氨基酸序列如下：METAsnArgIleTyrSerLeuArgTyrSerAlaValAlaArgGlyPheIleAlaIleSerGluPheAlaArgLysCysValHisLysSerValArgArgLeuCysPheProValLeuLeuLeuIleProValLeuPheSerAlaGlySerLeuAlaGlyThrValAsnAsnGluLeuGlyTyrGlnLeuPheArgAsp PheAlaGluAsnLysGlyMETPheArgProGlyAlaThrAsnIleAlaIleTyrAsnLysGlnGlyGluPheValGlyThrLeuAspLysAlaAlaMETProAspPheSerAlaValAspSerGluIleGlyValAlaThrLeuIleAsnProGlnTyrIleAlaSerValLysHisAsnGlyGlyTyrThrAsnValSerPheGlyAspGlyGluAsnArgTyrAsnIleValAspArgAsnAsnAlaProSerLeuAspPheHisAlaProArgLeuAspLysLeuValThrGluValAlaProThrAlaValThrAlaGlnGlyAlaValAlaGlyAlaTyrLeuAspLysGluArgTyrProValPheTyrArgLeuGlySerGlyThrGlnTyrIleLysAspSerAsnGlyGlnLeuThrLysMETGlyGlyAlaTyrSerTrpLeuThrGlyGlyThrValGlySerLeuSerSerTyrGlnAsnGlyGluMETIleSerThrSerSerGlyLeuValPheAspTyrLysLeuAsnGlyAlaMETProIleTyrGlyGluAlaGlyAspSerGlySerProLeuPheAlaPheAspThrValGlnAsnLysTrpValLeuValGlyValLeuThrAlaGlyAsnGlyAlaGlyGlyArgGlyAsnAsnTrpAlaValIleProLeuAspPheIleGlyGlnLysPheAsnGluAspAsnAspAlaProValThrPheArgThrSerGluGlyGlyAlaLeuGluTrpSerPheAsnSerSerThrGlyAlaGlyAlaLeuThrGlnGlyThrThrThrTyrAlaMETHisGlyGlnGlnGlyAsnAspLeuAsnAlaGlyLysAsnLeuIlePheGlnGlyGlnAsnGlyGlnIleAsnLeuLysAspSerValSerGlnGlyAlaGlySerLeuThrPheArgAspAsnTyrThrValThrThrSerAsnGlySerThrTrpThrGlyAlaGlyIleValValAspAsnGlyValSerValAsnTrpGlnValAsnGlyValLysGlyAspAsnLeuHisLysIleGlyGluGlyThrLeuThrValGlnGlyThrGlyIleAsnGluGlyGlyLeuLysValGlyAspGlyLysValValLeuAsnGlnGlnAlaAspAsnLysGlyGlnValGlnAlaPheSerSerValAsnIleAlaSerGlyArgProThrValValLeuThrAspGluArgGlnValAsnProAspThrValSerTrpGlyTyrArgGlyGlyThrLeuAspValAsnGlyAsnSerLeuThrPheHisGlnLeuLysAlaAlaAspTyrGlyAlaValLeuAlaAsnAsnValAspLysArgAlaThrIleThrLeuAspTyrAlaLeuArgAlaAspLysValAlaLeuAsnGlyTrpSerGluSerGlyLysGlyThrAlaGlyAsnLeuTyrLysTyrAsnAsnProTyrThrAsnThrThrAspTyrPheIleLeuLysGlnSerThrTyrGlyTyrPheProThrAspGlnSerSerAsnAlaThrTrpGluPheValGlyHisSerGlnGlyAspAlaGlnLysLeuValAlaAspArgPheAsnThrAlaGlyTyrLeuPheHisGlyGlnLeuLysGlyAsnLeuAsnValAspAsnArgLeuProGluGlyValThrSerAlaLeuValMETAspGlyAlaAlaAspIleSerGlyThrPheThrGlnGluAsnGlyArgLeuThrLeuGlnGlyHisProValIleHisAlaTyrAsnThrGlnProValAlaAspLysLeuAlaAlaSerGlyAspHisSerValLeuThrGlnProThrSerPheSerGlnGluAspTrpGluAsnArgSerPheThrPheAspArgLeuSerLeuLysAsnThrAspPheGlyLeuGlyArgAsnAlaThrLeuAsnThrThrIleGlnAla AspAsnSerSerValThrLeuGlyAspSerArgValPheIleAspLysAsnAspGlyGlnGlyThrAlaPheThrLeuGluGluGlyThrSerValAlaThrLysAspAlaAspLysSerValPheAsnGlyThrValAsnLeuAspAsnGlnSerValLeuAsnIleAsnAspIlePheAsnGlyGlyIleGlnAlaAsnAsnSerThrValAsnIleSerSerAspSerAlaValLeuGlyAsnSerThrLeuThrSerThrAlaLeuAsnLeuAsnLysGlyAlaAsnAlaLeuAlaSerGlnSerPheValSerAspGlyProValAsnIleSerAspAlaThrLeuSerLeuAsnSerArgProAspGluValSerHisThrLeuLeuProValTyrAspTyrAlaGlySerTrpAsnLeuLysGlyAspAspAlaArgLeuAsnValGlyProTyrSerMETLeuSerGlyAsnIleAsnValGlnAspLysGlyThrValThrLeuGlyGlyGluGlyGluLeuSerProAspLeuThrLeuGlnAsnGlnMETLeuTyrSerLeuPheAsnGlyTyrArgAsnIleTrpSerGlySerLeuAsnAlaProAspAlaThrValSerMETThrAspThrGlnTrpSerMETAsnGlyAsnSerThrAlaGlyAsnMETLysLeuAsnArgThrIleValGlyPheAsnGlyGlyThrSerProPheThrThrLeuThrThrAspAsnLeuAspAlaValGlnSerAlaPheValMETArgThrAspLeuAsnLysAlaAspLysLeuValIleAsnLysSerAlaThrGlyHisAspAsnSerIleTrpValAsnPheLeuLysLysProSerAsnLysAspThrLeuAspIleProLeuValSerAlaProGluAlaThrAlaAspAsnLeuPheArgAlaSerThrArgValValGlyPheSerAspValThrProIleLeuSerValArgLysGluAspGlyLysLysGluTrpValLeuAspGlyTyrGlnValAlaArgAsnAspGlyGlnGlyLysAlaAlaAlaThrPheMETHisIleSerTyrAsnAsnPheIleThrGluValAsnAsnLeuAsnLysArgMETGlyAspLeuArgAspIleAsnGlyGluAlaGlyThrTrpValArgLeuLeuAsnGlySerGlySerAlaAspGlyGlyPheThrAspHisTyrThrLeuLeuGlnMETGlyAlaAspArgLysHisGluLeuGlySerMETAspLeuPheThrGlyValMETAlaThrTyrThrAspThrAspAlaSerAlaAspLeuTyrSerGlyLysThrLysSerTrpGlyGlyGlyPheTyrAlaSerGlyLeuPheArgSerGlyAlaTyrPheAspValIleAlaLysTyrIleHisAsnGluAsnLysTyrAspLeuAsnPheAlaGlyAlaGlyLysGlnAsnPheArgSerHisSerLeuTyrAlaGlyAlaGluValGlyTyrArgTyrHisLeuThrAspThrThrPheValGluProGlnAlaGluLeuValTrpGlyArgLeuGlnGlyGlnThrPheAsnTrpAsnAspSerGlyMETAspValSerMETArgArgAsnSerValAsnProLeuValGlyArgThrGlyValValSerGlyLysThrLeuSerGlyLysAspTrpSerLeuThrAlaArgAlaGlyLeuHisTyrGluPheAspLeuThrAspSerAlaAspValHisLeuLysAspAlaAlaGlyGluHisGlnIleAsnGlyArgLysAspSerArgMETLeuTyrGlyValGlyLeuAsnAlaArgPheGlyAspAsnThrArgLeuGlyLeuGluValGluArgSerAlaPheGlyLysTyrAsnThrAspAspAlaIleAsnAlaAsnIleArgTyrSerPhe。 
本发明的具体工艺步骤和工艺条件还有： 
制备方法中的步骤A是选用OMEGA公司提供的质粒提取试剂盒(Plasmid Mini Kit I D6943-01*)，并按照其说明书进行，提取出O78血清型大肠杆菌的质粒DNA。 
步骤B是选用全式金公司提供的PCR试剂盒，并按照其说明书进行；通过PCR扩增来获得目的基因。设计上游引物为zx1：5’-atgaacagaatttatt-3’，下游引物为zx2：5’-gaatgaataacgaa-3’为特异引物，以O78血清型大肠杆菌质粒DNA为模板，采用PCR扩增方法获得。 
PCR反应体系如下： 
Figure BDA00003199306600071
PCR反应条件为：96℃预变性5分钟，94℃变性1分钟；50℃退火30-60秒，72℃延伸1-2分钟，30个循环；72℃延伸10分钟后，置于4℃冰箱保存。 
本发明中的引物由申请人自行设计，主要基于产物的特异性好、引物精简不冗长、不易引起错配等技术考虑。 
本发明中的tsh-Ser基因和大肠杆菌O1血清型的温度敏感血凝素tsh基因很相似。经过序列比对，该基因与国际基因库上收录的从美国火鸡体内分离得到的大肠杆菌O1血清型基因序列的同源性为99％，序列比对表明：第1900位的碱基由G突变为A，氨基酸国际基因库中的氨基酸序列由甘氨酸(Gly)突变为丝氨酸(Ser)。后续的实验表明，碱基突变之后该基因对大肠杆菌病产生很好的预防作用。 
tsh-Ser基因与tsh基因同源性、广谱性及免疫效果比较对照 
Figure BDA00003199306600081
由上表可知如下结果： 
1、本发明中的tsh-Ser基因和O1血清型来源的tsh基因相比具有较高的同源性(99％)。 
2、本发明中的tsh-Ser基因和O1血清型来源的tsh基因相比，能够对更多血清型的大肠杆菌(O1、O2、O57、O65、O78、O85、O92血清型)产生免疫。 
3、与O1血清型来源的tsh基因相比，本发明中的tsh-Ser基因对更多血清型的大肠杆菌(O1、O2、O57、O65、O78、O85、O92血清型)的免疫效果均为100％。 
本发明与现有技术相比所具有的实质性特点和显著技术进步在于： 
1、通过PCR方法从致病性大肠杆菌O78血清型上获得了基因tsh-Ser，与发致病性大肠杆菌O1血清型上获得的tsh基因相比，该基因的一个碱基突变之后，其密码子所代表的氨基酸由甘氨酸突变为丝氨酸。 
2、由于基因tsh-Ser发生突变导致蛋白结构和功能产生了变化。使该基因对6种血清型大肠杆菌(O1、O2、O57、O65、O78、O85、O92血清型)均能产生免疫性，所以具有较高的广谱性。 
3、基因tsh-Ser对6种血清型大肠杆菌(O1、O2、O57、O65、O78、O85、O92血清型)的免疫效果均为100％，因此该基因对易感大肠杆菌病的动物具有更好的免疫效果，相比现有的tsh基因具有更为广阔的工业开发前景。 
附图说明
本发明的附图有： 
图1致病性大肠杆菌O78血清型来源的基因经PCR后，该tsh-Ser基因 的1％琼脂糖凝胶电泳图。 
图1中M为DL8000Marker(指示范围为8000，5000，3000，1500，1000，500)；扩增得到的tsh片段长度4134bp，与预期大小相符。 
图2PCR反应两端条件的反应产物经1％琼脂糖凝胶电泳图。 
图2中DL8000Marker(指示范围为8000，5000，3000，1500，1000，500)；扩增得到的片段长度4134bp，与预期大小相符。 
具体实施方式
附图给出了本发明的实施例，以下结合附图对本发明的实施例作进一步描述，但不作为对本发明的限定，本发明的保护范围以权利要求记载的内容为准，任何依据说明书做出的等效技术手段替换，均不脱离本发明的保护范围。 
实施例1 
A、用营养肉汤活化培养O78血清型大肠杆菌，37℃，200转/分钟，培养18小时，获得O78血清型大肠杆菌培养物；利用OMEGA试剂盒(Plasmid Mini Kit I D6943-01*)并按照试剂盒中所规定的操作步骤提取质粒DNA，以提取的质粒DNA作为PCR反应的模板； 
B、PCR反应体系如下： 
C、预防大肠杆菌病的基因tsh-Ser的制备方法，以5’-atgaacagaatt tatt-3’为上游引物zx1，以5’-gaatgaataacgaa-3’为下游引物zx2，以O78血清型大肠杆菌为模板，采用PCR扩增方法获得。PCR反应条件为：96℃预变性5分钟，94℃变性1分钟；50℃退火30秒，72℃延伸1分钟，30个循 环；72℃延伸10分钟后，置于4℃冰箱保存。 
PCR产物经过1％琼脂糖凝胶电泳，电泳图如图2(左边第一条泳道)所示。 
实施例2 
A、用营养肉汤活化培养O78血清型大肠杆菌，37℃，200转/分钟，培养18小时，获得O78血清型大肠杆菌培养物；利用OMEGA试剂盒(Plasmid Mini Kit I D6943-01*)并按照试剂盒中所规定的操作步骤提取质粒DNA，以提取的质粒DNA作为PCR反应的模板； 
B、PCR反应体系如下： 
Figure BDA00003199306600101
C、预防大肠杆菌病的基因tsh-Ser的制备方法，以5’-atgaacagaattta tt-3’为上游引物zx1，以5’-gaatgaataacgaa-3’为下游引物zx2，以O78血清型大肠杆菌质粒DNA为模板，采用PCR扩增方法获得。PCR反应条件为：96℃预变性5分钟，94℃变性1分钟；50℃退火45秒，72℃延伸1.5分钟，30个循环；72℃延伸10分钟后，置于4℃冰箱保存。 
PCR产物经过1％琼脂糖凝胶电泳，电泳图如图1所示。 
实施例3 
A、用营养肉汤活化培养O78血清型大肠杆菌，37℃，200转/分钟，培养18小时，获得O78血清型大肠杆菌培养物；利用OMEGA试剂盒(Plasmid Mini Kit I D6943-01*)并按照试剂盒中所规定的操作步骤提取质粒DNA，以提取的质粒DNA作为PCR反应的模板； 
B、PCR反应体系如下： 
Figure BDA00003199306600102
C、预防大肠杆菌病的基因tsh-Ser的制备方法，以5’-atgaacagaatt tatt-3’为上游引物zx1，以5’-gaatgaataacgaa-3’为下游引物zx2，以O78血清型大肠杆菌为模板，采用PCR扩增方法获得。PCR反应条件为：96℃预变性5分钟，94℃变性1分钟；50℃退火60秒，72℃延伸2分钟，30个循环；72℃延伸10分钟后，置于4℃冰箱保存。 
PCR产物经过1％琼脂糖凝胶电泳，电泳图如图2(左边第二条泳道)所示。 
实施例1-3得到的DNA序列和氨基酸序列如前述。由实施例1、实施例2和实施例3的PCR产物经过1％琼脂糖凝胶电泳图可知：当PCR的退火温度在30-60秒、延伸时间范围分别在1-2分钟时，当PCR的退火温度在45秒、延伸时间范围在1.5分钟时，PCR产物的特异性最好，没有出现拖尾现象；当PCR的退火温度范围在30秒和60秒、延伸时间分别在1.5分钟向1分钟和2分钟延伸时，PCR产物的特异性降低，出现拖尾现象。拖尾现象的出现会使PCR产物和开环的质粒进行连接时降低形成阳性重组质粒的成功率。 
Figure IDA00003199307600021
Figure IDA00003199307600031
Figure IDA00003199307600041
Figure IDA00003199307600061

Claims (4)

1.预防大肠杆菌病的基因tsh-Ser，其特征在于其DNA序列为：
atgaacagaatttattctcttcgctacagcgctgtggcccggggctttattgccatatctgagtttgctaggaaatgtgttcataagtctgtcagacgtctgtgtttcccggttttattactgatcccggtactattctctgcaggaagtcttgcgggaacggtcaataatgaactcgggtatcagttatttcgtgattttgctgaaaataaggggatgttccgcccgggggcaacgaatatcgctatttataataagcagggagaatttgtcggtacgctggataaggcagctatgcctgatttcagtgctgtggattcggaaatcggtgtggcgacactgataaacccgcagtatatcgccagcgtgaaacataacgggggatatacaaacgttagctttggtgatggtgaaaaccgttacaatatcgtggaccggaataatgcgccgtcactggattttcatgccccccggctggataaactggtgacagaggttgcccctactgcggtgacggcgcagggggcagtggctggcgcatatctggataaggagcgctatcctgttttttatcgtctggggtctggtactcagtatattaaggacagtaacggacagctgacaaaaatgggaggtgcatattcctggctgaccggcgggactgtcggtagcctgtcatcctatcagaatggagaaatgattagcaccagttcaggtctggtttttgattacaaacttaatggtgcaatgcccatttatggcgaggccggtgacagcggttcgcctttatttgcttttgatactgttcagaataaatgggtgctggtcggtgttcttactgcggggaatggcgcggggggcaggggaaataactgggctgttattccactggattttatcgggcagaaatttaatgaagacaatgatgccccggtcacgttcagaacatcggaaggtggtgcactggagtggagctttaacagcagtaccggagctggtgcgctgacacagggaaccaccacatatgccatgcacgggcagcagggaaatgacctgaatgctggtaagaacctgatatttcaggggcagaatggtcagattaaccttaaggattcggtttctcagggggcgggttccctgacgttccgtgataattacacagtaacaacctctaacggaagtacctggaccggtgccggtattgttgtggacaacggggtgtccgtaaactggcaggttaatggtgttaagggcgataacctgcataaaattggtgaaggtacgctgacggtacagggtacaggtattaatgaaggtggcctgaaggtcggggacggaaaggttgtactgaaccagcaggcggacaataaaggacaggtgcaggcgttcagcagtgttaatattgccagtggccggccgaccgtggtactgactgatgagcggcaggtaaatccggataccgtctcatggggatatcgtgggggcacactggatgttaatggtaacagtctgacgtttcatcagttgaaggcggcagattatggtgccgtgctggcgaataacgttgataaacgggccactatcacgctggactatgccctgcgggctgacaaagtagcactgaatggctggtcggaatcaggtaaaggaactgccggaaatttatataaatacaataacccgtacacaaatacgacggattacttcatcctgaagcagagcacctatggttatttccccacggaccagagcagcaacgccacctgggagtttgtggggcacagtcagggggatgcgcagaaactggtagctgaccgtttcaatactgcagggtatctgtttcacggacaactgaaaggcaatctgaatgtggacaatcgcctgcctgaaggcgttaccagtgctctggtgatggacggagctgcggatatctccggtacattcacccaggaaaacgggcgtctgacgctgcaggggcatccggttatccatgcatacaatactcagcctgtggctgacaaactggctgccagtggagaccattcggttctgactcagcctacgtcattcagtcaggaggactgggagaaccgcagttttacctttgacaggctgtcactgaagaacactgattttggtcttggtcgcaatgccacactgaacacaaccatccaggcagataactccagcgtcacgctgggcgacagccgggtatttatcgacaaaaacgatggccagggaacagcctttacccttgaagaaggcacatctgttgcaactaaagatgcagataaaagtgtcttcaacggcaccgtcaacctggataatcagtcagtgctgaatatcaatgatatattcaatggcggaatacaggcgaacaacagtaccgtgaatatctcctcagacagtgccgttctggggaactcaacactgaccagtaccgccctgaatctgaacaagggagcaaatgctctggccagtcagagttttgtttctgacggtccagtgaatatttctgatgccaccctgagtctgaacagccgtcctgatgaggtatctcacacacttttacctgtatacgattatgccggttcatggaacctgaagggagacgatgcccgcctgaacgtggggccgtacagtatgttgtcaggtaatatcaatgttcaggataaagggactgtcaccctcggaggggaaggggaactgagtcctgacctgactcttcagaatcagatgttgtacagcctgtttaacgggtaccgcaatatctggagcgggagcctgaatgcaccggatgccaccgtcagcatgacagacacccagtggtcgatgaacggaaactccacggcaggaaatatgaaacttaaccggacaatagtcggttttaacgggggaacatcaccgttcacgacactgacaacagataatctggacgcggttcagtcagcatttgtcatgcgtacagaccttaacaaggcagacaaactggtgataaacaagtcggcaacaggtcatgacaacagcatctgggttaacttcctgaaaaaaccttctaacaaggacacgcttgatattccactggtcagcgcacctgaagcgacagctgataatctgttcagggcatcaacacgggttgtgggattcagtgatgtcacccccatccttagtgtcagaaaagaggacgggaaaaaagagtgggtcctcgatggttaccaggttgcacgtaacgacggccagggtaaggctgccgccacattcatgcacatcagctataacaacttcatcactgaagttaacaacctgaacaaacgcatgggcgatttgagggatattaatggcgaagccggtacgtgggtgcgtctgctgaacggttccggctctgctgatggcggtttcactgaccactataccctgctgcagatgggggctgaccgtaagcacgaactgggaagtatggacctgtttaccggcgtgatggccacctacactgacacagatgcgtcagcagacctgtacagcggtaaaacaaaatcatggggtggtggtttctatgccagtggtctgttccggtccggcgcttactttgatgtgattgccaaatatattcacaatgaaaacaaatatgacctgaactttgccggagctggtaaacagaacttccgcagccattcactgtatgcaggtgcagaagtcggataccgttatcatctgacagatacgacgtttgttgaacctcaggcggaactggtctggggaagactgcagggccaaacatttaactggaacgacagtggaatggatgtctcaatgcgtcgtaacagcgttaatcctctggtaggcagaaccggcgttgtttccggtaaaaccctcagtggtaaggactggagtctgacagcccgtgccggcctgcattatgagttcgatctgacggacagtgctgacgttcatctgaaggatgcagcgggagaacatcagattaatggcagaaaagacagtcgtatgctttacggtgtggggttaaatgcccggtttggcgacaatacgcgtctggggctggaagttgaacgctctgcatttggtaaatacaacacagatgatgcgataaacgctaatattcgttattcattctga。
2.预防大肠杆菌病的基因tsh-Ser的制备方法，其特征在于以5’-atgaacagaatttatt-3’为上游引物zx1，以5’-gaatgaataacgaa-3’为下游引物zx2，以O78血清型大肠杆菌的质粒DNA为模板，采用PCR扩增方法获得。
3.根据权利要求2所述的预防大肠杆菌病的基因tsh-Ser的制备方法，其特征在于其工艺条件如下：
A、活化培养O78血清型大肠杆菌，并提取质粒DNA，以提取的质粒DNA作为PCR反应的模板；
B、PCR反应体系如下：
Figure FDA00003199306500031
C、PCR反应条件：96℃预变性5分钟，94℃变性1分钟；50℃退火30-60秒，72℃延伸1-2分钟，30个循环；72℃延伸10分钟后，置于4℃冰箱保存，即得扩增后的温度敏感血凝素基因tsh-Ser。
4.利用预防大肠杆菌病的基因tsh-Ser编码的蛋白，其特征在于其序列如下：
METAsnArgIleTyrSerLeuArgTyrSerAlaValAlaArgGlyPheIleAlaIleSerGluPheAlaArgLysCysValHisLysSerValArgArgLeuCysPheProValLeuLeuLeuIleProValLeuPheSerAlaGlySerLeuAlaGlyThrValAsnAsnGluLeuGlyTyrGlnLeuPheArgAspPheAlaGluAsnLysGlyMETPheArgProGlyAlaThrAsnIleAlaIleTyrAsnLysGlnGlyGluPheValGlyThrLeuAspLysAlaAlaMETProAspPheSerAlaValAspSerGluIleGlyValAlaThrLeuIleAsnProGlnTyrIleAlaSerValLysHisAsnGlyGlyTyrThrAsnValSerPheGlyAspGlyGluAsnArgTyrAsnIleValAspArgAsnAsnAlaProSerLeuAspPheHisAlaProArgLeuAspLysLeuValThrGluValAlaProThrAlaValThrAlaGlnGlyAlaValAlaGlyAlaTyrLeuAspLysGluArgTyrProValPheTyrArgLeuGlySerGlyThrGlnTyrIleLysAspSerAsnGlyGlnLeuThrLysMETGlyGlyAlaTyrSerTrpLeuThrGlyGlyThrValGlySerLeuSerSerTyrGlnAsnGlyGluMETIleSerThrSerSerGlyLeuValPheAspTyrLysLeuAsnGlyAlaMETProIleTyrGlyGluAlaGlyAspSerGlySerProLeuPheAlaPheAspThrValGlnAsnLysTrpValLeuValGlyValLeuThrAlaGlyAsnGlyAlaGlyGlyArgGlyAsnAsnTrpAlaValIleProLeuAspPheIleGlyGlnLysPheAsnGluAspAsnAspAlaProValThrPheArgThrSerGluGlyGlyAlaLeuGluTrpSerPheAsnSerSerThrGlyAlaGlyAlaLeuThrGlnGlyThrThrThrTyrAlaMETHisGlyGlnGlnGlyAsnAspLeuAsnAlaGlyLysAsnLeuIlePheGlnGlyGlnAsnGlyGlnIleAsnLeuLysAspSerValSerGlnGlyAlaGlySerLeuThrPheArgAspAsnTyrThrValThrThrSerAsnGlySerThrTrpThrGlyAlaGlyIleValValAspAsnGlyValSerValAsnTrpGlnValAsnGlyValLysGlyAspAsnLeuHisLysIleGlyGluGlyThrLeuThrValGlnGlyThrGlyIleAsnGluGlyGlyLeuLysValGlyAspGlyLysValValLeuAsnGlnGlnAlaAspAsnLysGlyGlnValGlnAlaPheSerSerValAsnIleAlaSerGlyArgProThrValValLeuThrAspGluArgGlnValAsnProAspThrValSerTrpGlyTyrArgGlyGlyThrLeuAspValAsnGlyAsnSerLeuThrPheHisGlnLeuLysAlaAlaAspTyrGlyAlaValLeuAlaAsnAsnValAspLysArgAlaThrIleThrLeuAspTyrAlaLeuArgAlaAspLysValAlaLeuAsnGlyTrpSerGluSerGlyLysGlyThrAlaGlyAsnLeuTyrLysTyrAsnAsnProTyrThrAsnThrThrAspTyrPheIleLeuLysGlnSerThrTyrGlyTyrPheProThrAspGlnSerSerAsnAlaThrTrpGluPheValGlyHisSerGlnGlyAspAlaGlnLysLeuValAlaAspArgPheAsnThrAlaGlyTyrLeuPheHisGlyGlnLeuLysGlyAsnLeuAsnValAspAsnArgLeuProGluGlyValThrSerAlaLeuValMETAspGlyAlaAlaAspIleSerGlyThrPheThrGlnGluAsnGlyArgLeuThrLeuGlnGlyHisProValIleHisAlaTyrAsnThrGlnProValAlaAspLysLeuAlaAlaSerGlyAspHisSerValLeuThrGlnProThrSerPheSerGlnGluAspTrpGluAsnArgSerPheThrPheAspArgLeuSerLeuLysAsnThrAspPheGlyLeuGlyArgAsnAlaThrLeuAsnThrThrIleGlnAlaAspAsnSerSerValThrLeuGlyAspSerArgValPheIleAspLysAsnAspGlyGlnGlyThrAlaPheThrLeuGluGluGlyThrSerValAlaThrLysAspAlaAspLysSerValPheAsnGlyThrValAsnLeuAspAsnGlnSerValLeuAsnIleAsnAspIlePheAsnGlyGlyIleGlnAlaAsnAsnSerThrValAsnIleSerSerAspSerAlaValLeuGlyAsnSerThrLeuThrSerThrAlaLeuAsnLeuAsnLysGlyAlaAsnAlaLeuAlaSerGlnSerPheValSerAspGlyProValAsnIleSerAspAlaThrLeuSerLeuAsnSerArgProAspGluValSerHisThrLeuLeuProValTyrAspTyrAlaGlySerTrpAsnLeuLysGlyAspAspAlaArgLeuAsnValGlyProTyrSerMETLeuSerGlyAsnIleAsnValGlnAspLysGlyThrValThrLeuGlyGlyGluGlyGluLeuSerProAspLeuThrLeuGlnAsnGlnMETLeuTyrSerLeuPheAsnGlyTyrArgAsnIleTrpSerGlySerLeuAsnAlaProAspAlaThrValSerMETThrAspThrGlnTrpSerMETAsnGlyAsnSerThrAlaGlyAsnMETLysLeuAsnArgThrIleValGlyPheAsnGlyGlyThrSerProPheThrThrLeuThrThrAspAsnLeuAspAlaValGlnSerAlaPheValMETArgThrAspLeuAsnLysAlaAspLysLeuValIleAsnLysSerAlaThrGlyHisAspAsnSerIleTrpValAsnPheLeuLysLysProSerAsnLysAspThrLeuAspIleProLeuValSerAlaProGluAlaThrAlaAspAsnLeuPheArgAlaSerThrArgValValGlyPheSerAspValThrProIleLeuSerValArgLysGluAspGlyLysLysGluTrpValLeuAspGlyTyrGlnValAlaArgAsnAspGlyGlnGlyLysAlaAlaAlaThrPheMETHisIleSerTyrAsnAsnPheIleThrGluValAsnAsnLeuAsnLysArgMETGlyAspLeuArgAspIleAsnGlyGluAlaGlyThrTrpValArgLeuLeuAsnGlySerGlySerAlaAspGlyGlyPheThrAspHisTyrThrLeuLeuGlnMETGlyAlaAspArgLysHisGluLeuGlySerMETAspLeuPheThrGlyValMETAlaThrTyrThrAspThrAspAlaSerAlaAspLeuTyrSerGlyLysThrLysSerTrpGlyGlyGlyPheTyrAlaSerGlyLeuPheArgSerGlyAlaTyrPheAspValIleAlaLysTyrIleHisAsnGluAsnLysTyrAspLeuAsnPheAlaGlyAlaGlyLysGlnAsnPheArgSerHisSerLeuTyrAlaGlyAlaGluValGlyTyrArgTyrHisLeuThrAspThrThrPheValGluProGlnAlaGluLeuValTrpGlyArgLeuGlnGlyGlnThrPheAsnTrpAsnAspSerGlyMETAspValSerMETArgArgAsnSerValAsnProLeuValGlyArgThrGlyValValSerGlyLysThrLeuSerGlyLysAspTrpSerLeuThrAlaArgAlaGlyLeuHisTyrGluPheAspLeuThrAspSerAlaAspValHisLeuLysAspAlaAlaGlyGluHisGlnIleAsnGlyArgLysAspSerArgMETLeuTyrGlyValGlyLeuAsnAlaArgPheGlyAspAsnThrArgLeuGlyLeuGluValGluArgSerAlaPheGlyLysTyrAsnThrAspAspAlaIleAsnAlaAsnIleArgTyrSerPhe。
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