CN101838627A - 重组猪霍乱沙门氏菌及二价基因工程疫苗与制备方法 - Google Patents
重组猪霍乱沙门氏菌及二价基因工程疫苗与制备方法 Download PDFInfo
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Abstract
本发明属于动物细菌基因工程技术领域，具体涉及一种不含抗性标记的表达猪II型圆环病毒主要抗原Cap蛋白的重组猪霍乱沙门氏菌株的构建、二价基因工程疫苗及其制备方法与应用。本发明得到一株不含抗性标记的表达猪II型圆环病毒主要抗原位点的重组猪霍乱沙门氏菌C501(pYA-Δ41ORF2)，其保藏号为CCTCC M209314。该株缺失了猪霍乱沙门氏菌生长必需的asd基因，并含有能在该株中表达asd基因和猪II型圆环病毒Cap蛋白抗原位点基因的质粒。公开了利用该重组株制备猪霍乱沙门氏菌和猪II型圆环病毒二价基因工程疫苗的方法及应用。本发明的二价基因工程疫苗可以刺激猪产生抵抗猪霍乱沙门氏菌和猪II型圆环病毒的保护性免疫反应，有效防止猪霍乱沙门氏菌和猪II型圆环病毒的感染。
Description
技术领域
本发明属于动物细菌基因工程技术领域，具体涉及一种不含抗性标记的表达猪II型圆环病毒Cap蛋白的重组猪霍乱沙门氏菌活疫苗株的构建及应用。
背景技术
猪霍乱沙门氏菌(Salmonella choleraesuis)是导致2-4月龄仔猪副伤寒的主要病原，感染猪后会引起急性败血症、慢性坏死性肠炎、顽固性下痢等典型症状，常引起断奶仔猪大批发病，如伴发或继发感染其它疾病或治疗不及时，死亡率较高，造成重大损失。动物生前感染沙门氏菌或其产品受到污染，可使人发生食物中毒，因此该病原在公共卫生学上也具有重要意义。
人们最早用灭活的全菌疫苗来预防沙门氏菌引起的伤寒，由于灭活苗具有容易引起全身及局部反应、只能激发体液免疫、不能提供交叉保护、需多次加强免疫等缺点，此类疫苗的应用受到限制。随后，人们发现弱毒沙门氏菌在诱导体液和细胞免疫反应方面比灭活的或亚单位疫苗更为有效。于是，各种沙门氏菌弱毒苗在世界范围内得到了广泛应用，取得了良好的预防效果。我国在60年代初，房晓文等将抗原性良好的猪霍乱沙门氏菌强毒株接种含有醋酸铊的培养基中传数百代后，选出一株免疫原性好的弱毒株，命名为C500。全国推广使用C500后，我国仔猪副伤寒得到有效控制(黄昌炳等，仔猪副伤寒弱毒通气培养冻干苗口服免疫研究.国农业科学，1981，6：89～94；康凯.仔猪副伤寒活疫苗.中国兽药杂志，2003，37：49)。
近30年来，人们开始关注使用减毒沙门氏菌作为载体表达各种外源抗原以预防重大人、畜传染病研究领域。应用各种基因工程方法构建沙门氏菌减毒株和利用减毒沙门氏菌作为活载体表达外源抗原研制多价疫苗等已成为该领域的研究热点。减毒沙门氏菌作为疫苗载体具有诸多的优点：(1)产生细胞毒性T细胞的杀伤反应(CTL反应)；(2)减毒沙门氏菌因侵入淋巴系统，能更有效地激发宿主的体液和细胞免疫；(3)通过自然感染途径如口服或鼻内接种，可以激发系统和粘膜免疫反应；(4)具有免疫佐剂作用；(5)免疫具有持续性；(6)具有联合免疫作用，可以稳定表达原核和真核基因，本身不致病，且可作为伤寒疫苗；(7)所表达的靶抗原不需提纯，制备简单，可直接用于免疫保护试验，显著降低了疫苗的制作成本；(8)接种简单方便，易于操作等(Curtiss，R.，III，T.Doggett，A.Nayak，and J.Srinivasan.1996.Strategies for the use of live recombinant avirulent bacterial vaccines for mucosal immunization，p.499-511.In H.Kiyono and M.F.Kagnoff(ed.)，Essentials of mucosal immunology.Academic Press，San Diego，Calif；S.Spreng，G.Dietrich and G.Weidinger.2006.Rational design of Salmonella-based vaccination strategies.Methods38：133-143；Sirard，J.C.，F.Niedergang，and J.P.Kraehenbuhl.1999.Live attenuated Salmonella：a paradigm ofmucosal vaccines.Immunol.Rev.171：5-26)。重组沙门氏菌疫苗的发展常需要使用选择标记，由于以前普遍采用携带抗性基因的表达质粒，抗性基因成为体外转化筛选转化子和质粒在体外遗传的的主要标记。但是，抗生素是临床上治疗细菌感染的主要药物，由此引起的最大恐慌是抗性基因在环境病原微生物中传播，从而减弱治疗这些病原感染的药物效果。因此，抗生素抗性质粒选择系统不被人们所接受。如今，人们已经研制出多种不含抗生素抗性标记的沙门氏菌疫苗系统，如将外源抗原稳定整合到沙门氏菌染色体，或用能筛选重组菌但不需要抗生素的质粒平衡系统(Balanced-lethal Plasmid Stabilisation System)。其中，应用最多的系统是asd质粒载体平衡致死系统。沙门氏菌的asd基因编码天冬氨酸β-半乳糖脱氢酶(aspartateL-semialdehyde dehydrogenase)，是二氨基庚二酸(diaminopimelic acid，DAP)生物合成途径中的必需酶，而DAP是革兰氏阴性菌细胞壁主要化学成分肽聚糖四肽侧链的一个组分，asd缺失株在无外源DAP条件下不能形成完好的细胞壁，最终溶菌死亡。由于哺乳动物组织中不含DAP，因此突变菌在不含DAP的培养基中或动物体内均被降解。将目的基因插入含asd的质粒，转化asd宿主菌后可形成互补，asd质粒丢失的asd沙门氏菌在体内死亡，只有含有asd质粒的沙门氏菌才能存活，并稳定的在体内体外表达外源抗原。由于用asd质粒载体平衡致死系统代替了抗生素抗性标记，因此也更加安全。(Nakayama，K.，S.M.Kelly，andR.Curtiss III.1988.Coe N E and R L Wood.The effect of exposure to a ΔcrpΔcya mutant of Salmonellatyphimurium on the subsequent colonization of swine by the wild-type parent strain.Vet Microbiol，1992，31：207-220.Hassan，J O，and R Curtiss III.Control of colonization by virulent Salmonella typhimurium by oralimmunization of chickens with avirulent ΔcrpΔcya S.typhimurium.Res.Microbiol，1990，141：839-850.Kelly SM，Bosecker B A，Curtiss R III.Characterization and protective properties of attenuated mutants of Salmonellacholeraesuis.Infect Immun，1992，60：4881-4890 Construction of an Asd+ expression-cloning vector：stablemaintenance and high level expression of cloned genes in a Salmonella vaccine strain.Bio/Technology 6：693-697；Kang，H.Y.，J.Srinivasan，and R.Curtiss III.2002.Immune responses to recombinant pneumococcal PspAantigen delivered by live attenuated Salmonella enterica serovar Typhimurium vaccine.Infect.Immun.70：1739-1749.)。
猪圆环病毒(Porcine circovirus，PCV)的发现是在1974年，Tischer等(1974)在PK-15细胞系ATCC-CCL31的传代过程中，发现了与细小病毒和乳多空病毒类似的颗粒，这种颗粒状物质不引起细胞病变，曾在当时被认为是一种细胞污染物。Tischer等(1982)用超速离心提取病毒粒子和病毒核酸，通过电镜观察核酸状态，首次揭示了这种污染PK-15细胞系的物质实际上是一种单链环状DNA病毒，并将其命名为PCV。
用该病毒感染试验动物，发现只有猪产生特异性抗体，但无临床症状和病理变化，而兔、鼠、牛均为血清学阴性。大量的血清学调查结果显示，许多国家的猪群中PCV抗体呈阳性，PCV感染十分普遍。尤其1991年在加拿大出现了断奶猪和育肥猪生长缓慢、呼吸急迫、消瘦、贫血、黄疸，剖检有淋巴结肿大、肝硬变、多灶性粘液性气管炎、间质性肺炎和肾炎等，当时认为是一种新发的猪病，将之命名为断奶仔猪多系统衰竭综合征(Post-weaning pigs multisystemic wasting syndrome，PMWS)(Fenaux et al.，2000)。随后，该病迅速在全世界相继发生，我国也于2000年报道了该病的发生(郎洪武等，2000)。通过临床和实验室的研究表明，引起断奶仔猪多系统衰竭综合征的主要病原是猪圆环病毒，但并不是唯一的病原(Allan et al.，1998)。同时还发现这种病原与传统的持续感染PK-15细胞的PCV，彼此间抗原性相似，但也有很大的区别，所以根据PCV在致病性、抗原性及核苷酸序列上的差异性，将从PMWS病猪体内分离到的有致病性的PCV称为PCV2，而将污染PK-15细胞且无致病性的PCV称为PCV1(Allan et al.，1998a)。
PCV基因组为单股环状负链DNA，PCV1基因组全长为1759bp，PCV2基因组全长为1767或1768bp。两种基因型病毒的基因组同源性为76％，同基因型的PCV内部基因组变异很小，PCV1分离株间基因组的同源性大于99％，PCV2分离株间基因组的同源性大于96％。PCV的基因组共编码11个开放阅读框架(Openreading frames，ORFs)，阅读框之间相互重叠，使病毒可以充分利用有限的遗传物质传递大量的遗传信息。病毒的ORF1、ORF5、ORF7、ORF10在病毒DNA链上，而ORF2、ORF3、ORF4、ORF6、ORF8、ORF9、ORF11在互补链上，在这11个开放阅读框架中，功能上重要的是ORF1和ORF2。ORF1编码Rep和Rep’蛋白，与病毒基因组的复制有关。ORF2编码病毒的核衣壳(Cap)蛋白，是病毒唯一的结构蛋白。通过序列分析发现，PCV1和PCV2的ORF1间核酸同源性为83％，氨基酸同源性为86％，两种基因型病毒的ORF1基因间的差异比较小，Rep蛋白间存在交叉反应，而ORF2基因变异较大，核酸同源性为67％，氨基酸同源性为65％(Morozov et al.，1998)，Cap蛋白间不存在交叉反应，因此ORF2基因可作为鉴别诊断的依据。有研究发现，在PCV1和PCV2的保守区段都包含有Poly(A)信号，在正链上，PCV1的Poly(A)分别位于：314nt-319nt、973nt-978nt；PCV2的Poly(A)分别位于：327nt-332nt、983nt-998nt；在负链上，PCV1有两个Poly(A)信号分别位于：1015nt-1030nt、1184nt-1189nt；而PCV2只有一个Poly(A)信号位于1022nt-1027nt(Hamel et al.，1998)。ORF3基因为近年来新研究的具有重要功能的基因，它不参与病毒的复制过程，编码病毒的非结构蛋白，可以引起细胞凋亡，在PCV2致病机理中具有重要作用(Liu et al.，2005)。
ORF2基因编码病毒的Cap蛋白，含有234个氨基酸，分子量大小为27.8kDa。由基因组的互补链合成。Cap蛋白转录起始位点在1238位核苷酸，转录产物内含有由119个核苷酸(1238nt-1120nt)组成的非编码前导序列，Cap启动子精确定位于1353nt-1168nt核苷酸区域，与Rep基因有重叠(Mankertz et al.，1998)。Cap蛋白在圆环病毒感染细胞后6-12小时即可表达，在12-24小时定位于细胞核中(Meerts et al.，2005)。ORF2基因在圆环病毒中变异比较大，同源性约为70％。Cap蛋白端的41个氨基酸序列高度保守，富含碱性氨基酸(如精氨酸)，编码病毒的核定位信号，与病毒在细胞核内的定位有关，其中第12-18位和第34-41位氨基酸残基对ORF2的定位起着至关重要的作用(Liu et al.，2001b)。Mahe等(2000)发现PCV中共存在5个抗原位点，有1个位于ORF1中(185 aa-211 aa)，其余4个都在ORF2中，有2个是PCV2特异性的(B-121，B-133)，有1个与PCV1和PCV2抗血清都反应(B-146)，另有1个与PCV1和PCV2抗血清(B-152)都不反应。虽然PCV1和PCV2的Cap蛋白有交叉反应的抗原表位存在，但其ORF2之间却无交叉反应存在，可能是在整个蛋白中引起交叉反应的抗原表位处于不易被接触到的位置。Truong等(2001)用免疫相关线性B-细胞表位及ELISA方法也验证了ORF2的69-83位和117-131(B-133位)位氨基酸残基是特异性抗原决定簇，其中B-133是PCV2血清学检测的标志性抗原决定簇之一，用于PCV2的血清学诊断。
ORF2基因决定PCV2的毒力，ORF2基因的变异导致各地PCV2分离株在毒力上的不同，ORF2基因可能与病毒增殖滴度有一定关系。Fenaux等(2003)利用病毒感染性克隆将PCV1的ORF2与PCV2的ORF2基因进行互换，发现病毒的毒力大大下降。
虽然PCV1和PCV2的Cap蛋白有引起交叉反应的抗原表位存在，但二者的ORF2之间却不存在交叉反应，所以关于PCV2诊断和基因工程疫苗的研究，都集中在ORF2基因或Cap蛋白上。Mahe等(2000)提出可以利用PCV2-ORF2多肽或完整蛋白作为诊断抗原建立区别PCV1和PCV2的鉴别诊断方法。Blanchard等(2003)利用PCV2的Cap蛋白作为抗原建立检测PCV2特异性抗体间接ELISA，与IFA相比其准确率可达90％以上，并在临床检测中取得了良好的效果。Kamstrup等(2004)研究了ORF2基因的DNA疫苗，Blanchard等(2003b)用杆状病毒表达的Cap蛋白免疫猪，取得了理想的免疫效果。
重组沙门氏菌活疫苗自然感染或试验感染诱导抗任何异源血清型沙门氏菌保护的同时，沙门氏菌因侵入淋巴系统，能更有效地激发宿主产生针对异源抗原的体液和细胞免疫反应等优点。这样，重组弱毒活疫苗也许是解决当前猪传染性胃肠炎病毒商品化疫苗不足的一种可行方法。构建沙门氏菌重组菌株的传统方法存在很多缺陷。如以前普遍采用携带抗性基因的表达质粒，因存在生物安全问题而不被人们所接受。asd质粒载体平衡致死系统具有表达量高、外源基因表达稳定、不需纯化无抗生素抗性标记等优点，同时鉴于猪霍乱沙门氏菌C500的良好免疫原性，将其开发为表达猪圆环病毒主要抗原位点基因的重组活疫苗具有广阔的应用前景。
发明内容
本发明的目的在于克服现有技术存在的缺陷，其第一个目的是获得表达猪II型圆环病毒主要抗原位点的重组猪霍乱沙门氏菌株，该重组菌含有猪II型圆环病毒主要免疫性抗原Cap蛋白。本发明的第二个目的是利用上述的重组菌获得一种猪霍乱沙门氏菌株制备猪霍乱沙门氏菌和猪II型圆环病毒二价基因工程疫苗；本发明的第三个目的是上述重组菌在制备猪霍乱沙门氏菌和猪II型圆环病毒二价基因工程疫苗中的应用。
本发明通过以下技术方案实现：
申请人通过基因工程的方法，获得一种不含抗性标记的表达猪II型圆环病毒主要抗原位点的重组的猪霍乱沙门氏菌(Salmonella choleraesuis)C501/pYA-Δ41ORF2，该菌株于2009年12月24日送交位于湖北省武汉市武汉大学内的中国典型培养物保藏中心(CCTCC)保藏，其保藏编号为CCTCCNO：M209314。
一种重组猪霍乱沙门氏菌C501/pYA-Δ41ORF2的制备方法，它包括下列步骤：
1)PCV2去核定位信号ORF2的克隆：以申请人所在的华中农业大学农业微生物国家重点实验室制备的重组质粒pMD-PCV2(李文洁，何启盖，等.中国部分地区猪圆环病毒2型的基因型分析[J].畜牧兽医学报，2009，40(9)：1358-1362)为模版扩增Δ41ORF2片段，片段大小为582bp，连接pMD-18T，转化DH5α感受态细胞，挑取阳性克隆经PCR和酶切鉴定表明构建正确。经测序Δ41ORF2序列与GeneBank上发布的序列同源性在98％以上，确实无碱基错配；
2)重组质粒pYA-Δ41ORF2的构建：回收Δ41ORF2基因的PCR产物，并用限制性内切酶Saul I和Hind III双酶切，纯化回收后，连接用同样方法处理过的pYA3493载体，转化E.coli χ6097感受态细胞，获得重组表达质粒pYA-Δ41ORF2。PCR和酶切结果表明重组表达质粒pYA-Δ41ORF2构建。序列测定结果表明构建的pYA-Δ41ORF2重组质粒中插入的Δ41ORF2片段与GeneBank上发布的序列同源性在98％以上，未发生任何碱基突变。
3)构建重组菌株C501(pYA-Δ41ORF2)：将构建好的重组质粒pYA-Δ41ORF2，电转化至猪霍乱沙门氏菌ΔasdC500缺失株感受态细胞，得到保藏编号为CCTCC NO：M209314的重组猪霍乱沙门氏菌C501/pYA-Δ41ORF2。
申请人获得一种合成的猪II型圆环病毒主要免疫性抗原Cap蛋白主要抗原位点的基因片段ORF2(核苷酸序列)，其特征在于，它的核苷酸序列如序列表所示(其制备方法见实施例所示)。
申请人利用所述的重组猪霍乱沙门氏菌C501/pYA-Δ41ORF2菌液制备成功一种防治猪霍乱沙门氏菌病和猪圆环病毒病的二价基因工程疫苗。
上述的不含抗性标记的表达猪II型圆环病毒Cap蛋白主要抗原位点的重组猪霍乱沙门氏菌株缺失了沙门氏菌基因组中必需的细胞壁形成相关基因asd基因(需要含asd基因的质粒存在于菌体内或外源添加DAP的培养基上才能正常生长、繁殖)，同时该重组菌株含有外源质粒pYA-Δ41ORF2(含有asd基因)，保留了亲本菌株C500作为猪霍乱沙门氏菌弱毒疫苗株的免疫特性。质粒pYA-Δ41ORF2能在该菌株中表达asd基因与猪II型圆环病毒Cap蛋白的抗原位点基因。其中，asd基因表达产物提供沙门氏菌株必需的细胞壁形成相关成份。Cap蛋白的抗原位点基因表达产物提供良好的针对猪II型圆环病毒的免疫原性。
本发明的不含抗性标记的表达猪II型圆环病毒主要抗原位点的重组猪霍乱沙门氏菌株C501/pYA-Δ41ORF2，该基因工程菌株C501/pYA-Δ41ORF2衍生于在中国已经使用了50年以上的商品化猪霍乱沙门氏菌弱毒疫苗株C500。本发明所述的重组猪霍乱沙门氏菌株C501/pYA-Δ41ORF2，缺失了C500菌株基因组中的asd基因，同时添加了含有猪II型圆环病毒Cap蛋白的抗原位点基因的质粒pYA-Δ41ORF2。由于需要pYA-Δ41ORF2的存在重组菌株才能存活，因此大大提高了质粒pYA-Δ41ORF2(也就是Cap蛋白抗原位点基因)在重组菌株中的稳定性。Cap蛋白抗原位点基因在重组菌株中的稳定表达，使之具有针对猪II型圆环病毒很好的免疫保护力。
本发明的基本构建方法是：利用本申请人所在的农业部微生物国家重点实验室构建好的asd基因缺失的猪霍乱沙门氏菌弱毒疫苗株C500ΔasdΔcrp缺失株(参见文献：徐引弟等，猪霍乱沙门氏菌C500株ΔcrpΔasd缺失株平衡致死载体系统的构建及鉴定.生物工程学报，2006，5(3)：366-371)，同时回收含有猪II型圆环病毒Cap蛋白抗原位点的质粒pYA-Δ41ORF2。将质粒pYA-Δ41ORF2转入C500asd基因缺失株中，由于生长的需要质粒pYA-Δ41ORF2与C500asd基因缺失株互补而稳定共存，形成我们发明的不含抗性标记的表达猪II型圆环病毒Cap蛋白主要抗原位点的重组C501(pYA-Δ41ORF2)菌株。通过大量的生物学实验数据证明本发明制备的重组菌株可用于制备针对猪霍乱沙门氏菌病和猪圆环病毒病的二价基因工程疫苗。
本发明的主要优点是：
1、本发明的重组菌株表达的猪II型圆环病毒Cap蛋白中主要抗原位点是猪II型圆环病毒主要的免疫原性基因片段，具有良好的免疫保护性。由于猪II型圆环病毒危害日益严重，而目前市场上还没有各种商品化猪II型圆环病毒疫苗。因此，用该基因工程菌株制成的疫苗具有广阔的市场应用前景。
2、本发明的重组菌株衍生于在中国已经使用了50年以上的商品化猪霍乱沙门氏菌弱毒疫苗株C500，完全保留了C500针对猪霍乱沙门氏菌的免疫效力。而且，该重组菌株毒力比C500稍弱，具有更好的生物安全性。
3、本发明的重组菌株可以同时提供针对猪霍乱沙门氏菌和猪II型圆环病毒两种病原的保护力。
4、本发明的重组菌株不含抗性标记，完全符合疫苗生物安全性要求。
更详细的技术方案见实施例所述。
附图说明
序列表SEQ ID NO：1是本发明克隆的(插入的)猪II型圆环病毒去核定位信号的Cap蛋白的核苷酸序列。
图1：是本发明制备的重组质粒pYA-Δ41ORF2的物理图谱。
图2：是本发明制备的重组质粒pYA-Δ41ORF2的PCR鉴定电泳图谱。
图中M：DNA Marker(DL2000) 1：ddH2O；2：pYA-Δ41ORF2；3：pYA-Δ41ORF2
图3：是本发明制备的重组质粒pYA-Δ41ORF2的酶切鉴定结果。
图中M1：DNA Marker(DL 2000，DL 15,000) 1：pYA3493/Saul I+Hind III2And 3：pYA-Δ41ORF2/Saul I+Hind III
图4：是本发明中一种不含抗性标记的表达猪II型圆环病毒Cap蛋白主要抗原位点的重组猪霍乱沙门氏菌活疫苗菌株的遗传稳定性实验结果。图中M：DNA marker(DL 2,000)；1：ddH2O阴性对照；2：重组质粒阳性对照；3-8：1-50代重组菌株。
图5：重组菌株C501(pYA-Δ41ORF2)、空质粒对照菌株C500(pYA3493)与亲本菌株C500的生长曲线。
图6：是本发明制备的重组菌株C501(pYA-Δ41ORF2)构建的流程图。
图7：是本发明中一种不含抗性标记的表达猪II型圆环病毒Cap蛋白主要抗原位点的重组猪霍乱沙门氏菌活疫苗菌株C501(pYA-Δ41ORF2)的SDS-PAGE和Western-blot分析结果。
图7A是用SDS-PAGE方法检测融合蛋白Cpro-Δ41ORF2，图中1为C501(pYA3493)；2-7为重组菌株C501(pYA-Δ41ORF2)；箭头所示为融合表达蛋白Cpro-Δ41ORF2。
图7B是用Western-blot方法检测融合蛋白Cpro-Δ41ORF2，图中1-2为C501(pYA-Δ41ORF2)；图中3为C501(pYA3493)
图8：是本发明中重组沙门氏菌所用载体的图谱。
图9：是沙门氏菌血清抗体水平。
图10：是重组猪霍乱沙门氏菌中猪II型圆环病毒Cap蛋白IgG抗体水平。
具体实施方式
以下结合说明书附图对本发明作进一步的说明。
实施例1 猪II型圆环病毒去核定位信号ORF2蛋白基因片段的扩增
引物设计(用于基因克隆和分子检测)
根据Gene Bank发布的猪2型圆环病毒序列(登录号：AY035820)，设计1对可扩增去核定位信号ORF2基因的引物(见表1)，从本实验室公布的pMD-PCV2质粒(李文洁，何启盖，等.中国部分地区猪圆环病毒2型的基因型分析[J].畜牧兽医学报，2009，40(9)：1358-1362)中扩增去核定位信号ORF2基因，扩增后的片段大小为582bp，引物上下游分别引入Saul I和Hind III酶切位点。该引物均由上海生工生物工程技术服务有限公司合成。
表1 本发明所用的引物
实施例2 重组质粒pYA-Δ41ORF2的构建与鉴定
回收Δ41ORF2基因的PCR产物，并用限制性内切酶Saul I和Hind III双酶切，纯化回收后，连接用限制性内切酶Saul I和Hind III双酶切处理过的pYA3493载体(美国华盛顿大学Dr.Roy Curtiss III教授惠赠)，转化E.coli χ6097感受态细胞，获得重组质粒pYA-Δ41ORF2。PCR和酶切结果表明重组表达质粒pYA-Δ41ORF2构建成功。序列测定结果表明构建的pYA-Δ41ORF2重组质粒中插入的Δ41ORF2片段与GenBank上发布的序列同源性均在98％以上，未发生任何碱基突变。重组表达质粒pYA-Δ41ORF2的物理图谱和酶切鉴定结果(见图1、图3)。
实施例3 表达Cap蛋白基因片段的猪霍乱沙门氏菌重组菌株C501(pYA-Δ41ORF2)的构建与鉴定
用BioRad GenePμlser II电转仪按电压2.0Kv，时间4ms，电容25F和脉冲电阻200Ω的参数将重组质粒pYA-Δ41ORF2电转化ΔasdC500缺失株感受态细胞，在DAP阴性平板上筛选阳性重组子。阳性克隆因为含有与asd基因互补的平衡表达质粒而具有表达DAP的能力，所以培养基中不需再添加DAP。对重组菌株进行PCR鉴定，可以扩增出Δ41ORF2基因581bp(图2)，结果表明重组菌构建正确，申请人将其命名为C501(pYA-Δ41ORF2)。
实施例4表达Cap蛋白基因片段的猪霍乱沙门氏菌重组菌株C501(pYA-Δ41ORF2)的生物学特性
1、C501(pYA-Δ41ORF2)重组菌株的表达特性分析
SDS-PAGE结果显示，本发明的重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)在约26kDa处有明显的表达带，Western-blot分析表明，重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)所表达的蛋白能够与猪2型圆环病毒单克隆抗体发生特异性反应，而空质粒对照菌株C501(pYA3493)则不能发生反应(图7B)。
2、重组菌株C501(pYA-Δ41ORF2)的表型鉴定
与亲本株C500相同，本发明的重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)能够利用麦芽糖，在加麦芽糖的麦康凯琼脂平板上呈红色菌落，在LB平板上长成无色、光滑、湿润的乳白色菌落。生化鉴定结果表明，C501(pYA3493)和C501(pYA-Δ41ORF2)与亲本菌C500均不产H2S，不能利用乳糖、蔗糖、阿拉伯糖、半乳糖醇和尿素，但是能利用麦芽糖、葡萄糖、鼠李糖、甘露糖和木糖作为唯一碳源。这些结果表明本发明的重组菌株C501(pYA-Δ41ORF2)的生化特性与亲本菌株C500一致，符合猪霍乱沙门氏菌的典型表型特征。
3、重组菌株C501(pYA-Δ41ORF2)的生长特性研究
从重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)、空质粒对照菌株C500(pYA3493)与亲本菌株C500的生长曲线(图5)可以看出，重组菌株C501(pYA-Δ41ORF2)生长速度与亲本菌株C500相似，而空质粒对照菌株C500(pYA3493)的生长速度比亲本菌株C500相对较慢。
4、重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)的遗传稳定性
将重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)在LB液体培养基中连续传50代，分别取含有相同的CFU第1、10、20、30、40和50代的培养物为模板，进行PCR检测Δ41ORF2基因在C501中的遗传稳定性。结果表明，第1、10、20、30、40和50代均能扩增出581bp的条带，其亮度没有差别(图4)。分别取以上代次的含有相同CFU的培养物进行Western-blot，均能与抗PCV2-ORF2得单克隆抗体反应，结果表明重组菌C501(pYA-Δ41ORF2)能稳定性的分泌表达(图6A)。上述结果表明重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)能够稳定遗传。
实施例5 重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)在小鼠体内的免疫效力检测
1、小鼠的免疫程序：
使用6-7周龄的BALB/c小鼠作为免疫效力评价，根据试验要求分为3组，分别为本发明制备的重组猪霍乱沙门氏菌菌株C501(pYA-Δ41ORF2)疫苗组、猪霍乱沙门氏菌C500亲本菌株疫苗组和非免疫空白对照组。免疫途径为口服0.2mL(含5.1×109CFU活菌量)细菌液或LB培养基，14天后加强免疫1次。分别于免疫前0天、首免后1、2、3、4周每只小鼠尾静脉负压采血，收集血清，采用间接ELISA法检测沙门氏菌血清抗体水平(见图9)(ELISA，操作方法参照徐引弟.猪霍乱沙门氏菌C500株crp-、asd-缺失株平衡致死系统的构建及应用.[博士学位论文].武汉：华中农业大学图书馆，2006.)。同时使用武汉科前生物制品有限责任公司生产的商品ORF2蛋白包被ELISA板(生产批号：091005，生产日期：091015)，按间接ELISA方法(方法参照柳忠辉等，免疫学常用实验技术，北京：科学出版社，2002)检测重组Cap蛋白IgG抗体水平(见图10)。
实施例7 重组猪霍乱沙门氏菌菌C501(pYA-Δ41ORF2)二价基因工程疫苗在猪体内的免疫效力
自2009年7月份以来，先后在A、B、C和D猪场口服免疫四批的本发明制备的重组猪霍乱沙门氏菌菌疫苗。A猪场未使用本发明制备的重组猪霍乱沙门氏菌菌疫苗(简称二价基因工程疫苗)前，仔猪病死率约为50％，口服本发明的二价基因工程疫苗后，存活率为90％。其他各场口服本发明的二价基因工程疫苗后，也收到与A场相似的效果。
序列表
<110>华中农业大学
<120>重组猪霍乱沙门氏菌及二价基因工程疫苗与制备方法
<130>
<141>2009-12-20
<160>1
<170>PatentIn version 3.1
<210>1
<211>582
<212>DNA
<213>猪圆环病毒(Porcine circovirus)
<220>
<221>gene
<222>(1)..(582)
<223>
<400>1
aatggcatct tcaacacccg cctctcccgc accttcggat atactatcaa gcgaaccaca 60
gtcaaaacgc cctcctgggc ggtggacatg atgagattca atattaatga ctttcttccc 120
ccaggagggg gctcaaaccc ccgctctgtg ccctttgaat actacagaat aagaaaggtt 180
aaggttgaat tctggccctg ctccccgatc acccagggtg acaggggagt gggctccagt 240
gctgttattc tagatgataa ctttgtaaca aaggccacag ccctcaccta tgacccctat 300
gtaaactact cctcccgcca taccataacc cagcccttct cctaccactc ccgctacttt 360
acccccaaac ctgtcctaga ttccactatt gattacttcc aatcaaacaa caaaagaaat 420
cagctggggc tgagactaca aactgctgga aatgtagacc acgtaggcct cggaactgcg 480
ttcgaaaaca gtatatacga ccaggaatac aatatccgtg taaccatgta tgtacaattc 540
cgggaattta atcttaaaga ccccccactt aaccctaagt ga 582
Claims (4)
1.一株表达猪II型圆环病毒主要抗原Cap蛋白的重组猪霍乱沙门氏菌(Salmonellacholeraesuis)C501(pYA-Δ41ORF2)，保藏在中国典型培养物保藏中心，其保藏编号为：CCTCCNO：M209314。
2.一种重组猪霍乱沙门氏菌C501(pYA-Δ41ORF2)的制备方法，它包括下列步骤：
1)扩增猪II型圆环病毒Cap蛋白去核定位信号基因：以重组质粒pMD-PCV2为模版扩增Δ41ORF2片段，连接pMD-18T，转化DH5α感受态细胞，挑取阳性克隆进行PCR扩增，得到PCR产物；
2)构建重组质粒pYA-Δ41ORF2：将回收的Δ41ORF2基因的PCR产物用Sal I和Hind III双酶切，纯化回收后，连接用Sal I和Hind III双酶切的pYA3493载体，转化E.coli χ 6097感受态细胞，获得重组质粒pYA-Δ41ORF2；
3)构建重组菌株C501(pYA-Δ41ORF2)：将构建好的重组质粒pYA-Δ41ORF2，电转化至猪霍乱沙门氏菌Δasd C500缺失株感受态细胞，得到保藏编号为CCTCC M209314的重组猪霍乱沙门氏菌C501/pYA-Δ41ORF2。
3.一种二价基因工程疫苗，其特征是，将保藏编号为CCTCC NO：M209314的重组猪霍乱沙门氏菌C501/pYA-Δ41ORF2菌液制备成猪霍乱沙门氏菌和猪II型圆环病毒的二价基因工程疫苗。
4.权利要求1所述的重组猪霍乱沙门氏菌在制备猪霍乱沙门氏菌与猪II型圆环病毒二价基因工程疫苗中的应用。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732472A (zh) * | 2011-04-08 | 2012-10-17 | 华中农业大学 | 表达猪肺炎支原体p65蛋白的减毒猪霍乱沙门氏菌的构建与应用 |
| CN103421731A (zh) * | 2013-01-31 | 2013-12-04 | 华中农业大学 | 一种副猪嗜血杆菌减毒沙门氏菌苗 |
| CN103421732A (zh) * | 2013-01-31 | 2013-12-04 | 华中农业大学 | 表达副猪嗜血杆菌表面抗原的猪霍乱沙门氏菌减毒疫苗 |
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2009
- 2009-12-25 CN CN200910273372A patent/CN101838627A/zh active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732472A (zh) * | 2011-04-08 | 2012-10-17 | 华中农业大学 | 表达猪肺炎支原体p65蛋白的减毒猪霍乱沙门氏菌的构建与应用 |
| CN103421731A (zh) * | 2013-01-31 | 2013-12-04 | 华中农业大学 | 一种副猪嗜血杆菌减毒沙门氏菌苗 |
| CN103421732A (zh) * | 2013-01-31 | 2013-12-04 | 华中农业大学 | 表达副猪嗜血杆菌表面抗原的猪霍乱沙门氏菌减毒疫苗 |
| CN103421731B (zh) * | 2013-01-31 | 2015-10-28 | 华中农业大学 | 一种副猪嗜血杆菌减毒沙门氏菌苗 |
| CN103421732B (zh) * | 2013-01-31 | 2015-10-28 | 华中农业大学 | 表达副猪嗜血杆菌表面抗原的猪霍乱沙门氏菌减毒疫苗 |
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