CN111094546A - 粗糙型肠炎沙门氏菌的获取及其用于禽流感疫苗的基因改造 - Google Patents
粗糙型肠炎沙门氏菌的获取及其用于禽流感疫苗的基因改造 Download PDFInfo
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Abstract
本发明涉及一种已删除waaL基因以获得粗糙表型(3934vac DwaaL)的肠炎沙门氏菌3934vac菌株,获得方法以及出于降低毒性并保持其作为疫苗的免疫原性为目的而使用的寡核苷酸。本发明的另一方面涉及肠炎沙门氏菌3934vac DwaaL菌株,即粗糙型,已经修改以表达禽腺病毒I型纤维的基因,以及获得表达Ava‑I纤维基因的肠炎沙门氏菌3034vac DwaaL菌株的流程。本发明还包括通过在减毒且非致病性肠炎沙门氏菌菌株的染色体中Ava‑I纤维的插入和整合而开发的抗AvA‑I病毒的全新、活体、充足、有效和无害的禽类疫苗的开发。
Description
技术领域
本发明属于动物健康领域。具体来说,本发明涉及通过参与脂多糖合成的基因缺失而获得的减毒和粗糙表面型肠炎沙门氏菌肠炎沙门氏菌(肠炎沙门氏菌或SE)菌株的开发。在本文中,它还包括通过插入并整合到对Ava-I纤维抗原进行编码的染色体中的过程,将该菌株用作抗Ava-I病毒的疫苗载体。已使用的SE载体经过基因修饰,可作为用于表达AvA-I病毒纤维的免疫优势基因的载体,目的在于刺激针对禽类HCI(通过包涵体的病毒性肝炎)有效而持久的免疫应答。产生的菌株是完全安全的,因为它不含对第二个环状di-GMP信使的信号传导途径的蛋白质进行编码的十二个基因,西格玛RpoS因子及WaaL蛋白。因此,建议将该菌株作为一种针对Ava-I病毒的新型有效且安全的重组活疫苗,适合于禽类疫苗接种。
该疫苗的所有成分都是根据功效、安全性和成本三个优先因素进行专门设计和开发的。
发明背景
预计未来8到10年,家禽业将增长30%(APA,2008);增长伴随着决于有效和安全疫苗的成功健康计划。HCI/SHP是一种疾病,可导致感染的禽类高达50%的死亡率,在秘鲁是一个潜在问题,因为尽管进行了疫苗接种仍在爆发,该问题尚未解决,因为用于制作疫苗的技术在20年内都是相同的。
最近,加入可用细菌基因组的基因操作技术,对发病机理和免疫应答机制更深入的了解,为作为异源基因载体的减毒细菌菌株的开发提供了新方法。因此,肠致病细菌,例如李斯特菌、沙门氏菌、志贺氏菌、霍乱弧菌、小肠结肠炎耶尔森菌或百日咳博德特氏菌已被用作疫苗的有效载体。在他们当中,沙门氏菌给出了最有希望的结果。因此,基于减毒鼠伤寒沙门氏菌菌株的疫苗载体已被用于产生对抗病毒、细菌和原生动物病原体的保护性免疫反应,并被用作进行抗癌治疗的载体。在禽类的特殊情况下,该类型疫苗已评估为传染性支气管炎病毒,禽流感病毒,禽呼肠孤病毒和产气荚膜梭菌的基因载体。所有这些工作都与以下事实相符:这些疫苗能够激发持久的保护性免疫反应,还可以安全地避免引起疾病的风险。综上所述,将减毒的SE菌株用作载体,抗AvA-I重组活疫苗的开发将为家禽业的成功带来大力支持。优点如下:(1)通过防止逆转的等位基因交换的机制定向诱变的几个过程的强烈减毒不引入外源DNA并将所得菌株转化为完全安全的疫苗载体;(2)AvA-I抗原的表达量可以通过基因操作来调节;(3)可以口服接种给药;(4)这强烈刺激先天性和适应性免疫系统;(5)转而将提供对抗SE的保护。尽管使用细菌载体开发疫苗一直是是众多研究的目标,但迄今为止还没有基于SE经过许可的商业疫苗。因此,此类疫苗的开发将在这一研发领域树立良好的先例,并且在国内和国际市场上不会有竞争。
本发明中使用的菌株已由索拉诺等人于2002年进行分离及描述。这项研究报告了一种新的筛选方法,该方法基于来鉴定在生物膜形成上有缺陷的肠炎沙门氏菌突变体的钙氟荧光琼脂平板上菌落的荧光。这些结果不仅证实了已经得到描述的对鼠伤寒沙门氏菌和其他种类多细胞行为的调节的基因需求,而且还揭示了生物膜形成过程的新方面,例如合成纤维素酶可水解的胞外多糖所必需的名为bcsABZC及bcsEFG操纵子的新的遗传因素。BcsC和bcsE基因的非极性突变以及互补实验表明,两个操纵子对于在LB和ATM培养基中,在肠炎沙门氏菌和鼠伤寒沙门氏菌中的纤维素生物合成都是必不可少的。这项研究还表明,肠炎沙门氏菌产生的生物膜由不同的成分组成,这表明生物膜的组成和调节取决于环境条件。此外,结果表明纤维素酶不在肠炎沙门氏菌的致病性中,尽管在这种多糖的生产中不足的突变体对氯处理更为敏感,这表明纤维素的产生和生物膜的形成将是肠炎沙门氏菌在表面存活的一个重要的因素。在之后的研究中,索拉诺等人(2009,包括参考文献)进行了一项充满雄心的基因方法,在上述相同菌株中删除了利用双鸟苷酸环化酶的酶促活性(DGC,具有GGDEF结构域的蛋白质)对蛋白质进行编码的12个基因。这些酶合成了次级信使c-di-GMP,此分子在例如生物膜或毒性形成的关键细菌生物学过程中的参与已在众多种类中进行了广泛的研究。在SE菌株的特定情况下,c-di-GMP的总消耗与生物膜形成能力的丧失,鼠模型的轻微衰减以及对极端环境条件更高的敏感性有关。
作为上述基因方法的结果,专利文献ES 2324084B1描述了一种方法的发明,该方法允许在或获取的C-di-GMP合成中不足的革兰氏阴性细菌和沙门氏菌菌株的染色体中产生多重修改。这项发明还涉及通过所述方法获得的两种肠炎沙门氏菌突变株,其中通过GGDEF域对蛋白质进行编码的十二个基因被删除(突变体ΔXII::Km和突变体ΔXII::Clo)并用作表达载体,免疫治疗剂及在代谢研究使用。
GarcíaOna(2011),论文:一种用作减毒活疫苗的肠炎沙门氏菌无毒菌株的分析,研究了文件ES 2324084B1中包含的几种衍生菌株在开发能够诱导免疫应答,减少了集约化养猪场中粪便中器官的定殖和沙门氏菌的释放的疫苗方面的潜力。这项工作得出结论,用于开发疫苗的候选沙门氏菌菌株将是作为肠炎沙门氏菌3934ΔXII*被了解的菌株,该菌株已删除了通过GGDEF域对蛋白质进行编码的12个基因,以及在包括转录调节子RpoS染色体区域中一个额外的缺失。该菌株无毒,在环境中存活的能力降低,并且由于它不含外源DNA,因此不会归类为GMO(转基因生物)。使用鼠类感染模型进行的口服免疫试验表明,菌株107ufc单剂量(菌落形成单位)的免疫能够诱导针对沙门氏菌感染的保护性免疫反应,这表明该菌株可能是适合用作候选减毒活疫苗。
拉塔莎等人2016年进行了目的在于构建新的减毒活沙门氏菌疫苗以下研究:首先,分析了环状di-GMP(c-di-GMP)的缺失对沙门氏菌毒力的影响。如前所述,C-di-GMP是一种次级细胞内信使,控制广泛的细菌过程,包括生物膜的形成和毒力因子的合成,以及通过STING受体调节宿主的先天免疫应答。本文描述的结果表明,从基因组中删除了对二鸟苷酸环化酶蛋白进行编码的十二个基因并且因此无法合成c-di-GMP的沙门氏菌的多重突变体,在系统性鼠感染模型中表现出中等程度的衰减。rpoS基因的进一步受控突变(不同于肠炎沙门氏菌3934ΔXII*菌株,其中sigma因子的损失是由于自然反选造成的)导致了协同衰减作用,从而产生了称为ΔXIII的高度减毒的菌株。该菌株具有足够的免疫原性,可以保护小鼠免受鼠伤寒沙门氏菌强毒株的口服致死性攻击。ΔXIII的免疫原性取决于体液和细胞反应的激活,其特征在于调理抗体的产生以及显著水平IFN-γ,TNF-A,IL-2,IL-17及IL-10的诱导。ΔXIII能够形成生物膜,并且在干燥条件下无法存活,这表明它很容易从环境中去除。此外,ΔXIII具有DIVA(从疫苗接种动物区分感染)特性,可以区分受感染和接种疫苗的动物。为了方便术语的使用,DXII菌株也称为3934vac。
此外,Kong等人2011的论文描述了消除O抗原和核心糖对肠炎沙门氏菌血清型鼠伤寒毒力和免疫原性的影响。在这项研究中,在野生型鼠伤寒沙门氏菌的waaG(rfaG)、waaL(rfaI)、rfaH、waaJ(rfaJ)、wbaP(rfbP)、waaL(rfaL)或wzy(rfc)基因中进行了通过删除进行的非极性突变。确认LPS的结构后,分析了每种突变体在体外和体内的几种特性,并且与口服BALB/c小鼠的野生型亲本菌株相比,所有突变体均表现出明显的减毒和较低的入侵能力。此外,在waaG和waaL携带突变的菌株在派伊尔斑块和肝脏的定殖中缺乏,经鼻内施用突变体DwaaL后,这种不足得到了部分补偿。在供应肺炎球菌抗原PspA的减毒疫苗株的情况下,测试的所有突变均导致对抗PspA和沙门氏菌抗原的免疫反应降低。这些结果表明,不可逆转的外核截断不是开发口服沙门氏菌活疫苗的可行选择,而保留一个O抗原单位的wzy突变体将足以激发对口服、鼻内或腹膜内注射同源或异源抗原的最佳保护性免疫。
发明概述
本发明涉及一种减毒肠炎沙门氏菌菌株,该菌株已通过基因工程技术修改以删除waaL基因,因此表型粗糙。本发明的另一方面涉及所述的waaL基因突变肠炎沙门氏菌菌株,其染色体上同样携带在其染色体上对AVA-I纤维抗原进行编码的基因。该菌株除了表型粗糙且无毒之外,还具有针对Ava-I的保护作用。另外,本发明包括获得表型粗糙的减毒肠炎沙门氏菌菌株,并将在其染色体上对AVA-I纤维抗原进行编码的基因进行整合的流程,包括用于获得所述菌株的质粒和方法。
为了降低基于3934vac基因背景(SE DXIII)的菌株的毒性并保持其免疫原性,本发明的一方面涉及waaL基因缺失。该基因的突变导致表型粗糙,因为脂多糖仅由核心和脂质A组成,失去了合成O抗原的能力。这种突变的另一个优势是可以区分通过肠炎沙门氏菌和鼠伤寒沙门氏菌的野生型菌株接种疫苗并进行自然定殖的动物。
为了粗糙突变体的产生,使用了基于等位基因交换的实验设计,产生了带有两个waaL基因的相邻5和3'同源区域的热敏质粒。为了通过同源重组进行等位基因交换,首先将在3'和5'区域(AB和CD片段)的大约500bp侧翼waaL的两个片段克隆到该质粒中。通过电穿孔将构建的质粒导入3934vac菌株中(携带该质粒的沙门氏菌菌株在20μg/mL氯霉素的下于28℃中生长)。菌株转化后,通过在42℃(质粒复制的非许可温度)下进行培养,选择几个克隆以将质粒整合到染色体中。确认通过PCR载体整合入染色体后,进行切除或第二次重组。在第二步中,质粒丢失,产生了所需的缺失或恢复了基因的野生副本(这些基因事件的大致比例为50-50%)。第二种重组是通过在无抗生素和蔗糖存在的情况下于30℃下进行培养而实现的,因为该质粒带有致死性sacB基因,并且该糖在培养基中的存在反选仍保留有质粒的克隆。通过PCR和随后的测序确认发生等位基因交换的克隆。应该注意的是,这种方法产生的突变体对抗生素或外源DNA的痕迹不具有抗性。
为了将表达盒整合到允许在SE菌株中产生纤维抗原的染色体中,进行了与前述相似的方法。在这种情况下,除了重组侧翼区域外,质粒还包含整合到染色体中的表达盒。该表达盒被克隆到携带ST64B前噬菌体的Sb13基因的大约500bp的AB和CD区域的整合质粒中。克隆进行设计,使Ava-I纤维表达盒位于两个有缺陷的前噬菌体基因的片段之间(携带该质粒的沙门氏菌菌株在28℃下在20μg/mL氯霉素下生长)。通过电穿孔转化质粒后,几个克隆将在42℃下经历数次生长通道(质粒复制的非容许温度——得益于与waaL相邻的AB和CD同源区域,质粒得以整合)。通过PCR检查质粒已经整合入染色体后,在28℃下在无抗生素并且具有蔗糖存在下进行切除或第二次重组。如上所述,该反选择系统允许识别在质粒中丢失的克隆。最后一步再次包括通过PCR选择已出现在前噬菌体ST64B的sb13基因中的纤维表达盒插入的克隆。
本发明涉及的所得菌株是肠沙门氏菌的修改突变株,携带waaL基因缺失,因此表型粗糙,并表达来自自身染色体的Ava-I纤维基因。
出于本发明的目的,肠炎沙门氏菌是肠道沙门氏菌肠炎血清型的缩写。
另外,对于本发明来说,应当认为PCR是聚合酶链式反应,寡核苷酸是PCR中使用的引物。
发明详述
获得减毒沙门氏菌肠炎(ASE)
设计和构建允许在肠炎沙门氏菌(SE)染色体中引入异种抗原的系统。
活疫苗或疫苗载体必须是安全有效的,具有避免转化为毒性风险的完全受控的基因型和表型。另外,该菌株必须在减毒程度和免疫原性之间保持平衡,在宿主生物体中停留足够长的时间,以引起针对同源和/或异源抗原的保护性免疫应答。在这种情况下,产生的禽类肠炎沙门氏菌(ASE)菌株的主要特征是禽类的急剧衰减。另外,ASE菌株不能形成生物膜,并且在环境中的存活率较低,避免与接种动物可以排泄该菌株时期有关的任何风险。与大多数商业疫苗(例如9R)不同,其基因型和表型完全受控,并且不具有抗生素抗性基因。减毒活细菌的安全性已在其他模型中经过验证,例如9R疫苗,其报告科学地排除了转化为原始强毒形式潜在的可能性(冈本等人,2010,巴西家禽科学杂志)。
因此,本发明使用肠炎沙门氏菌3934菌株(保存在西班牙典型培养物保藏中心(STCC),访问号STCC 7236,通过基因工程技术,对二鸟苷酸环化酶进行编码的十二个基因、rpoS基因和waaL基因已被删除。进行最后一个突变的目的是为了获得一种可在繁殖动物中提供保护的表型粗糙的疫苗株(肠炎沙门氏菌3934vac DwaaL)。此外,本发明的另一个方面包括携带可赋予禽类抵抗包涵体肝炎免疫力的Ava-I纤维基因(肠炎沙门氏菌3934vacDwaaL-纤维)表达盒表型粗糙的疫苗株。
粗糙突变的产生
为了降低毒性并保持基于3934vac基因背景的菌株的免疫原性,删除waaL基因。该基因的突变导致表型粗糙,因为脂多糖仅由核心和脂质A组成,失去了合成O抗原的能力。该突变的另一个优势是可以通过肠炎沙门氏菌和鼠伤寒沙门氏菌的野生株区分接种疫苗和自然定殖的动物。
详细方法
所使用的方法是构建整合载体pKO:waaL,在重组的第一步整合此载体pKO:waaL,然后在重组的第二步中进行整合载体的切除(图1)
1.构建整合载体pKO:waaL
为了构建整合质粒pKO::waaL,通过PCR分别扩增520bp(寡核苷酸A和B)和504bp(寡核苷酸C和D)的waaL基因的两个侧翼片段。从凝胶中提纯PCR产物,并在标准克隆载体中独立克隆,之后分解为NotI XhoI(片段AB)和XhoI BglII(片段CD)。两个分解的片段均从凝胶中提纯并亚克隆到pKO质粒中,其序列为SEQ ID NO:1,分解至NotI BlgII,产生了载体pKO::waaL。pKO质粒带有一个抗氯霉素、热敏源、lacZ选择系统和sacB的盒体(图2)
序列为SEQ ID NO:2的pKO::waaL构建在大肠杆菌菌株中进行,并通过PCR、微量制备和分解进行验证。
表1显示了用于构建pDEST::waaL载体的寡核苷酸。序列(5'-3')。
下划线标出限制位点。
表1:用于在肠炎沙门氏菌3934中waaL基因缺失的寡核苷酸
2.自杀载体pKO::waaL的整合(第一次重组)
一旦构建并验证了pKO::waaL质粒(图3),就可以通过微量制备从大肠杆菌菌株中进行提取,并在肠炎沙门氏菌3934vac菌株中进行电穿孔,其整合在42℃下通过成长推动。该温度不允许质粒的复制,并且得益于与waaL相邻的同源区域AB和CD,发生了质粒的整合。来自在44℃下孵化的多个抗氯霉素的克隆,通过带有寡核苷酸E-F和E-G对的PCR进行测试,以验证整合。在第一种情况下,PCR必须是阴性,在第二种情况下,在AB片段中发生重组的那些克隆,所得的PCR将为1282bp。
表2:用于验证pKO::waaL整合的寡核苷酸
3.整合载体pKO::waaL的切除(第二次重组)
为了第二次重组以及pKO质粒的丢失,几个整合的克隆在没有抗生素的情况下在30℃的液体培养基中生长。24小时后,5份系列稀释液铺在补充有5%蔗糖的固体培养基上。
在此培养基中,只有那些通过第二次重组失去质粒的克隆才生长,取决于发生的片段,导致亲本基因型的再生或waaL基因的完全缺失。两个等位基因都可以使用带有寡核苷酸E和F通过PCR进行区分(上述序列)
在亲本菌株3934vac中,带有寡核苷酸E和F的PCR产生于2328bp的片段,来自基因型3934vac的扩增子序列为SEQ ID NO:7,而在已出现缺失的克隆中,该片段具有1106bp的大小,其扩增子序列为SEQ ID NO:8
3934vacDwaal或3934vacR菌株可以在没有抗生素的情况下在37℃下生长,因为它不携带任何选择盒。
肠炎沙门氏菌3934vac的Waa区(rfa)和肠炎沙门氏菌3934vacR的Waa区(rfa)分别在图4和图5中可见。
用于在DwaaL突变体验证的寡核苷酸为:SEQ ID NO:9和SEQ IDNO:10
带有在3934VacR菌株中上的AVA-I纤维基因的盒体的构建和插入
带有在3934VacR染色体上的AvA-I纤维基因表达盒的构建和插入
在3934vacR菌株中的基因插入是在染色体水平上进行的。这种策略使异源基因的表达稳定,消除了质粒分离的问题以及使用抗生素作为选择标记的需要。这些步骤在图6中进行总结。
1.整合载体pKO:sb13-纤维的构建
在此第一阶段,构建整合质粒以将表达盒整合到有缺陷的前噬菌体ST64B的sb13基因中(所有SE菌株在其染色体中都有它)(图6)。按照与上述相似的方法,通过寡核苷酸对H-I和J-K扩增AB(427bp)和CD(597bp)区域。从凝胶中提纯PCR产物,并独立克隆到标准克隆载体中,然后用通过SmaI sphI(片段AB)和spHI SalI(片段CD)分解。两个分解的片段均从凝胶中提纯,然后亚克隆到自杀质粒pKO中,分解至SmaI SalI,产生载体pKO::sb13(图7),其序列为SEQ ID NO:12
使用的寡核苷酸
片段AB:
寡核苷酸H(SEQ ID NO:13)
寡核苷酸I(SEQ ID NO:14)
片段CD:
寡核苷酸J(SEQ ID NO:15)
寡核苷酸K(SEQ ID NO:16)
一旦构建pKO::sb13质粒,盒体进行了组装,将引起纤维抗原的表达。该步骤分几个阶段进行:(i)从带有寡核苷酸L-M合成质粒中扩增<终止子(43bp)-Pr.启动子.RBS(114bp)-clyA(915bp)>,(ii)使用具有一对寡核苷酸N-O的pET28::纤维质粒作为模板,扩增纤维抗原(1597bp)的编码区,(iii)重叠PCR以通过外部寡核苷酸L-O融合两个片段。
寡核苷酸L(SEQ ID NO:17)
寡核苷酸M(SEQ ID NO:18)
寡核苷酸N(SEQ ID NO:19)
寡核苷酸O(SEQ ID NO:20)
纤维表达盒具有序列SEQ ID NO:21
得到的片段(2,702bp)从凝胶中提纯,并通过在先前分解成spHI的载体pKO::sb13中的同源末端组装进行克隆,获得载体pKO::sb13-纤维,其序列为SEQ ID NO:22
2.自杀载体pKO::sb13-纤维的整合(第一次重组)
一旦构建并验证pKO::sb13-纤维质粒,即可通过微量制备从大肠杆菌菌株中进行提取,并在肠炎沙门氏菌3934vacR(或3934vac DwaaL)菌株中进行电穿孔,其整合在42℃下通过成长推动。该温度不允许质粒的复制,并且得益于与sb13基因同源的同源区域AB和CD,质粒的整合发生。来自在44℃下孵化的多个抗氯霉素的克隆,通过带有寡核苷酸P-Q和P-M对的PCR进行测试,以验证整合。在第一种情况下,PCR必须是阴性,在第二种情况下,在AB片段中发生重组的那些克隆,所得的PCR将为1607bp。
使用的寡核苷酸为:
寡核苷酸P(SEQ ID NO:23)
寡核苷酸Q(SEQ ID NO:24)
寡核苷酸M(SEQ ID NO:25)
-整合载体pKO::ab13-纤维的切除(第二次重组)
为了第二次重组以及pKO质粒的丢失,将纤维抗原表达盒插入染色体中,几个整合的克隆在30℃没有抗生素的情况下在液体培养基中成长。24小时后,5份系列稀释液铺在补充有5%蔗糖的固体培养基上。只有那些通过第二次重组失去质粒的克隆才在此培养基中生长,取决于发生的片段,导致亲本基因型的再生或在负责纤维抗原的产生的表达盒的缺陷型前噬菌体ST64B的sb13基因中插入。之后的克隆都可以使用寡核苷酸P和Q通过PCR进行区分(上述序列)
在亲本菌株3934vacR中,带有寡核苷酸P和Q的PCR产生片1314bp的片段,而在已产生3934vacR::纤维(Ava-I)菌株的克隆中,纤维表达盒的整合具有3792bp bp的大小。
基因背景3934vacR中的序列为SEQ ID NO:25
基因背景3934vacR::纤维(Ava-I)中的序列sb13为SEQ ID NO:26
通过ASE纤维蛋白表达的检测
为了证实纤维抗原的异源表达,使用了SDS-PAGE和蛋白质印迹技术。通过在LB培养基中培养细菌来表达异源蛋白质,之后蛋白质将从细胞质、周质中提取并进行分泌。从表达获得的样本通过SDS-PAGE在10%聚丙烯酰胺凝胶中分离。将蛋白质转移到硝酸纤维素膜上,并使用IgG-HRP(与辣根过氧化物酶相连的抗体)作为缀合物间接检测蛋白质,然后使用DAB(3,3'二氨基联苯胺四氢氯化物)作为底物进行检测。
因此,通过蛋白质印迹获得的结果验证并令人满意地证实了在37℃和28℃的生长条件下在肠炎沙门氏菌3934Vac菌株中纤维抗原的表达。虽然没有一个条带(A、B和C)与ClyA-FiberAVAI-His融合物的预计理论分子量完全匹配(85.4kDa),但带A可能与融合物整个大小匹配,而带B和C是蛋白质降解的产物。其中ClyA代表ClyA基因的序列;fiberAVAI代表AVA-I的纤维基因序列,His代表6个连续组氨酸的序列。
3934菌株和粗糙型疫苗衍生物的表型研究。
为了研究不同菌株的表型,将其铺在刚果红琼脂上,这允许以简单的方式区分携带纤维基因的沙门氏菌菌株。与通常会产生纤维素、菌毛和其他附属物,会产生RDAR–红色干燥和粗糙的基因型的野生菌株不同,而在本发明中产生的突变体具有锯型表型(光滑白色)。当由于waaL基因的突变,菌株产生截短的脂多糖时,这种表型尤其明显。在半固体琼脂中差异也很明显,尽管该表型可能会因培养板的确切组成和湿度而发生变化(图8)。
重组疫苗的评估
免疫和挑战测试
疫苗接种物的准备。
接种物从能充分表达AvA-I纤维基因的RAvA-SE菌株中进行准备。将细菌接种在XLD培养基中,并在37℃下孵化24小时。将典型的菌落转移至LB培养基,在250rpm的搅拌下(机械搅拌器)将培养物在37℃下孵化18小时。通过以5000g离心10分钟收集细菌细胞,并根据所需浓度在PBS中连续稀释。
用于挑战测试的AvA-I接种物的准备。
该试验中使用的AvA-I分离物是从秘鲁伊卡查恰一个农场的HCI(包括机体肝炎)暴发中获得的,特点为通过RFLP-PCR的AvA-I血清型4。将分离物通过绒毛尿囊膜(CAM)接种在10天大的SPF卵中,孵化9天,然后收集肝脏进行均质化,并通过PCR确定病毒载量。在接种物鸡胚(IDCE 50/mL)中的的平均感染剂量使用里德和穆恩克公式计算。均质材料在-20℃下储存至挑战使用。
保护测试
在这一点上,动物试验将允许评估候选疫苗RAvA-SE的免疫原性。为此,建立了如下所示类似的时间表。根据给药剂量和给药途径确定工作组数。如果产生了一种以上的候选疫苗,则将遵循相同的方案。每个治疗组由30只动物组成,该数量根据涉及在整个实验中以评估细菌在内部器官中定殖的家禽动物牺牲的时间表进行决定。
按照以下时间表遵循标准化疫苗接种时间模型:
表3:疫苗接种时间表
LMS:微生物学和血清学实验室
LBMG:分子生物学和基因组学实验室
使用SPF Hyline 1天大的鸡,按照上述方案进行了疫苗接种。接种疫苗后,对鸡进行临床症状观察。每个实验组都放在单独的笼子里。在第21天,用1010copies/mL的AvA-I血清型IV,相当于105IDCE50(鸡胚中的平均感染剂量),进行肌肉注射。其中一组采用AvA-I(肌肉内)和野生肠炎沙门氏菌(口服107copies/mL)。按照时间表定期取样,包括粪便、泄殖腔拭子、肝脏和脾脏,以对RAvA-SE、野生SE和AvA-I进行检测和量化。还根据保护性抗体测量的时间表考虑血样。挑战后第7天,所有鸡都被实施安乐死。
在鸡身上肠炎减毒沙门氏菌排泄和定殖的测定
动物试验可以确定减毒肠炎沙门氏菌菌株(ASE)具有设计特性:与野生菌株相比,无毒,定殖能力降低,具有持久性和排泄。
工作时间的设计具体说明:治疗组、给药途径、所用细菌的浓度、要评估的样品类型和采样天数。每种实验包括15只动物,该数字包括在时间表中指定日期实施安乐死的动物,以评估细菌在内部器官中的定殖。对根据实验进行分组的1天大的禽类,使用1mL无菌注射器进行ASE菌株接种。提供食物和自由饮水,每天监测禽类的临床体征。保持相对湿度和适当的明暗循环条件。实验结束时,对禽类实施电击安乐死。
根据实验设计,将样品送至实验室进行相应的微生物、血清和分子分析。
用于沙门氏菌检测和量化的微生物检测。
根据确定的样本运输时间表,可以确定整个实验过程中ASE细菌的排泄和组织定殖。
根据确定的样本运输时间表,在整个实验过程中,对所评估的不同组禽类的粪便样品、直肠拭子、脾脏和肝脏进行分析。隔离过程按照流程图所示实施(图9)。
样本中的细菌载量得以确定。
图片列表
图1:用于3934vacR构建的等位基因交换方法。
图2:整合质粒pKO
图3:整合质粒pKO::waaL
图4:肠炎沙门氏菌3934vac的Waa区域(rfa)
图5:肠炎沙门氏菌3934vacR的Waa区域(rfa)
图6:pKOB::sb13质粒
图7:pKOB::sb13纤维质粒
图8:沙门氏菌菌株在刚果红琼脂平板(左)和半固体琼脂(右)上的表型分化。
图9:沙门氏菌检测和微生物量化的流程图。此外,TSI:三糖铁琼脂;LIA:赖氨酸铁琼脂。
图10:clyA-fiberFAdV-His蛋白的蛋白质印迹结果。
图11:带有阳性对照A和B,clyA-fiberFAdV-His蛋白的SDS-PAGE结果。
图12:在37℃或28℃的LB培养基中生长后获得的蛋白质提取物的SDS-PAGE和蛋白质印迹的考马斯染色。此外,MW:分子量标记,wt:肠炎沙门氏菌3934,Δ:肠炎沙门氏菌3934Vac;Δ-fiber:带有在染色体上具有ClyA-fiberFAdV-His融合的肠炎沙门氏菌3934Vac,Δ-pfiber:从质粒表达ClyA-fiberFAdV-His融合的肠炎沙门氏菌3934Vac。已经指示在蛋白质印迹中获得的特别带(A、B和C)。
图13:PCR产物的电泳分析表明存在带有waaL缺失的克隆。为了选择携带缺失的克隆,使用构建体外部的寡核苷酸扩增细菌染色体。此外,M=分子量标记,wt=野生株,c=菌落。预期大小:Wt=2328bp,ΔwaaL=1112bp
图14:使用的寡核苷酸汇总表
本发明的优选实施方案:示例
本文提供的以下示例用于说明本发明的性质。这些示例仅出于说明目的,不应解释为发明的限制。
示例1:通过免疫印迹,在肠炎沙门氏菌3934Vac菌株中纤维抗原的表达。
蛋白质提取
我们在用于ClyA纤维的5mL LB培养基中放置1个菌落,该ClyA纤维在染色体上有插入。在37℃下以200rpm过夜孵化。
我们以110000rpm离心5分钟,然后加入裂解缓冲液1(Tris HCL 10Mm、EDTA 5mM、NaCl 50mM)+蛋白酶抑制剂40μL(3g/mL)+50μL溶菌酶(10mg/mL),以及每个样品的SDS缓冲液(50μL简易缓冲液2x+50μL尿素8M+5μL B-巯基乙醇50μL样本),进行均质化并在100℃下加热5分钟,然后放在冰上5分钟,取20μL进行上样。以1:30为每种凝胶使用0.1Amps。
在蛋白质印迹的结果中观察到clyA-fiberFAdV-His蛋白质带。使用了标记;左侧为梯状P7709V(175KDa),右侧为ab 116029(245KDa)(图10)。
另外,当使用两个阳性His+对照,但首字母A和B按重量计时,A约为60kDa,B约为30kDa。这些结果让我们得出结论,clyA-fiberFAdV-His融合体得到表达,分子量约为85.4kDa。
(图11)
对野生株:肠炎沙门氏菌3934、Δ:肠炎沙门氏菌3934Vac;和Δ-fiber:在染色体上具ClyA-fiberFAdV-His融合体的肠炎沙门氏菌3934Vac,Δ-pfiber:从质粒表达cly-AfiberFAdV-His融合体的肠炎沙门氏菌3934Vac。
PCR产物的电泳分析揭示了带有waaL缺失的克隆的存在(图13)
出于本发明的目的,肠炎沙门氏菌3934vac菌株在实验模型中的使用表明它可以在鼠类甚至禽类模型中起作用,
在我们的试验中,我们观察到,将接种到禽类中后,效果并不理想,因为无论如何都会发展感染;为了获得免疫,在肠炎沙门氏菌3934vac的waaL基因进行了删除,从而获得了免疫。同样,用3934vac sb13::clyA-fiberFAdVΔwaaL菌株进行的挑战试验表明,它赋予抗沙门氏菌和I型禽腺病毒免疫,可防止出现包涵体肝炎。
微生物保存
根据用于专利程序的微生物保存布达佩斯条约的规定,SG-9R sb:ClyA-FIBER6His菌株、3934vac-粗糙型突变体和3934vac-Fiber粗糙型突变体菌株已在以下日期保存在西班牙典型培养物保藏中心(西班牙巴伦西亚帕特纳),并且为他们分配了以下保存编号:
内容 | 保存日期 | 访问号 |
SG-9Rsb:ClyA-FIBER 6His | 2017年4月5日 | CECT 9331 |
3934vac-粗糙型突变体 | 2017年4月5日 | CECT 9332 |
3934vac-纤维粗糙型突变体 | 2017年4月5日 | CECT 9333 |
本发明不限于该专利中所保存的微生物的范围,因为它们代表了本发明一个方面的具体说明。与本发明中描述的那些在功能上等同的任何微生物或质粒都包括在本发明内。
序列表
<110> 兽医制药股份公司(FARMACOLÓGICOS VETERINARIOS S.A.C)
<120> 粗糙型肠炎沙门氏菌的获取及其用于禽流感疫苗的基因改造
<130> PN19038199P
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 7999
<212> DNA
<213> 人工序列
<220>
<223> pKO
<220>
<221> misc_feature
<222> (1039)..(1039)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (3427)..(3427)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (3979)..(3980)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (3983)..(3983)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (5891)..(5892)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (7966)..(7966)
<223> n是a, c, g, 或t
<400> 1
ccctttcgtc ttcgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc 60
tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga 120
tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt 180
ttttgagtta tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg 240
atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc 300
agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac 360
cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat 420
gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag 480
tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag 540
tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta 600
cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc 660
caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt 720
cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct 780
ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga 840
attacaacag tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg 900
gtgcccttaa acgcctggtg ctacgcctga ataagtgata ataagcggat gaatggcaga 960
aattcgaaag caaattcgac ccggtcgtcg gttcagggca gggtcgttaa atagccgctt 1020
atgtctattg ctggtttant cggtaccccc tttagcatat tatgttgcca actgtcggaa 1080
cgagacttct ctaaaccttc ccggcttcat catgctctct taacactgct acatcaaccc 1140
cttgaatcct caattcacct cctcgaaaac atttcccatc aatcatattc tggtatatct 1200
tatcttctgg cagtgacagc gtcacttcgt aatcattatg attgatgata atcaaatact 1260
tccattcatc agtctctctt tgttgcacct ctacattttc agctacttca agaatgggat 1320
taatatgatg tttagcgaaa acctgttcta aaagcctgcc taaataatta ctatctggat 1380
aagtaccgac gtaaatcccc tctcctttac cgtagcagtt acgtgtaacc gccggaagtc 1440
ctgcatacca atccccctta aatgtcgcta gaggttctgc cccttctaat cggattatgt 1500
ccgcccacgt cgtacaatca tattccccat cgttactata tattttgttt acctttgttt 1560
ctgggtatgg tacaaattcc tcaacaaaaa tccccaaaat atctcgcaga ggaccaggat 1620
atccgcctag atgtacacgg tcattttcat ctacaatgcc actgaagaaa ctgacaatca 1680
aagtgccacc gttagcaaca aattgccgta agttttcatc ttctccctct ttaaccatat 1740
ataacattgg agcaataact actttgtatt ttgttagatc atcagatggc cttacaaaat 1800
cgacagcaat attacgttta tataattccc tataataagc ttcaactata ggaatatatc 1860
ttagtttatt atgtggtttg gaacttagtt cgacagccca ccagttttcc caatcaaaaa 1920
tgatcgcgac ctctgccttg attctagatc cgaccaaaca atctaacttt ttcagctctt 1980
gccctaactg tgtaacttcc ctataaattc tattattctc gttcaaaaag tggggcacca 2040
ttgcaccgtg gaatttttca gctcctgctc tactttgacg ccactggaaa aacataatac 2100
catctgcacc acgggcaata gttgcataac tccatagacg cattacacct ggcggttttg 2160
gaacattaat atcgcgccag ttaacatgtg aggttacctg ctccatcaaa ataaacggtt 2220
gaccttttct taaactacgc ataaggtcat tcatcatggc gtgctgaatt ggcaagccct 2280
ctctgggatc aggatatgag tcccatgtca caatatctac atgctgagcc cattgaaaat 2340
agtttaacgg tttgaatgaa cccatgaaat tagttgatac tggaatatct ggtgttacct 2400
cacgtaaaat ttccttttct gttaaaaaca acttgagaat tgagtcattc ataaaacggt 2460
agtaatcaag ttcttgggat ggattaataa aagttggtgc ctttctaggg ggattaattt 2520
catcccaatg attgtatcgc tgtccccaaa agtttgtacc ccaacgttca tttaattcat 2580
cgattgtttt atatctttcc tttagccact ttctaaacgc gacagcacaa ttctcacaaa 2640
aacacttgga aacgtgacat gcatactcat tattaacatg ccacattttg agtgccggat 2700
gatttttata ccgttctgct atagccctca caagtctctt tatgtgcgta attaattgag 2760
gatgattagg acaataatgt tgtctactgc caaacgagag aatgacaccg ctttcatcga 2820
tcggcaaaga atctggatac ttttttacaa accaagctgg agtagttgca gtcgccgtcc 2880
ccaagttaat ataaacaccg tggtcatata gtatatctat aaccttgtct agccattcaa 2940
agtcgaacac tccatcagac ggttcgatct tgctccaact gaaaatccct aaagatacta 3000
aattcacccc cgctttttgc atcaacttag catcttcata ccaaatttcc tctggccatt 3060
gctctgggtt ataatctcct ccgtaacaaa ttgaggataa cacattcata attattcccc 3120
ctagctaatt ttcgtttaat tataaattaa gttaaaattt aggtacctac gtaggatcga 3180
tccgatcctt aattatatta tagtcccaat ataatcattt atcaactctt ttacacttaa 3240
atttcctaat atttgtaaat atttttttga ataaaaaaat ctagctaatg ttacgttaca 3300
cattaactag acagatctat cgatgcatgc catggtaccc gggagctcga attctagaag 3360
cttctgcaga cgcgtcgacg tcatatggat cctctagagc ggccgcgatc ctctagagtc 3420
gaccggngaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 3480
tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 3540
tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 3600
tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 3660
gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 3720
agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 3780
cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 3840
agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg 3900
tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 3960
aaatatgtat ccgctcatnn cangatcctt tttaacccat cacatatacc tgccgttcac 4020
tattatttag tgaaatgaga tattatgata ttttctgaat tgtgattaaa aaggcaactt 4080
tatgcccatg caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt 4140
ttagttcttt aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg 4200
aaaatagacc agttgcaatc caaacgagag tctaatagaa tgaggtcgaa aagtaaatcg 4260
cgcgggtttg ttactgataa agcaggcaag acctaaaatg tgtaaagggc aaagtgtata 4320
ctttggcgtc accccttaca tattttaggt ctttttttat tgtgcgtaac taacttgcca 4380
tcttcaaaca ggagggctgg aagaagcaga ccgctaacac agtacataaa aaaggagaca 4440
tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag tattaacctt tactaccgca 4500
ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa cgaaccaaaa gccatataag 4560
gaaacatacg gcatttccca tattacacgc catgatatgc tgcaaatccc tgaacagcaa 4620
aaaaatgaaa aatatcaagt tcctgaattc gattcgtcca caattaaaaa tatctcttct 4680
gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa acgctgacgg cactgtcgca 4740
aactatcacg gctaccacat cgtctttgca ttagccggag atcctaaaaa tgcggatgac 4800
acatcgattt acatgttcta tcaaaaagtc ggcgaaactt ctattgacag ctggaaaaac 4860
gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa atgattctat cctaaaagac 4920
caaacacaag aatggtcagg ttcagccaca tttacatctg acggaaaaat ccgtttattc 4980
tacactgatt tctccggtaa acattacggc aaacaaacac tgacaactgc acaagttaac 5040
gtatcagcat cagacagctc tttgaacatc aacggtgtag aggattataa atcaatcttt 5100
gacggtgacg gaaaaacgta tcaaaatgta cagcagttca tcgatgaagg caactacagc 5160
tcaggcgaca accatacgct gagagatcct cactacgtag aagataaagg ccacaaatac 5220
ttagtatttg aagcaaacac tggaactgaa gatggctacc aaggcgaaga atctttattt 5280
aacaaagcat actatggcaa aagcacatca ttcttccgtc aagaaagtca aaaacttctg 5340
caaagcgata aaaaacgcac ggctgagtta gcaaacggcg ctctcggtat gattgagcta 5400
aacgatgatt acacactgaa aaaagtgatg aaaccgctga ttgcatctaa cacagtaaca 5460
gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca aatggtacct gttcactgac 5520
tcccgcggat caaaaatgac gattgacggc attacgtcta acgatattta catgcttggt 5580
tatgtttcta attctttaac tggcccatac aagccgctga acaaaactgg ccttgtgtta 5640
aaaatggatc ttgatcctaa cgatgtaacc tttacttact cacacttcgc tgtacctcaa 5700
gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa acagaggatt ctacgcagac 5760
aaacaatcaa cgtttgcgcc aagcttcctg ctgaacatca aaggcaagaa aacatctgtt 5820
gtcaaagaca gcatccttga acaaggacaa ttaacagtta acaaataaaa acgcaaaaga 5880
aaatgccgat nnccggttta ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg 5940
cctcaggctg tctatgtgtg actgttgagc tgtaacaagt tgtctcaggt gttcaatttc 6000
atgttctagt tgctttgttt tactggtttc acctgttcta ttaggtgtta catgctgttc 6060
atctgttaca ttgtcgatct gttcatggtg aacagcttta aatgcaccaa aaactcgtaa 6120
aagctctgat gtatctatct tttttacacc gttttcatct gtgcatatgg acagttttcc 6180
ctttgatatc taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact 6240
gatagataca agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta 6300
gtgtggttcg ttgtttttgc gtgagccatg agaacgaacc attgagatca tgcttacttt 6360
gcatgtcact caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat 6420
cgtgtagtgt ttttcttagt ccgttacgta ggtaggaatc tgatgtaatg gttgttggta 6480
ttttgtcacc attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc 6540
tatctagttc aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca 6600
atttcatatt gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa 6660
gccttttaaa ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc 6720
taatctctat atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat 6780
aaatcctcat agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga 6840
atttttttaa ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt 6900
cgcttgagaa cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat 6960
tcctgatttc cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag 7020
cattttccct actgatgttc atcatctgag cgtattggtt ataagtgaac gataccgtcc 7080
gttctttcct tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta 7140
gcttggtttc atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa 7200
caactaattc agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga 7260
gatgggctag tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat 7320
tctgctagac ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta 7380
gacctttgtg tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag 7440
aataaaaaaa gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag 7500
ttccgcagta ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt 7560
aaaaccctaa aggcttaagt agcaccctcg caagctcggg caaatcgctg aatattcctt 7620
ttgtctccga ccatcaggca cctgagtcgc tgtctttttc gtgacattca gttcgctgcg 7680
ctcacggctc tggcagtgaa tgggggtaaa tggcactaca ggcgcctttt atggattcat 7740
gcaaggaaac tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg 7800
gcgggtctgc tatgtggtgc tatctgactt tttgctgttc agcagttcct gccctctgat 7860
tttccagtct gaccacttcg gattatcccg tgacaggtca ttcagactgg ctaatgcacc 7920
cagtaaggca gcggtatcat caacaggctt acccgtctta ctgtcnggat cgacgctctc 7980
ccttatgcga ctcctgcat 7999
<210> 2
<211> 8926
<212> DNA
<213> 人工序列
<220>
<223> pKO::waaL
<220>
<221> misc_feature
<222> (34)..(34)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (2108)..(2109)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (4017)..(4017)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (4020)..(4021)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (4573)..(4573)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (7888)..(7888)
<223> n是a, c, g, 或t
<400> 2
atgcaggagt cgcataaggg agagcgtcga tccngacagt aagacgggta agcctgttga 60
tgataccgct gccttactgg gtgcattagc cagtctgaat gacctgtcac gggataatcc 120
gaagtggtca gactggaaaa tcagagggca ggaactgctg aacagcaaaa agtcagatag 180
caccacatag cagacccgcc ataaaacgcc ctgagaagcc cgtgacgggc ttttcttgta 240
ttatgggtag tttccttgca tgaatccata aaaggcgcct gtagtgccat ttacccccat 300
tcactgccag agccgtgagc gcagcgaact gaatgtcacg aaaaagacag cgactcaggt 360
gcctgatggt cggagacaaa aggaatattc agcgatttgc ccgagcttgc gagggtgcta 420
cttaagcctt tagggtttta aggtctgttt tgtagaggag caaacagcgt ttgcgacatc 480
cttttgtaat actgcggaac tgactaaagt agtgagttat acacagggct gggatctatt 540
ctttttatct ttttttattc tttctttatt ctataaatta taaccacttg aatataaaca 600
aaaaaaacac acaaaggtct agcggaattt acagagggtc tagcagaatt tacaagtttt 660
ccagcaaagg tctagcagaa tttacagata cccacaactc aaaggaaaag gactagtaat 720
tatcattgac tagcccatct caattggtat agtgattaaa atcacctaga ccaattgaga 780
tgtatgtctg aattagttgt tttcaaagca aatgaactag cgattagtcg ctatgactta 840
acggagcatg aaaccaagct aattttatgc tgtgtggcac tactcaaccc cacgattgaa 900
aaccctacaa ggaaagaacg gacggtatcg ttcacttata accaatacgc tcagatgatg 960
aacatcagta gggaaaatgc ttatggtgta ttagctaaag caaccagaga gctgatgacg 1020
agaactgtgg aaatcaggaa tcctttggtt aaaggctttg agattttcca gtggacaaac 1080
tatgccaagt tctcaagcga aaaattagaa ttagttttta gtgaagagat attgccttat 1140
cttttccagt taaaaaaatt cataaaatat aatctggaac atgttaagtc ttttgaaaac 1200
aaatactcta tgaggattta tgagtggtta ttaaaagaac taacacaaaa gaaaactcac 1260
aaggcaaata tagagattag ccttgatgaa tttaagttca tgttaatgct tgaaaataac 1320
taccatgagt ttaaaaggct taaccaatgg gttttgaaac caataagtaa agatttaaac 1380
acttacagca atatgaaatt ggtggttgat aagcgaggcc gcccgactga tacgttgatt 1440
ttccaagttg aactagatag acaaatggat ctcgtaaccg aacttgagaa caaccagata 1500
aaaatgaatg gtgacaaaat accaacaacc attacatcag attcctacct acgtaacgga 1560
ctaagaaaaa cactacacga tgctttaact gcaaaaattc agctcaccag ttttgaggca 1620
aaatttttga gtgacatgca aagtaagcat gatctcaatg gttcgttctc atggctcacg 1680
caaaaacaac gaaccacact agagaacata ctggctaaat acggaaggat ctgaggttct 1740
tatggctctt gtatctatca gtgaagcatc aagactaaca aacaaaagta gaacaactgt 1800
tcaccgttag atatcaaagg gaaaactgtc catatgcaca gatgaaaacg gtgtaaaaaa 1860
gatagataca tcagagcttt tacgagtttt tggtgcattt aaagctgttc accatgaaca 1920
gatcgacaat gtaacagatg aacagcatgt aacacctaat agaacaggtg aaaccagtaa 1980
aacaaagcaa ctagaacatg aaattgaaca cctgagacaa cttgttacag ctcaacagtc 2040
acacatagac agcctgaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat 2100
aaaccggnna tcggcatttt cttttgcgtt tttatttgtt aactgttaat tgtccttgtt 2160
caaggatgct gtctttgaca acagatgttt tcttgccttt gatgttcagc aggaagcttg 2220
gcgcaaacgt tgattgtttg tctgcgtaga atcctctgtt tgtcatatag cttgtaatca 2280
cgacattgtt tcctttcgct tgaggtacag cgaagtgtga gtaagtaaag gttacatcgt 2340
taggatcaag atccattttt aacacaaggc cagttttgtt cagcggcttg tatgggccag 2400
ttaaagaatt agaaacataa ccaagcatgt aaatatcgtt agacgtaatg ccgtcaatcg 2460
tcatttttga tccgcgggag tcagtgaaca ggtaccattt gccgttcatt ttaaagacgt 2520
tcgcgcgttc aatttcatct gttactgtgt tagatgcaat cagcggtttc atcacttttt 2580
tcagtgtgta atcatcgttt agctcaatca taccgagagc gccgtttgct aactcagccg 2640
tgcgtttttt atcgctttgc agaagttttt gactttcttg acggaagaat gatgtgcttt 2700
tgccatagta tgctttgtta aataaagatt cttcgccttg gtagccatct tcagttccag 2760
tgtttgcttc aaatactaag tatttgtggc ctttatcttc tacgtagtga ggatctctca 2820
gcgtatggtt gtcgcctgag ctgtagttgc cttcatcgat gaactgctgt acattttgat 2880
acgtttttcc gtcaccgtca aagattgatt tataatcctc tacaccgttg atgttcaaag 2940
agctgtctga tgctgatacg ttaacttgtg cagttgtcag tgtttgtttg ccgtaatgtt 3000
taccggagaa atcagtgtag aataaacgga tttttccgtc agatgtaaat gtggctgaac 3060
ctgaccattc ttgtgtttgg tcttttagga tagaatcatt tgcatcgaat ttgtcgctgt 3120
ctttaaagac gcggccagcg tttttccagc tgtcaataga agtttcgccg actttttgat 3180
agaacatgta aatcgatgtg tcatccgcat ttttaggatc tccggctaat gcaaagacga 3240
tgtggtagcc gtgatagttt gcgacagtgc cgtcagcgtt ttgtaatggc cagctgtccc 3300
aaacgtccag gccttttgca gaagagatat ttttaattgt ggacgaatcg aattcaggaa 3360
cttgatattt ttcatttttt tgctgttcag ggatttgcag catatcatgg cgtgtaatat 3420
gggaaatgcc gtatgtttcc ttatatggct tttggttcgt ttctttcgca aacgcttgag 3480
ttgcgcctcc tgccagcagt gcggtagtaa aggttaatac tgttgcttgt tttgcaaact 3540
ttttgatgtt catcgttcat gtctcctttt ttatgtactg tgttagcggt ctgcttcttc 3600
cagccctcct gtttgaagat ggcaagttag ttacgcacaa taaaaaaaga cctaaaatat 3660
gtaaggggtg acgccaaagt atacactttg ccctttacac attttaggtc ttgcctgctt 3720
tatcagtaac aaacccgcgc gatttacttt tcgacctcat tctattagac tctcgtttgg 3780
attgcaactg gtctattttc ctcttttgtt tgatagaaaa tcataaaagg atttgcagac 3840
tacgggccta aagaactaaa aaatctatct gtttcttttc attctctgta ttttttatag 3900
tttctgttgc atgggcataa agttgccttt ttaatcacaa ttcagaaaat atcataatat 3960
ctcatttcac taaataatag tgaacggcag gtatatgtga tgggttaaaa aggatcntgn 4020
natgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4080
atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta 4140
aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 4200
aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 4260
ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 4320
ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 4380
aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 4440
gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 4500
gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc 4560
cattcgccat tcnccggtcg actctagagg atcgcggccg cgcgattatg ccatcgcaaa 4620
acattttctg cattgccagc aggcggtcag cgatccatat gcggtatttt tacatgccac 4680
gacccgcgat gataaacact ggccggaagc aaactggcgc gagcttatcg gtatggtggg 4740
caacaccgga ttacggataa aacttccctg gggcgcgcct catgaggagg cccgggctaa 4800
acgactggcc gaaggctttg actatgtgga tgtgttaccg cgcatgagcc tggaggaggt 4860
cgccagagtg ctggctggcg caaaatttgt cgtatcggtt gataccggcc tgagccatct 4920
caccgccgcg ctcgacagac cgaatattac gctatatggc ccaacggacc ctgggttaat 4980
tggaggttat gggaagaacc aaatggcatg ctgctcacca gaaaaaaacc tggcgaattt 5040
agatgccaca agcgtatttg gaaagattca ttaaagagac actgtctcat cccaaaccta 5100
ttgtggactc gaggcgctga tacttattac ggtatcagcg cgttttccat cgtccggact 5160
caatcactta tcaaaccagc ttttcatctg ttcctcgaaa cgctgagcta cattttccca 5220
actgtatttt gaaaacacca gggattttgc tttttcggca atctggtggc gttccttatc 5280
agcaagcgca cggttaatat cattaattat actgtcgctc gacataggtt ctgcgaggtg 5340
atagcccgtt atgccatcta acacaaattc gctaatcccc ccttttttgc tggcaagaac 5400
cgcttttcct gctgccatcg cttctacagc caccatgcaa aatgcttctt caacctgaga 5460
tggcacaata accagatcgg ctatatgata gaagttatgc atctggtcag gagattgccc 5520
cccagccata atacaatcag ttccaatctc ttttgcggcg tccagtactt tcttttgata 5580
ctctgctttt tcacccttgc ggcttagatc ttctgtctag ttaatgtgta acgtaacatt 5640
agctagattt ttttattcaa aaaaatattt acaaatatta ggaaatttaa gtgtaaaaga 5700
gttgataaat gattatattg ggactataat ataattaagg atcggatcga tcctacgtag 5760
gtacctaaat tttaacttaa tttataatta aacgaaaatt agctaggggg aataattatg 5820
aatgtgttat cctcaatttg ttacggagga gattataacc cagagcaatg gccagaggaa 5880
atttggtatg aagatgctaa gttgatgcaa aaagcggggg tgaatttagt atctttaggg 5940
attttcagtt ggagcaagat cgaaccgtct gatggagtgt tcgactttga atggctagac 6000
aaggttatag atatactata tgaccacggt gtttatatta acttggggac ggcgactgca 6060
actactccag cttggtttgt aaaaaagtat ccagattctt tgccgatcga tgaaagcggt 6120
gtcattctct cgtttggcag tagacaacat tattgtccta atcatcctca attaattacg 6180
cacataaaga gacttgtgag ggctatagca gaacggtata aaaatcatcc ggcactcaaa 6240
atgtggcatg ttaataatga gtatgcatgt cacgtttcca agtgtttttg tgagaattgt 6300
gctgtcgcgt ttagaaagtg gctaaaggaa agatataaaa caatcgatga attaaatgaa 6360
cgttggggta caaacttttg gggacagcga tacaatcatt gggatgaaat taatccccct 6420
agaaaggcac caacttttat taatccatcc caagaacttg attactaccg ttttatgaat 6480
gactcaattc tcaagttgtt tttaacagaa aaggaaattt tacgtgaggt aacaccagat 6540
attccagtat caactaattt catgggttca ttcaaaccgt taaactattt tcaatgggct 6600
cagcatgtag atattgtgac atgggactca tatcctgatc ccagagaggg cttgccaatt 6660
cagcacgcca tgatgaatga ccttatgcgt agtttaagaa aaggtcaacc gtttattttg 6720
atggagcagg taacctcaca tgttaactgg cgcgatatta atgttccaaa accgccaggt 6780
gtaatgcgtc tatggagtta tgcaactatt gcccgtggtg cagatggtat tatgtttttc 6840
cagtggcgtc aaagtagagc aggagctgaa aaattccacg gtgcaatggt gccccacttt 6900
ttgaacgaga ataatagaat ttatagggaa gttacacagt tagggcaaga gctgaaaaag 6960
ttagattgtt tggtcggatc tagaatcaag gcagaggtcg cgatcatttt tgattgggaa 7020
aactggtggg ctgtcgaact aagttccaaa ccacataata aactaagata tattcctata 7080
gttgaagctt attataggga attatataaa cgtaatattg ctgtcgattt tgtaaggcca 7140
tctgatgatc taacaaaata caaagtagtt attgctccaa tgttatatat ggttaaagag 7200
ggagaagatg aaaacttacg gcaatttgtt gctaacggtg gcactttgat tgtcagtttc 7260
ttcagtggca ttgtagatga aaatgaccgt gtacatctag gcggatatcc tggtcctctg 7320
cgagatattt tggggatttt tgttgaggaa tttgtaccat acccagaaac aaaggtaaac 7380
aaaatatata gtaacgatgg ggaatatgat tgtacgacgt gggcggacat aatccgatta 7440
gaaggggcag aacctctagc gacatttaag ggggattggt atgcaggact tccggcggtt 7500
acacgtaact gctacggtaa aggagagggg atttacgtcg gtacttatcc agatagtaat 7560
tatttaggca ggcttttaga acaggttttc gctaaacatc atattaatcc cattcttgaa 7620
gtagctgaaa atgtagaggt gcaacaaaga gagactgatg aatggaagta tttgattatc 7680
atcaatcata atgattacga agtgacgctg tcactgccag aagataagat ataccagaat 7740
atgattgatg ggaaatgttt tcgaggaggt gaattgagga ttcaaggggt tgatgtagca 7800
gtgttaagag agcatgatga agccgggaag gtttagagaa gtctcgttcc gacagttggc 7860
aacataatat gctaaagggg gtaccganta aaccagcaat agacataagc ggctatttaa 7920
cgaccctgcc ctgaaccgac gaccgggtcg aatttgcttt cgaatttctg ccattcatcc 7980
gcttattatc acttattcag gcgtagcacc aggcgtttaa gggcaccaat aactgcctta 8040
aaaaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct 8100
gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac 8160
cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat 8220
attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa 8280
catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc 8340
ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga 8400
aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac 8460
cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag 8520
aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc 8580
cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc 8640
aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt 8700
ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag 8760
tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt 8820
tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc 8880
tgcgaagtga tcttccgtca caggtattta ttcgaagacg aaaggg 8926
<210> 3
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸A
<400> 3
gcggccgcgc gattatgcca tcgcaa 26
<210> 4
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸B
<400> 4
ctcgagtcca caataggttt gggat 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸C
<400> 5
ctcgaggcgc tgatacttat tacgg 25
<210> 6
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸C
<400> 6
agatctaagc cgcaagggtg aaaa 24
<210> 7
<211> 2328
<212> DNA
<213> 人工序列
<220>
<223> 3934vac
<400> 7
tgggatacga taaaccgcag tcgcagggcg attatgccat cgcaaaacat tttctgcatt 60
gccagcaggc ggtcagcgat ccatatgcgg tatttttaca tgccacgacc cgcgatgata 120
aacactggcc ggaagcaaac tggcgcgagc ttatcggtat ggtgggcaac accggattac 180
ggataaaact tccctggggc gcgcctcatg aggaggcccg ggctaaacga ctggccgaag 240
gctttgacta tgtggatgtg ttaccgcgca tgagcctgga ggaggtcgcc agagtgctgg 300
ctggcgcaaa atttgtcgta tcggttgata ccggcctgag ccatctcacc gccgcgctcg 360
acagaccgaa tattacgcta tatggcccaa cggaccctgg gttaattgga ggttatggga 420
agaaccaaat ggcatgctgc tcaccagaaa aaaacctggc gaatttagat gccacaagcg 480
tatttggaaa gattcattaa agagacactg tctcatccca aacctattgt ggagaaaaga 540
tgctaaccac atcattaacg ttaaataaag agaaatggaa accgatctgg aataaagcgc 600
tggtttttct ttttgttgcc acgtattttc tggatggtat tacgcgttat aaacatttga 660
taatcatact tatggttatc accgcgattt atcaggtctc acgctcaccg aaaagtttcc 720
cccctctttt caaaaatagc gtattttata gcgtagcagt attatcatta atccttgttt 780
attccatact catatcgcca gatatgaaag aaagcttcaa ggaatttgaa aatacggtac 840
tggagggttt cttattatat actttattaa ttcccgtact attaaaagat gaaacaaaag 900
aaacggttgc gaaaatagta cttttctcct ttttaacaag tttaggactt cgctgccttg 960
cagagagtat tctgtatatc gaggactata ataaagggat tatgccattc ataagctatg 1020
cgcatcgaca tatgtccgat tccatggttt tcttatttcc agcattattg aatatttggc 1080
tgtttagaaa aaatgcaatt aagttggttt ttttggtgct tagcgccatc taccttttct 1140
ttatcctggg aaccctatcg cgaggggcat ggttggcggt gcttatagta ggtgttctgt 1200
gggcaatact gaaccgccaa tggaagttaa taggagttgg tgccatttta ttagccatta 1260
tcggcgcttt ggttatcact caacataata acaaaccaga cccagaacat ttactgtata 1320
aattacagca gacagatagc tcatatcgtt atactaacgg aacccagggc accgcgtgga 1380
tactgattca ggaaaacccg atcaagggct acggctatgg taatgatgtg tatgatggtg 1440
tttataataa acgcgttgtc gattatccaa cgtggacctt taaagaatct atcggtccgc 1500
ataataccat tctgtacatc tggtttagtg caggcatatt gggtctggcg agcctggcct 1560
atttatatgg cgctatcatc agggaaacag ccagctctac cttcaggaaa gtagagataa 1620
gcccctacaa tgctcatctc ttgctatttt tatctttcgt cggtttttat atcgttcgtg 1680
gcaattttga acaggtcgat attgctcaaa ttggtatcat tactggtttt ctgttggcgc 1740
taagaaatag ataaaaaaaa cgcgctgata cttattacgg tatcagcgcg ttttccatcg 1800
tccggactca atcacttatc aaaccagctt ttcatctgtt cctcgaaacg ctgagctaca 1860
ttttcccaac tgtattttga aaacaccagg gattttgctt tttcggcaat ctggtggcgt 1920
tccttatcag caagcgcacg gttaatatca ttaattatac tgtcgctcga cataggttct 1980
gcgaggtgat agcccgttat gccatctaac acaaattcgc taatcccccc ttttttgctg 2040
gcaagaaccg cttttcctgc tgccatcgct tctacagcca ccatgcaaaa tgcttcttca 2100
acctgagatg gcacaataac cagatcggct atatgataga agttatgcat ctggtcagga 2160
gattgccccc cagccataat acaatcagtt ccaatctctt ttgcggcgtc cagtactttc 2220
ttttgatact ctgctttttc acccttgcgg cttgcataag gatcgccaac aacgacaagt 2280
ttaatattac ttcttaaggt acgtaatttt ttgaacgcct gcaaaagc 2328
<210> 8
<211> 1106
<212> DNA
<213> 人工序列
<220>
<223> 3934vacDwaaL o 3934vacR
<400> 8
tgggatacga taaaccgcag tcgcagggcg attatgccat cgcaaaacat tttctgcatt 60
gccagcaggc ggtcagcgat ccatatgcgg tatttttaca tgccacgacc cgcgatgata 120
aacactggcc ggaagcaaac tggcgcgagc ttatcggtat ggtgggcaac accggattac 180
ggataaaact tccctggggc gcgcctcatg aggaggcccg ggctaaacga ctggccgaag 240
gctttgacta tgtggatgtg ttaccgcgca tgagcctgga ggaggtcgcc agagtgctgg 300
ctggcgcaaa atttgtcgta tcggttgata ccggcctgag ccatctcacc gccgcgctcg 360
acagaccgaa tattacgcta tatggcccaa cggaccctgg gttaattgga ggttatggga 420
agaaccaaat ggcatgctgc tcaccagaaa aaaacctggc gaatttagat gccacaagcg 480
tatttggaaa gattcattaa agagacactg tctcatccca aacctattgt ggactcgagg 540
cgctgatact tattacggta tcagcgcgtt ttccatcgtc cggactcaat cacttatcaa 600
accagctttt catctgttcc tcgaaacgct gagctacatt ttcccaactg tattttgaaa 660
acaccaggga ttttgctttt tcggcaatct ggtggcgttc cttatcagca agcgcacggt 720
taatatcatt aattatactg tcgctcgaca taggttctgc gaggtgatag cccgttatgc 780
catctaacac aaattcgcta atcccccctt ttttgctggc aagaaccgct tttcctgctg 840
ccatcgcttc tacagccacc atgcaaaatg cttcttcaac ctgagatggc acaataacca 900
gatcggctat atgatagaag ttatgcatct ggtcaggaga ttgcccccca gccataatac 960
aatcagttcc aatctctttt gcggcgtcca gtactttctt ttgatactct gctttttcac 1020
ccttgcggct tgcataagga tcgccaacaa cgacaagttt aatattactt cttaaggtac 1080
gtaatttttt gaacgcctgc aaaagc 1106
<210> 9
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸E
<400> 9
tgggatacga taaaccgc 18
<210> 10
<211> 18
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸F
<400> 10
gcttttgcag gcgttcaa 18
<210> 11
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸G
<400> 11
ctccgtaaca aattgaggat 20
<210> 12
<211> 6663
<212> DNA
<213> 人工序列
<220>
<223> pKO::sb13
<220>
<221> misc_feature
<222> (608)..(608)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (1160)..(1161)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (1164)..(1164)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (3072)..(3073)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (5147)..(5147)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (6219)..(6219)
<223> n是a, c, g, 或t
<400> 12
cagacgcctg ctgatgaact ggctgcaagc cgtactgccc gtgctgcggt ttttatccgt 60
aacgatccgg cgcgcccgac ccagaccggg gagctggtgg acatgctgcc ggcaccgaaa 120
ggcaaacgtt tcacgacgac tgaacagcag acgttacttt cccacggtgt ggcaacggcg 180
tatgtggaaa gcggcgtgct gcgtattcag cgggatatca cgacgtacag gaaaaatgcg 240
tatggtgtgg cggataacag ctaccttgac agcgagacgc tgcataccag tgcttatgtg 300
ttgcgccgtc tgaaatctgt tattaccagt aaatacgggc gccataaact tgctaatgat 360
ggtacgcgtt tcgggtctgg tcaggccatt gtcacgcctg ccgttatccg tggtgagctg 420
ggatcaacat atcgccagat ggagcgggaa ggcatcgtgg aaaacttcga tctgttccag 480
caacatctga tagtggagcg taacgcgaac gattcgaacc gcctggatgt gctgtttccg 540
cctgattatg tcaatcagtt acgtgtgttt gcagtgctta accagttccg tctgcagtgt 600
cgaccggnga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 660
gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 720
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 780
ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 840
ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 900
gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 960
tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 1020
gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 1080
gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 1140
caaatatgta tccgctcatn ncangatcct ttttaaccca tcacatatac ctgccgttca 1200
ctattattta gtgaaatgag atattatgat attttctgaa ttgtgattaa aaaggcaact 1260
ttatgcccat gcaacagaaa ctataaaaaa tacagagaat gaaaagaaac agatagattt 1320
tttagttctt taggcccgta gtctgcaaat ccttttatga ttttctatca aacaaaagag 1380
gaaaatagac cagttgcaat ccaaacgaga gtctaataga atgaggtcga aaagtaaatc 1440
gcgcgggttt gttactgata aagcaggcaa gacctaaaat gtgtaaaggg caaagtgtat 1500
actttggcgt caccccttac atattttagg tcttttttta ttgtgcgtaa ctaacttgcc 1560
atcttcaaac aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac 1620
atgaacgatg aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc 1680
actgctggca ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa 1740
ggaaacatac ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca 1800
aaaaaatgaa aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa atatctcttc 1860
tgcaaaaggc ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc 1920
aaactatcac ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga 1980
cacatcgatt tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa 2040
cgctggccgc gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga 2100
ccaaacacaa gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt 2160
ctacactgat ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa 2220
cgtatcagca tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt 2280
tgacggtgac ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag gcaactacag 2340
ctcaggcgac aaccatacgc tgagagatcc tcactacgta gaagataaag gccacaaata 2400
cttagtattt gaagcaaaca ctggaactga agatggctac caaggcgaag aatctttatt 2460
taacaaagca tactatggca aaagcacatc attcttccgt caagaaagtc aaaaacttct 2520
gcaaagcgat aaaaaacgca cggctgagtt agcaaacggc gctctcggta tgattgagct 2580
aaacgatgat tacacactga aaaaagtgat gaaaccgctg attgcatcta acacagtaac 2640
agatgaaatt gaacgcgcga acgtctttaa aatgaacggc aaatggtacc tgttcactga 2700
ctcccgcgga tcaaaaatga cgattgacgg cattacgtct aacgatattt acatgcttgg 2760
ttatgtttct aattctttaa ctggcccata caagccgctg aacaaaactg gccttgtgtt 2820
aaaaatggat cttgatccta acgatgtaac ctttacttac tcacacttcg ctgtacctca 2880
agcgaaagga aacaatgtcg tgattacaag ctatatgaca aacagaggat tctacgcaga 2940
caaacaatca acgtttgcgc caagcttcct gctgaacatc aaaggcaaga aaacatctgt 3000
tgtcaaagac agcatccttg aacaaggaca attaacagtt aacaaataaa aacgcaaaag 3060
aaaatgccga tnnccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat 3120
gcctcaggct gtctatgtgt gactgttgag ctgtaacaag ttgtctcagg tgttcaattt 3180
catgttctag ttgctttgtt ttactggttt cacctgttct attaggtgtt acatgctgtt 3240
catctgttac attgtcgatc tgttcatggt gaacagcttt aaatgcacca aaaactcgta 3300
aaagctctga tgtatctatc ttttttacac cgttttcatc tgtgcatatg gacagttttc 3360
cctttgatat ctaacggtga acagttgttc tacttttgtt tgttagtctt gatgcttcac 3420
tgatagatac aagagccata agaacctcag atccttccgt atttagccag tatgttctct 3480
agtgtggttc gttgtttttg cgtgagccat gagaacgaac cattgagatc atgcttactt 3540
tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc agttaaagca 3600
tcgtgtagtg tttttcttag tccgttacgt aggtaggaat ctgatgtaat ggttgttggt 3660
attttgtcac cattcatttt tatctggttg ttctcaagtt cggttacgag atccatttgt 3720
ctatctagtt caacttggaa aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc 3780
aatttcatat tgctgtaagt gtttaaatct ttacttattg gtttcaaaac ccattggtta 3840
agccttttaa actcatggta gttattttca agcattaaca tgaacttaaa ttcatcaagg 3900
ctaatctcta tatttgcctt gtgagttttc ttttgtgtta gttcttttaa taaccactca 3960
taaatcctca tagagtattt gttttcaaaa gacttaacat gttccagatt atattttatg 4020
aattttttta actggaaaag ataaggcaat atctcttcac taaaaactaa ttctaatttt 4080
tcgcttgaga acttggcata gtttgtccac tggaaaatct caaagccttt aaccaaagga 4140
ttcctgattt ccacagttct cgtcatcagc tctctggttg ctttagctaa tacaccataa 4200
gcattttccc tactgatgtt catcatctga gcgtattggt tataagtgaa cgataccgtc 4260
cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt 4320
agcttggttt catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa 4380
acaactaatt cagacataca tctcaattgg tctaggtgat tttaatcact ataccaattg 4440
agatgggcta gtcaatgata attactagtc cttttccttt gagttgtggg tatctgtaaa 4500
ttctgctaga cctttgctgg aaaacttgta aattctgcta gaccctctgt aaattccgct 4560
agacctttgt gtgttttttt tgtttatatt caagtggtta taatttatag aataaagaaa 4620
gaataaaaaa agataaaaag aatagatccc agccctgtgt ataactcact actttagtca 4680
gttccgcagt attacaaaag gatgtcgcaa acgctgtttg ctcctctaca aaacagacct 4740
taaaacccta aaggcttaag tagcaccctc gcaagctcgg gcaaatcgct gaatattcct 4800
tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc 4860
gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca 4920
tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat 4980
ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga 5040
ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac 5100
ccagtaaggc agcggtatca tcaacaggct tacccgtctt actgtcngga tcgacgctct 5160
cccttatgcg actcctgcat ccctttcgtc ttcgaataaa tacctgtgac ggaagatcac 5220
ttcgcagaat aaataaatcc tggtgtccct gttgataccg ggaagccctg ggccaacttt 5280
tggcgaaaat gagacgttga tcggcacgta agaggttcca actttcacca taatgaaata 5340
agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa 5400
atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 5460
cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 5520
attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 5580
cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt 5640
gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 5700
acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 5760
tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 5820
aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 5880
gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 5940
gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 6000
gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 6060
tttttttaag gcagttattg gtgcccttaa acgcctggtg ctacgcctga ataagtgata 6120
ataagcggat gaatggcaga aattcgaaag caaattcgac ccggtcgtcg gttcagggca 6180
gggtcgttaa atagccgctt atgtctattg ctggtttant cggtacccgg gaactgtatg 6240
tcattgccgt acctgaatcc acaggcgcgg cagcaaccgt cgctttgacg gtaactggcg 6300
aagcgacgga aaccggaacg gtgaatgtct ataccggccg aacccgcgtt caggctcccg 6360
tgaccagcgg tgatgacgct gcggcggtgg ctgtgagcat taaggatgcg gtcaatgcaa 6420
accctgatct tccctttacg gcaacatcag aagcgggggt ggtgacactg actgcgcgcc 6480
acaaggggtt atatggaaat gaaattccgg tcactctcaa ttattacggc tttggcggtg 6540
gggaggtgtt accggcgggt gtgaatatta cggttgccag cggcgtgaag ggggctggtg 6600
cgccagctct taacgacgcg gtggcagcga tgggagatga gccgttcgat tatatcgggc 6660
atc 6663
<210> 13
<211> 26
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸H
<400> 13
cccgggaact gtatgtcatt gccgta 26
<210> 14
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸I
<400> 14
gcatgcccga tataatcgaa cggct 25
<210> 15
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸J
<400> 15
gcatgcagac gcctgctgat gaact 25
<210> 16
<211> 25
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸K
<400> 16
gtcgactgca gacggaactg gttaa 25
<210> 17
<211> 49
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸L
<400> 17
ttcgattata tcgggcatgc taaaacgaaa ggctcagtcg aaagactgg 49
<210> 18
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸M
<400> 18
ggagcatgac gtcaggaacc tcgaaaag 28
<210> 19
<211> 28
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸N
<400> 19
ctgacgtcat gctccgggcc cctaaaag 28
<210> 20
<211> 41
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸O
<400> 20
tcagcaggcg tctgcatgcc aaaaaacccc tcaagacccg t 41
<210> 21
<211> 2702
<212> DNA
<213> 人工序列
<220>
<223> 纤维表达盒
<400> 21
ttcgattata tcgggcatgc taaaacgaaa ggctcagtcg aaagactggg cctttcgttt 60
tataaatcta tcaccgcaag ggataaatat ctaacaccgt gcgtgttgac tattttacct 120
ctggcggtga taatggttgc atgtactaga attcattaaa gaggagaaag gtaccgcatg 180
attatgaccg gaatatttgc agaacaaact gtagaggtag ttaaaagcgc gatcgaaacc 240
gcagatgggg cattagatct ttataacaaa tacctcgacc aggtcatccc ctggaagacc 300
tttgatgaaa ccataaaaga gttaagccgt tttaaacagg agtactcgca ggaagcttct 360
gttttagttg gtgatattaa agttttgctt atggacagcc aggacaagta ttttgaagcg 420
acacaaactg tttatgaatg gtgtggtgtc gtgacgcaat tactctcagc gtatatttta 480
ctatttgatg aatataatga gaaaaaagca tcagcccaga aagacattct cattaggata 540
ttagatgatg gtgtcaagaa actgaatgaa gcgcaaaaat ctctcctgac aagttcacaa 600
agtttcaaca acgcttccgg aaaactgctg gcattagata gccagttaac taatgatttt 660
tcggaaaaaa gtagttattt ccagtcacag gtggatagaa ttcgtaagga agcttatgcc 720
ggtgctgcag ccggcatagt cgccggtccg tttggattaa ttatttccta ttctattgct 780
gcgggcgtga ttgaagggaa attgattcca gaattgaata acaggctaaa aacagtgcaa 840
aatttcttta ctagcttatc agctacagtg aaacaagcga ataaagatat cgatgcggca 900
aaattgaaat tagccactga aatagcagca attggggaga taaaaacgga aaccgaaaca 960
accagattct acgttgatta tgatgattta atgctttctt tattaaaagg agctgcaaag 1020
aaaatgatta acacctgtaa tgaataccaa caaagacacg gtaagaagac gcttttcgag 1080
gttcctgacg tcatgctccg ggcccctaaa agaagacatt ccgaaaacgg gaagcccgag 1140
accgaagcgg gaccttcccc ggctccaatc aagcgcgcca aacgcatggt gagagcatcc 1200
cagcttgacc tggtttatcc tttcgattac gtggccgacc ccgtcggagg gctcaacccg 1260
ccttttttgg gtggctccgg acccctagtg gaccagggcg gtcagcttac gctcaacgtc 1320
accgatccca tcatcatcaa gaacagatcg gtggacttgg cccacgaccc cagtctcgat 1380
gtcaacgccc aaggtcaact ggcggtggcc gttgaccccg aaggggccct ggacattacc 1440
cccgatggac tggacgtcaa ggtcgacgga gtgaccgtaa tggtcaacga tgactgggaa 1500
ctggccgtaa aagtcgaccc gtccggtgga ttggattcca ccgcgggtgg actgggggtc 1560
agcgtggacg acaccttgct cgtggatcag ggagaactgg gcgtacacct caaccaacaa 1620
ggacccatca ctgccgatag cagtggtatc gacctcgaga tcaatcctaa catgttcacg 1680
gtcaacacct cgaccggaag cggagtgctg gaactcaacc taaaagcgca gggaggcatc 1740
caagccgaca gttcgggagt gggcgtttcc gtggatgaaa gcctagagat tgtcaacaac 1800
acgctggaag tgaaaccgga tcccagcgga ccgctgacgg tctccgccaa tggcctaggg 1860
ctgaagtacg acactaatac cctagcggtg accgcgggcg ctttaaccgt ggtcggaggg 1920
gggagcgtct ccacacccat cgctactttt gtatcgggaa gtcccagcct caacacctac 1980
aatgccacga ccgtcaattc cagcgcgaac gccttctctt gcgcctacta ccttcaacag 2040
tggaacatac aggggctcct tgttacctcc ctctacttga aattggacag cgccaccatg 2100
gggaatcgcc ctggggacct caactccgcc aatgccaaat ggttcacctt ttgggtgtcc 2160
gcctatctcc agcaatgcaa cccctccggg attcaagcgg gaacggtcag cccctccacc 2220
gccaccctca cggactttga acccatggcc aataggagcg tgaccagccc atggacgtac 2280
tcggccaatg gatactatga accatccatc ggggaattcc aagtgttcag cccggtggta 2340
acaggtgcct ggaacccggg aaacataggg atccgcgtcc tcccagtgcc ggttacggcc 2400
tctggagacc gctacaccct tctatgctac agtttgcagt gcacgaactc gagcattttt 2460
aatccagcca acagcggaac tatgatcgtg ggacccgtgc tctacagctg tccagcagcc 2520
tccgtcccga agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct 2580
gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 2640
taaccccttg gggcctctaa acgggtcttg aggggttttt tggcatgcag acgcctgctg 2700
at 2702
<210> 22
<211> 9331
<212> DNA
<213> 人工序列
<220>
<223> pKO::sb13-纤维
<220>
<221> misc_feature
<222> (608)..(608)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (1160)..(1161)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (1164)..(1164)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (3072)..(3073)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (5147)..(5147)
<223> n是a, c, g, 或t
<220>
<221> misc_feature
<222> (6219)..(6219)
<223> n是a, c, g, 或t
<400> 22
cagacgcctg ctgatgaact ggctgcaagc cgtactgccc gtgctgcggt ttttatccgt 60
aacgatccgg cgcgcccgac ccagaccggg gagctggtgg acatgctgcc ggcaccgaaa 120
ggcaaacgtt tcacgacgac tgaacagcag acgttacttt cccacggtgt ggcaacggcg 180
tatgtggaaa gcggcgtgct gcgtattcag cgggatatca cgacgtacag gaaaaatgcg 240
tatggtgtgg cggataacag ctaccttgac agcgagacgc tgcataccag tgcttatgtg 300
ttgcgccgtc tgaaatctgt tattaccagt aaatacgggc gccataaact tgctaatgat 360
ggtacgcgtt tcgggtctgg tcaggccatt gtcacgcctg ccgttatccg tggtgagctg 420
ggatcaacat atcgccagat ggagcgggaa ggcatcgtgg aaaacttcga tctgttccag 480
caacatctga tagtggagcg taacgcgaac gattcgaacc gcctggatgt gctgtttccg 540
cctgattatg tcaatcagtt acgtgtgttt gcagtgctta accagttccg tctgcagtgt 600
cgaccggnga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 660
gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 720
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 780
ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 840
ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 900
gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 960
tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 1020
gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 1080
gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 1140
caaatatgta tccgctcatn ncangatcct ttttaaccca tcacatatac ctgccgttca 1200
ctattattta gtgaaatgag atattatgat attttctgaa ttgtgattaa aaaggcaact 1260
ttatgcccat gcaacagaaa ctataaaaaa tacagagaat gaaaagaaac agatagattt 1320
tttagttctt taggcccgta gtctgcaaat ccttttatga ttttctatca aacaaaagag 1380
gaaaatagac cagttgcaat ccaaacgaga gtctaataga atgaggtcga aaagtaaatc 1440
gcgcgggttt gttactgata aagcaggcaa gacctaaaat gtgtaaaggg caaagtgtat 1500
actttggcgt caccccttac atattttagg tcttttttta ttgtgcgtaa ctaacttgcc 1560
atcttcaaac aggagggctg gaagaagcag accgctaaca cagtacataa aaaaggagac 1620
atgaacgatg aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc 1680
actgctggca ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa 1740
ggaaacatac ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca 1800
aaaaaatgaa aaatatcaag ttcctgaatt cgattcgtcc acaattaaaa atatctcttc 1860
tgcaaaaggc ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc 1920
aaactatcac ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga 1980
cacatcgatt tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa 2040
cgctggccgc gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga 2100
ccaaacacaa gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt 2160
ctacactgat ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa 2220
cgtatcagca tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt 2280
tgacggtgac ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag gcaactacag 2340
ctcaggcgac aaccatacgc tgagagatcc tcactacgta gaagataaag gccacaaata 2400
cttagtattt gaagcaaaca ctggaactga agatggctac caaggcgaag aatctttatt 2460
taacaaagca tactatggca aaagcacatc attcttccgt caagaaagtc aaaaacttct 2520
gcaaagcgat aaaaaacgca cggctgagtt agcaaacggc gctctcggta tgattgagct 2580
aaacgatgat tacacactga aaaaagtgat gaaaccgctg attgcatcta acacagtaac 2640
agatgaaatt gaacgcgcga acgtctttaa aatgaacggc aaatggtacc tgttcactga 2700
ctcccgcgga tcaaaaatga cgattgacgg cattacgtct aacgatattt acatgcttgg 2760
ttatgtttct aattctttaa ctggcccata caagccgctg aacaaaactg gccttgtgtt 2820
aaaaatggat cttgatccta acgatgtaac ctttacttac tcacacttcg ctgtacctca 2880
agcgaaagga aacaatgtcg tgattacaag ctatatgaca aacagaggat tctacgcaga 2940
caaacaatca acgtttgcgc caagcttcct gctgaacatc aaaggcaaga aaacatctgt 3000
tgtcaaagac agcatccttg aacaaggaca attaacagtt aacaaataaa aacgcaaaag 3060
aaaatgccga tnnccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat 3120
gcctcaggct gtctatgtgt gactgttgag ctgtaacaag ttgtctcagg tgttcaattt 3180
catgttctag ttgctttgtt ttactggttt cacctgttct attaggtgtt acatgctgtt 3240
catctgttac attgtcgatc tgttcatggt gaacagcttt aaatgcacca aaaactcgta 3300
aaagctctga tgtatctatc ttttttacac cgttttcatc tgtgcatatg gacagttttc 3360
cctttgatat ctaacggtga acagttgttc tacttttgtt tgttagtctt gatgcttcac 3420
tgatagatac aagagccata agaacctcag atccttccgt atttagccag tatgttctct 3480
agtgtggttc gttgtttttg cgtgagccat gagaacgaac cattgagatc atgcttactt 3540
tgcatgtcac tcaaaaattt tgcctcaaaa ctggtgagct gaatttttgc agttaaagca 3600
tcgtgtagtg tttttcttag tccgttacgt aggtaggaat ctgatgtaat ggttgttggt 3660
attttgtcac cattcatttt tatctggttg ttctcaagtt cggttacgag atccatttgt 3720
ctatctagtt caacttggaa aatcaacgta tcagtcgggc ggcctcgctt atcaaccacc 3780
aatttcatat tgctgtaagt gtttaaatct ttacttattg gtttcaaaac ccattggtta 3840
agccttttaa actcatggta gttattttca agcattaaca tgaacttaaa ttcatcaagg 3900
ctaatctcta tatttgcctt gtgagttttc ttttgtgtta gttcttttaa taaccactca 3960
taaatcctca tagagtattt gttttcaaaa gacttaacat gttccagatt atattttatg 4020
aattttttta actggaaaag ataaggcaat atctcttcac taaaaactaa ttctaatttt 4080
tcgcttgaga acttggcata gtttgtccac tggaaaatct caaagccttt aaccaaagga 4140
ttcctgattt ccacagttct cgtcatcagc tctctggttg ctttagctaa tacaccataa 4200
gcattttccc tactgatgtt catcatctga gcgtattggt tataagtgaa cgataccgtc 4260
cgttctttcc ttgtagggtt ttcaatcgtg gggttgagta gtgccacaca gcataaaatt 4320
agcttggttt catgctccgt taagtcatag cgactaatcg ctagttcatt tgctttgaaa 4380
acaactaatt cagacataca tctcaattgg tctaggtgat tttaatcact ataccaattg 4440
agatgggcta gtcaatgata attactagtc cttttccttt gagttgtggg tatctgtaaa 4500
ttctgctaga cctttgctgg aaaacttgta aattctgcta gaccctctgt aaattccgct 4560
agacctttgt gtgttttttt tgtttatatt caagtggtta taatttatag aataaagaaa 4620
gaataaaaaa agataaaaag aatagatccc agccctgtgt ataactcact actttagtca 4680
gttccgcagt attacaaaag gatgtcgcaa acgctgtttg ctcctctaca aaacagacct 4740
taaaacccta aaggcttaag tagcaccctc gcaagctcgg gcaaatcgct gaatattcct 4800
tttgtctccg accatcaggc acctgagtcg ctgtcttttt cgtgacattc agttcgctgc 4860
gctcacggct ctggcagtga atgggggtaa atggcactac aggcgccttt tatggattca 4920
tgcaaggaaa ctacccataa tacaagaaaa gcccgtcacg ggcttctcag ggcgttttat 4980
ggcgggtctg ctatgtggtg ctatctgact ttttgctgtt cagcagttcc tgccctctga 5040
ttttccagtc tgaccacttc ggattatccc gtgacaggtc attcagactg gctaatgcac 5100
ccagtaaggc agcggtatca tcaacaggct tacccgtctt actgtcngga tcgacgctct 5160
cccttatgcg actcctgcat ccctttcgtc ttcgaataaa tacctgtgac ggaagatcac 5220
ttcgcagaat aaataaatcc tggtgtccct gttgataccg ggaagccctg ggccaacttt 5280
tggcgaaaat gagacgttga tcggcacgta agaggttcca actttcacca taatgaaata 5340
agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa 5400
atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 5460
cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 5520
attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 5580
cacattcttg cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt 5640
gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 5700
acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 5760
tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 5820
aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 5880
gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 5940
gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 6000
gtcggcagaa tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa 6060
tttttttaag gcagttattg gtgcccttaa acgcctggtg ctacgcctga ataagtgata 6120
ataagcggat gaatggcaga aattcgaaag caaattcgac ccggtcgtcg gttcagggca 6180
gggtcgttaa atagccgctt atgtctattg ctggtttant cggtacccgg gaactgtatg 6240
tcattgccgt acctgaatcc acaggcgcgg cagcaaccgt cgctttgacg gtaactggcg 6300
aagcgacgga aaccggaacg gtgaatgtct ataccggccg aacccgcgtt caggctcccg 6360
tgaccagcgg tgatgacgct gcggcggtgg ctgtgagcat taaggatgcg gtcaatgcaa 6420
accctgatct tccctttacg gcaacatcag aagcgggggt ggtgacactg actgcgcgcc 6480
acaaggggtt atatggaaat gaaattccgg tcactctcaa ttattacggc tttggcggtg 6540
gggaggtgtt accggcgggt gtgaatatta cggttgccag cggcgtgaag ggggctggtg 6600
cgccagctct taacgacgcg gtggcagcga tgggagatga gccgttcgat tatatcgggc 6660
atgctaaaac gaaaggctca gtcgaaagac tgggcctttc gttttataaa tctatcaccg 6720
caagggataa atatctaaca ccgtgcgtgt tgactatttt acctctggcg gtgataatgg 6780
ttgcatgtac tagaattcat taaagaggag aaaggtaccg catgattatg accggaatat 6840
ttgcagaaca aactgtagag gtagttaaaa gcgcgatcga aaccgcagat ggggcattag 6900
atctttataa caaatacctc gaccaggtca tcccctggaa gacctttgat gaaaccataa 6960
aagagttaag ccgttttaaa caggagtact cgcaggaagc ttctgtttta gttggtgata 7020
ttaaagtttt gcttatggac agccaggaca agtattttga agcgacacaa actgtttatg 7080
aatggtgtgg tgtcgtgacg caattactct cagcgtatat tttactattt gatgaatata 7140
atgagaaaaa agcatcagcc cagaaagaca ttctcattag gatattagat gatggtgtca 7200
agaaactgaa tgaagcgcaa aaatctctcc tgacaagttc acaaagtttc aacaacgctt 7260
ccggaaaact gctggcatta gatagccagt taactaatga tttttcggaa aaaagtagtt 7320
atttccagtc acaggtggat agaattcgta aggaagctta tgccggtgct gcagccggca 7380
tagtcgccgg tccgtttgga ttaattattt cctattctat tgctgcgggc gtgattgaag 7440
ggaaattgat tccagaattg aataacaggc taaaaacagt gcaaaatttc tttactagct 7500
tatcagctac agtgaaacaa gcgaataaag atatcgatgc ggcaaaattg aaattagcca 7560
ctgaaatagc agcaattggg gagataaaaa cggaaaccga aacaaccaga ttctacgttg 7620
attatgatga tttaatgctt tctttattaa aaggagctgc aaagaaaatg attaacacct 7680
gtaatgaata ccaacaaaga cacggtaaga agacgctttt cgaggttcct gacgtcatgc 7740
tccgggcccc taaaagaaga cattccgaaa acgggaagcc cgagaccgaa gcgggacctt 7800
ccccggctcc aatcaagcgc gccaaacgca tggtgagagc atcccagctt gacctggttt 7860
atcctttcga ttacgtggcc gaccccgtcg gagggctcaa cccgcctttt ttgggtggct 7920
ccggacccct agtggaccag ggcggtcagc ttacgctcaa cgtcaccgat cccatcatca 7980
tcaagaacag atcggtggac ttggcccacg accccagtct cgatgtcaac gcccaaggtc 8040
aactggcggt ggccgttgac cccgaagggg ccctggacat tacccccgat ggactggacg 8100
tcaaggtcga cggagtgacc gtaatggtca acgatgactg ggaactggcc gtaaaagtcg 8160
acccgtccgg tggattggat tccaccgcgg gtggactggg ggtcagcgtg gacgacacct 8220
tgctcgtgga tcagggagaa ctgggcgtac acctcaacca acaaggaccc atcactgccg 8280
atagcagtgg tatcgacctc gagatcaatc ctaacatgtt cacggtcaac acctcgaccg 8340
gaagcggagt gctggaactc aacctaaaag cgcagggagg catccaagcc gacagttcgg 8400
gagtgggcgt ttccgtggat gaaagcctag agattgtcaa caacacgctg gaagtgaaac 8460
cggatcccag cggaccgctg acggtctccg ccaatggcct agggctgaag tacgacacta 8520
ataccctagc ggtgaccgcg ggcgctttaa ccgtggtcgg aggggggagc gtctccacac 8580
ccatcgctac ttttgtatcg ggaagtccca gcctcaacac ctacaatgcc acgaccgtca 8640
attccagcgc gaacgccttc tcttgcgcct actaccttca acagtggaac atacaggggc 8700
tccttgttac ctccctctac ttgaaattgg acagcgccac catggggaat cgccctgggg 8760
acctcaactc cgccaatgcc aaatggttca ccttttgggt gtccgcctat ctccagcaat 8820
gcaacccctc cgggattcaa gcgggaacgg tcagcccctc caccgccacc ctcacggact 8880
ttgaacccat ggccaatagg agcgtgacca gcccatggac gtactcggcc aatggatact 8940
atgaaccatc catcggggaa ttccaagtgt tcagcccggt ggtaacaggt gcctggaacc 9000
cgggaaacat agggatccgc gtcctcccag tgccggttac ggcctctgga gaccgctaca 9060
cccttctatg ctacagtttg cagtgcacga actcgagcat ttttaatcca gccaacagcg 9120
gaactatgat cgtgggaccc gtgctctaca gctgtccagc agcctccgtc ccgaagcttg 9180
cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa 9240
aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct 9300
ctaaacgggt cttgaggggt tttttggcat g 9331
<210> 23
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸P
<400> 23
atcggttgat tatgcccgtc a 21
<210> 24
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 寡核苷酸Q
<400> 24
caacacataa gcactggtat 20
<210> 25
<211> 1314
<212> DNA
<213> 人工序列
<220>
<223> 3934vacR
<400> 25
atcggttgat tatgcccgtc agatttgcgg tgccggaagc cagctggccc gtatggtcgg 60
ggcgtaccgt aagaccgatc catttggcga actgtatgtc attgccgtac ctgaatccac 120
aggcgcggca gcaaccgtcg ctttgacggt aactggcgaa gcgacggaaa ccggaacggt 180
gaatgtctat accggccgaa cccgcgttca ggctcccgtg accagcggtg atgacgctgc 240
ggcggtggct gtgagcatta aggatgcggt caatgcaaac cctgatcttc cctttacggc 300
aacatcagaa gcgggggtgg tgacactgac tgcgcgccac aaggggttat atggaaatga 360
aattccggtc actctcaatt attacggctt tggcggtggg gaggtgttac cggcgggtgt 420
gaatattacg gttgccagcg gcgtgaaggg ggctggtgcg ccagctctta acgacgcggt 480
ggcagcgatg ggagatgagc cgttcgatta tatcggcctt ccgtttaacg acacggcatc 540
ggtgaactcg atggcaactg aaatgaatga ttccagcggt cgctggagtt atgtccggca 600
gttgtatggt cacgtttata cggcgaagac ggggactctg tcggagcttg tggccgcggg 660
tgaccagttt aacctgcagc acatcaccct ggcgggctat gagaaagaca cccagacgcc 720
tgctgatgaa ctggctgcaa gccgtactgc ccgtgctgcg gtttttatcc gtaacgatcc 780
ggcgcgcccg acccagaccg gggagctggt ggacatgctg ccggcaccga aaggcaaacg 840
tttcacgacg actgaacagc agacgttact ttcccacggt gtggcaacgg cgtatgtgga 900
aagcggcgtg ctgcgtattc agcgggatat cacgacgtac aggaaaaatg cgtatggtgt 960
ggcggataac agctaccttg acagcgagac gctgcatacc agtgcttatg tgttgcgccg 1020
tctgaaatct gttattacca gtaaatacgg gcgccataaa cttgctaatg atggtacgcg 1080
tttcgggtct ggtcaggcca ttgtcacgcc tgccgttatc cgtggtgagc tgggatcaac 1140
atatcgccag atggagcggg aaggcatcgt ggaaaacttc gatctgttcc agcaacatct 1200
gatagtggag cgtaacgcga acgattcgaa ccgcctggat gtgctgtttc cgcctgatta 1260
tgtcaatcag ttacgtgtgt ttgcagtgct taaccagttc cgtctgcagt cgac 1314
<210> 26
<211> 3792
<212> DNA
<213> 人工序列
<220>
<223> 3934vacR::纤维 (AVA-I)
<400> 26
atcggttgat tatgcccgtc agatttgcgg tgccggaagc cagctggccc gtatggtcgg 60
ggcgtaccgt aagaccgatc catttggcga actgtatgtc attgccgtac ctgaatccac 120
aggcgcggca gcaaccgtcg ctttgacggt aactggcgaa gcgacggaaa ccggaacggt 180
gaatgtctat accggccgaa cccgcgttca ggctcccgtg accagcggtg atgacgctgc 240
ggcggtggct gtgagcatta aggatgcggt caatgcaaac cctgatcttc cctttacggc 300
aacatcagaa gcgggggtgg tgacactgac tgcgcgccac aaggggttat atggaaatga 360
aattccggtc actctcaatt attacggctt tggcggtggg gaggtgttac cggcgggtgt 420
gaatattacg gttgccagcg gcgtgaaggg ggctggtgcg ccagctctta acgacgcggt 480
ggcagcgatg ggagatgagc cgttcgatta tatcgggcat gctaaaacga aaggctcagt 540
cgaaagactg ggcctttcgt tttataaatc tatcaccgca agggataaat atctaacacc 600
gtgcgtgttg actattttac ctctggcggt gataatggtt gcatgtacta gaattcatta 660
aagaggagaa aggtaccgca tgattatgac cggaatattt gcagaacaaa ctgtagaggt 720
agttaaaagc gcgatcgaaa ccgcagatgg ggcattagat ctttataaca aatacctcga 780
ccaggtcatc ccctggaaga cctttgatga aaccataaaa gagttaagcc gttttaaaca 840
ggagtactcg caggaagctt ctgttttagt tggtgatatt aaagttttgc ttatggacag 900
ccaggacaag tattttgaag cgacacaaac tgtttatgaa tggtgtggtg tcgtgacgca 960
attactctca gcgtatattt tactatttga tgaatataat gagaaaaaag catcagccca 1020
gaaagacatt ctcattagga tattagatga tggtgtcaag aaactgaatg aagcgcaaaa 1080
atctctcctg acaagttcac aaagtttcaa caacgcttcc ggaaaactgc tggcattaga 1140
tagccagtta actaatgatt tttcggaaaa aagtagttat ttccagtcac aggtggatag 1200
aattcgtaag gaagcttatg ccggtgctgc agccggcata gtcgccggtc cgtttggatt 1260
aattatttcc tattctattg ctgcgggcgt gattgaaggg aaattgattc cagaattgaa 1320
taacaggcta aaaacagtgc aaaatttctt tactagctta tcagctacag tgaaacaagc 1380
gaataaagat atcgatgcgg caaaattgaa attagccact gaaatagcag caattgggga 1440
gataaaaacg gaaaccgaaa caaccagatt ctacgttgat tatgatgatt taatgctttc 1500
tttattaaaa ggagctgcaa agaaaatgat taacacctgt aatgaatacc aacaaagaca 1560
cggtaagaag acgcttttcg aggttcctga cgtcatgctc cgggccccta aaagaagaca 1620
ttccgaaaac gggaagcccg agaccgaagc gggaccttcc ccggctccaa tcaagcgcgc 1680
caaacgcatg gtgagagcat cccagcttga cctggtttat cctttcgatt acgtggccga 1740
ccccgtcgga gggctcaacc cgcctttttt gggtggctcc ggacccctag tggaccaggg 1800
cggtcagctt acgctcaacg tcaccgatcc catcatcatc aagaacagat cggtggactt 1860
ggcccacgac cccagtctcg atgtcaacgc ccaaggtcaa ctggcggtgg ccgttgaccc 1920
cgaaggggcc ctggacatta cccccgatgg actggacgtc aaggtcgacg gagtgaccgt 1980
aatggtcaac gatgactggg aactggccgt aaaagtcgac ccgtccggtg gattggattc 2040
caccgcgggt ggactggggg tcagcgtgga cgacaccttg ctcgtggatc agggagaact 2100
gggcgtacac ctcaaccaac aaggacccat cactgccgat agcagtggta tcgacctcga 2160
gatcaatcct aacatgttca cggtcaacac ctcgaccgga agcggagtgc tggaactcaa 2220
cctaaaagcg cagggaggca tccaagccga cagttcggga gtgggcgttt ccgtggatga 2280
aagcctagag attgtcaaca acacgctgga agtgaaaccg gatcccagcg gaccgctgac 2340
ggtctccgcc aatggcctag ggctgaagta cgacactaat accctagcgg tgaccgcggg 2400
cgctttaacc gtggtcggag gggggagcgt ctccacaccc atcgctactt ttgtatcggg 2460
aagtcccagc ctcaacacct acaatgccac gaccgtcaat tccagcgcga acgccttctc 2520
ttgcgcctac taccttcaac agtggaacat acaggggctc cttgttacct ccctctactt 2580
gaaattggac agcgccacca tggggaatcg ccctggggac ctcaactccg ccaatgccaa 2640
atggttcacc ttttgggtgt ccgcctatct ccagcaatgc aacccctccg ggattcaagc 2700
gggaacggtc agcccctcca ccgccaccct cacggacttt gaacccatgg ccaataggag 2760
cgtgaccagc ccatggacgt actcggccaa tggatactat gaaccatcca tcggggaatt 2820
ccaagtgttc agcccggtgg taacaggtgc ctggaacccg ggaaacatag ggatccgcgt 2880
cctcccagtg ccggttacgg cctctggaga ccgctacacc cttctatgct acagtttgca 2940
gtgcacgaac tcgagcattt ttaatccagc caacagcgga actatgatcg tgggacccgt 3000
gctctacagc tgtccagcag cctccgtccc gaagcttgcg gccgcactcg agcaccacca 3060
ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 3120
caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt 3180
tttggcatgc cagacgcctg ctgatgaact ggctgcaagc cgtactgccc gtgctgcggt 3240
ttttatccgt aacgatccgg cgcgcccgac ccagaccggg gagctggtgg acatgctgcc 3300
ggcaccgaaa ggcaaacgtt tcacgacgac tgaacagcag acgttacttt cccacggtgt 3360
ggcaacggcg tatgtggaaa gcggcgtgct gcgtattcag cgggatatca cgacgtacag 3420
gaaaaatgcg tatggtgtgg cggataacag ctaccttgac agcgagacgc tgcataccag 3480
tgcttatgtg ttgcgccgtc tgaaatctgt tattaccagt aaatacgggc gccataaact 3540
tgctaatgat ggtacgcgtt tcgggtctgg tcaggccatt gtcacgcctg ccgttatccg 3600
tggtgagctg ggatcaacat atcgccagat ggagcgggaa ggcatcgtgg aaaacttcga 3660
tctgttccag caacatctga tagtggagcg taacgcgaac gattcgaacc gcctggatgt 3720
gctgtttccg cctgattatg tcaatcagtt acgtgtgttt gcagtgctta accagttccg 3780
tctgcagtcg ac 3792
Claims (9)
1.肠炎沙门氏菌3934vac突变株,其中包含waaL基因的缺失。
2.肠炎沙门氏菌3934vac突变株的产生方法,其中包括:
·构建一个整合载体,构建时通过PCR分别扩增waaL基因两侧的两个片段,一个520bp(寡核苷酸A和B),另一个504bp(寡核苷酸C和D),其中寡核苷酸A、B、C和D为序列SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6
·分别在pKOB载体中提纯和克隆PCR产物以进行扩增,以获得用于扩增的pKOB::DwaaLR1载体,然后酶促分解、提纯并结合到pDESTINATION载体中。
·将在大肠杆菌中产生的载体pDEST::waaL进行转化,并通过PCR、微量制备和分解进行验证;一旦构建完成,pDEST::waaL质粒将在肠炎沙门氏菌3934vac和3934vac sb13::clyA-fiberFAdV菌株中进行电穿孔,在42℃下的生长及之后在28℃下的切除促使其整合。
·通过带有结构外部的寡核苷酸(waaL E和F)的PCR双同源重组验证waaL基因的缺失,其中寡核苷酸E和F为SEQ ID NO:9和SEQ ID NO:10。
3.肠炎沙门氏菌重组禽类疫苗,其中包含肠炎沙门氏菌3934vac经过修改的突变株,waaL基因的缺失。
4.肠炎沙门氏菌3934vac突变株,其中包含AVA-I纤维基因的完整序列和waaL基因的缺失。
5.产生肠炎沙门氏菌3934vac突变株的方法,其中包括:
·拥有一个肠炎沙门氏菌3934菌株。
·通过同源重组的第一步插入选择盒,其中除抗生素抗性基因外,该盒在其侧翼区域还包含与前噬菌体ST64B互补的序列;在携带同源重组的第一步中选择盒的线性DNA存在下进行细菌电穿孔。
·同源重组第一步后,在含有抗生素的LB培养基中选择转化菌落。
·通过带有序列SEQ ID NO:23、SEQ ID NO:24和SEQ ID NO:25寡核苷酸的PCR,确认肠炎沙门氏菌3934染色体中盒体的存在。
·通过同源重组的第二步插入ClyA-fiberFAdV-His表达盒,其中表达盒替换选择盒,表达盒包含序列SEQ ID NO:21
·通过蛋白质印迹和SDS-PAGE验证序列SEQ ID NO:21的表达产物,选择肠炎沙门氏菌3934vac sb13::clyA-fiberFAdV菌株。
6.根据声明5的方法,其中同样包括:
·构建一个整合载体,其中两个位于waaL基因两侧的片段,一个为520bp基因(分别为序列SEQ ID NO:3和SEQ ID NO:4的寡核苷酸A和B),另一个为504bp基因(别为序列SEQ IDNO:5和SEQ ID NO:6的寡核苷酸C和D)通过PCR扩增以构建整合载体。
·分别在pKOB载体提纯和克隆PCR产物进行扩增,以获得载体pKOB::DwaaLR1进行扩增,然后酶促分解、提纯并结合到pDESTINATION载体中。
·将在大肠杆菌中产生的载体pDEST::waaL进行转化,并通过PCR、微量制备和分解进行验证;一旦构建完成,pDEST::waaL质粒将在肠炎沙门氏菌3934vac和3934vac sb13::clyA-fiberFAdV菌株中进行电穿孔,在42℃下的生长及之后在28℃下的切除促使其整合。
·通过带有结构外部的寡核苷酸SEQ ID NO:9和SEQ ID NO:10的PCR验证通过双同源重组的waaL基因的缺失。
7.肠炎沙门氏菌的重组禽类疫苗,其中包含带有AVA-I纤维基因序列和waaL基因缺失的肠炎沙门氏菌3934vac经过修改的突变株。
8.肠炎沙门氏菌3934vac突变株,其中包含表达盒SEQ ID NO:21和通过序列SEQ IDNO:8的存在验证的waaL的缺失。
9.肠炎沙门氏菌3934vac的突变株,其中包含序列SEQ ID NO:26和SEQ ID NO:8。
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