CN114854654B - 一种肠炎沙门氏菌活疫苗 - Google Patents
一种肠炎沙门氏菌活疫苗 Download PDFInfo
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Abstract
本发明提供一种禽肠炎沙门氏菌活疫苗,包含有抗原和佐剂,其中抗原是活的肠炎沙门氏菌(Salmonella enterica)R073△aroA株,其保藏编号为CCTCC M 2022376。所述的肠炎沙门氏菌R073ΔaroA株是通过Red/ET同源重组系统把肠炎沙门氏菌R073株的aroA基因缺失制备的。本发明所提供的肠炎沙门氏菌疫苗可以通过传统的方法进行使用,比如通过经济易操作的喷雾和饮用水进行大规模的施用,也可以通过肌肉注射或其他的方式进行施用。
Description
技术领域
本发明属于兽医生物制品技术领域,具体涉及一种肠炎沙门氏菌活疫苗。
背景技术
肠炎沙门氏菌(Salmonella enteritidis)属于无宿主特异性并且有侵害性的病原菌之一,宿主包括人以及各种动物。肠炎沙门氏菌不仅能引起家禽发病和死亡,造成严重的经济损失,而且被污染的家禽产品如禽蛋和禽肉能作为作为肠炎沙门氏菌的携带着严重危害人类健康。
肠炎沙门氏菌病发病时间快、死亡率高,目前养殖业中主要使用抗生素治疗细菌感染性疾病。但是在动物机体中长期使用低于治疗剂量的抗生素能大大加速耐药细菌的出现,如果耐药菌一旦在养殖动物中大范围传播,将使养殖动物成为很大的耐药基因储藏库。因此通过疫苗来预防,利用非抗生素方法防控沙门氏菌病将是未来发展的必然趋势,其中接种疫苗是有效防控沙门氏菌病发生和流行、减少禽产品污染及保证食品安全的重要途径。但是目前沙门氏菌病疫苗多为化学随机突变活或者自然传代弱毒活疫苗,遗传背景不清晰,具有毒力返强的危险。
随着分子生物学及DNA重组技术的快速发展,基因工程疫苗成为近年来的研究热点,基因缺失苗的优点显而易见,既能保障疫苗保护效果,又降低了毒力返强的可能性利。
发明内容
本发明的目的是提供一种禽肠炎沙门氏菌活疫苗,是在筛选获得了一种新型的肠炎沙门氏菌的基础上,通过删除aroA基因制备的弱毒株,使用该弱毒株来制备疫苗;该弱毒株保持了流行毒株肠炎沙门氏菌所具备的天然的免疫原性,同时毒性明显致弱,遗传背景清晰,缺失株不会返强,从而弥补现有技术的不足。
本发明所提供的肠炎沙门氏菌活疫苗,包含有抗原和佐剂,其中抗原是活的肠炎沙门氏菌(Salmonella enterica)R073△aroA株,其保藏编号为CCTCC M2022376,保藏地址:中国武汉武汉大学,中国典型培养物保藏中心,保藏日期为:2022年04月02日。
所述的肠炎沙门氏菌R073ΔaroA株是通过Red/ET同源重组系统把肠炎沙门氏菌R073株的aroA基因缺失制备的。
所述的疫苗佐剂为水性疫苗佐剂。
本发明所提供的疫苗还用于制备用于防治家禽的制品;
所述的家禽包括鸡、鸭、火鸡、鹅、矮脚鸡、鹌鹑、野鸡、鸽子等,优选的是商业上重要的家禽例如鸡、鸭、鹅、火鸡,更优选的是鸡和火鸡,特别优选的是鸡。
本发明所提供的肠炎沙门氏菌疫苗可以通过传统的方法进行使用,比如通过经济易操作的喷雾和饮用水进行大规模的施用,也可以通过肌肉注射或其他的方式进行施用。
附图说明
图1:aroA基因在肠炎沙门氏菌R073染色体中的位置图。
图2:肠炎沙门氏菌R073 aroA基因敲除路线图。
图3:PCR鉴定肠炎沙门氏菌R073 aroA基因敲除株图。
图4:肠炎沙门氏菌R073与R073△aroA生长曲线图。
图5:肠炎沙门氏菌R073ΔaroA与Salmonella vac E基因敲除株生长曲线图。
图6:肠炎沙门氏菌R073ΔaroA稳定性的测定图。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法,例如A newlogic for DNA engineering using recombination in Escherichia coli(zhang etal.,Nature genetics 1998),Rapid modification of bacterial artificialchromosomes by ET-recombination(Muyrers et al.,Nucleic acids research 1993)中所记载的方法。
细菌的aroA基因编码5-烯醇丙酮酰莽草酸-3-磷酸合成酶的合成,该酶对于细菌芳香族氨基酸等芳香族化合物的合成代谢具有十分重要的意义,该基因缺失后会阻断细菌体内的芳香族生物合成途径。
Red/ET同源重组技术来源于lamda噬菌体,可以用于肠炎沙门氏菌基因组的基因敲除、插入、点突变等,利用Red/ET同源重组技术构建肠炎沙门氏菌aroA基因缺失株,在其生物学特性和致病力基础上评估该基因缺失菌株作为疫苗候选株的潜力。
本发明所提供的肠炎沙门氏菌R073是从青岛某大型鸡场肠炎沙门氏菌发病鸡器官组织中筛选培养获得的一株强毒株;R073aroA是通过Red/ET同源重组系统得到的R073株aroA基因缺失株,导致毒力减弱,使用该菌来制备活疫苗,从而对鸡提供更完善的免疫保护。
下面结合实施例对本发明进行详细的描述。
实施例1:菌株的分离纯化及理化性质分析
1)2018年6月山东某个鸡养殖场发生了严重的肠炎沙门氏菌疫情,通过采集病死鸡的器官心脏、肝脏、脾脏等,置于采样袋中密封,带回实验室置于4℃冰盒保存备用。
2)把收集的鸡器官研磨后涂在显色培养基上,显示不同颜色的菌株,每3个显示不同颜色的菌株作为一组接种到LB液体培养基进行扩大培养,10000rpm离心收集菌体,然后把菌体用无菌的PBS溶液重悬,用比浊法将菌株的细菌浓度定为1×107CFU/ml。
3)强毒分离实验:
将分离得到的菌株分别在普通肉汤培养基中37℃培养24h,分别按菌数1.0×106/ml配成菌液,把40只1日龄的鸡随机分成4组,每组10只,每组鸡腹腔注射一种菌株的菌液0.2ml/只,对照组注射0.2ml/只PBS溶液,接种后隔离器中观察72h,记录鸡的死亡数并从其肝和脾中分离细菌,然后把存活的鸡解剖观察病变情况(表1)。
表1:肠炎沙门氏菌R070、R071、R072、R073攻击1日龄鸡实验表
4)使用细菌基因组DNA提取试剂盒提取细菌的DNA,利用16S rRNA细菌鉴定的通用引物1492F(5-CGG TTA CCT TGT TAC GAC TT-3)和27F(5-AGA GTT TGA TCM TGG CTC AG-3)进行高保真PCR扩增,琼脂糖凝胶电泳回收后切取目的条带、产物送到上海生工生物有限公司测序,并对测序结果进行NCBI blast分析,其中16S rRNA序列如下(SEQ ID NO:1):
AATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCNCTTCGCTNNNNAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTGGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCGCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGACGTAACGCGTTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACAGAAGAATCCAGAGATGGATTGGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGATTAGGTCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGNAACCTGCGGTTGGATCACCTCCTTA。
确定分离的菌为沙门氏菌,然后利用血清型检测证实所筛选的菌株为肠炎沙门氏菌。实验结果分离到的R073为肠炎沙门氏菌强毒株,命名肠炎沙门氏菌R073。
5)药物敏感试验
药敏试验采用纸片法,将培养好的菌株用生理盐水稀释为1×1010CFU/ml后,吸取0.1ml菌液均匀涂布于营养琼脂培养基上;用无菌镊子夹取药敏纸片贴于平板(6张/板,相隔不少于3cm),置于37℃培养24h后观察结果,用游标卡尺测量抑菌圈大小(表2)。药物敏感性实验表明肠炎沙门氏菌R073株对卡那霉素和氯霉素敏感,而对阿莫西林、链霉素、头孢咪唑等抗生素具有耐药性。
表2:分离出的肠炎沙门氏菌R073药物敏感性实验表
注:药敏试验判断标准:Φ抑菌圈直径,Ф>19mm为高度敏感,15mm≤Ф≤19mm为中度敏感,Ф<15mm为耐药。
实施例2:禽肠炎沙门氏菌R073的aroA基因的敲除和检测
1)根据NCBI GenBank上肠炎沙门氏菌的aroA基因序列,设计测序引物(表3),PCR扩增临床分离株R073 aroA基因,测序鉴定。
表3:测序引物的序列信息表
2)根据测序结果设计aroA基因敲除的引物(表4)。
表4:aroA基因敲除的引物序列表
3)构建重组质粒pSC101-BAD-gbaA-cm,测序正确后电转到肠炎沙门氏菌R073菌中。
4)阿拉伯糖诱导,把卡那霉素基因盒loxp-kan-loxp电转到肠炎沙门氏菌R073中置换aroA基因,涂在卡那霉素抗性平板上。筛选阳性克隆,提取基因组,PCR,测序鉴定。挑取正确的单克隆命名为R073-loxp-kan-loxp。
5)把重组质粒pSC101-BAD-Cre-cm电转进R073-loxp-kan-loxp菌中,涂在氯霉素平板上。
6)加入10μl 10%的阿拉伯糖诱导Cre酶的表达,Cre酶识别卡那基因盒两侧的loxp位点,从而删除中间的kan基因,过夜培养后涂在无抗的平板上,挑取单克隆提基因组检测。
7)通过PCR特异性检测以及DNA测序结果,验证肠炎沙门氏菌aroA缺失株是否构建成功。
8)测序验证正确的菌株进行多次传代,选取性状稳定、且生长速度快的缺失株为待筛选的疫苗株。
最终筛选获得的弱毒株命名为R073ΔaroA,于2022年4月2日保藏在位于武汉市武昌珞珈山的中国典型培养物保藏中心,保藏编号为CCTCC M 2022376。
实施例3:肠炎沙门氏菌R073ΔaroA生长曲线的测定
1)肠炎沙门氏菌R073ΔaroA株与肠炎沙门氏菌R073株的生长比较
挑取菌株肠炎沙门氏菌R073和肠炎沙门氏菌R073ΔaroA单克隆在液体LB的试管中37℃950rpm过夜培养,统一调整菌液OD600值为1.0,然后将等量的细菌培养物以1∶100(v/v)的比例转移至培养瓶中,在37℃220r/min条件下振荡培养。使用分光光度计每隔1h监测培养物的OD600,检测10个小时并记录结果,试验重复3次。
实验结果如图4所示,结果显示肠炎沙门氏菌R073ΔaroA株与野生菌相比生长速度并没有受到影响,因此可以保证了该基因缺失株可以用于大规模生长。
2)肠炎沙门氏菌R073ΔaroA株与商品化疫苗Salmonella vac E的生长曲线的测定
挑取菌株肠炎沙门氏菌R073ΔaroA株和Salmonella vac E单克隆在液体LB的试管中37℃950rpm过夜培养,统一调整菌液OD600值为1.0,然后将等量的细菌培养物以1∶100(v/v)的比例转移至培养瓶中,在37℃220r/min条件下振荡培养。使用分光光度计每隔1h监测培养物的OD600,检测10个小时并记录结果,试验重复3次。实验结果如图5所示,肠炎沙门氏菌R073ΔaroA株生长速度要比/>Salmonella vac E快,在同一时间的R073ΔaroA株的OD600要高于/>Salmonella vac E,因此本发明筛选保藏的R073ΔaroA株的生长速度快,同一时间的菌的浓度高,可以大大降低企业的生产成本。
将肠炎沙门氏菌R073ΔaroA株进行传代,并将每一代的菌株单菌落为模板,利用aroA-seq-F/R引物对进行PCR扩增,琼脂糖凝胶电泳分别获得预期的904bp大小的目的条带(图6),而且测序结果与预期结果一致。表明肠炎沙门氏菌基因缺失株均能够稳定遗传。
实施例4:肠炎沙门氏菌R073ΔaroA株的生物安全性评价
首先将肠炎沙门氏菌R073株和基因缺失株R073ΔaroA株划线接种到无抗LB固体平板培养基上,37℃培养箱中过夜,然后挑选单个菌落到接种到10ml无抗LB液体培养基,放置在小型振荡器中,37℃,200rpm培养过夜后,10000rpm离心1min后收集沉淀,沉淀用无菌PBS进行重悬,把菌悬液进行活菌计数,准备进行后续的动物口服感染实验。
选取30日龄的SPF鸡90只,随机分为9组,每组9只。将R073ΔaroA菌液稀释为1.0×106CFU/ml、1.0×107CFU/ml、1.0×108CFU/ml三种不同的梯度,分别接种10只试验SPF鸡1.0ml/只,另外3组口服分离的野生R073株1.0ml/只作为对照,3组口服商品化肠炎沙门氏菌活疫苗Salmonella vac E。
隔离器中观察14天,记录临床发病情况。结果表明口服攻毒的对照组野生R073株的SPF鸡在24小时内有一只鸡死亡,剩余四只鸡在72小时内全部死亡;而R073ΔaroA实验组1.0×106CFU/ml、1.0×107CFU/ml、1.0×108CFU/ml三种不同浓度均未导致SPF鸡发病(表5)。
表5:肠炎沙门氏菌R073ΔaroA株的安全性实验数据表
上述结果表明,肠炎沙门氏菌R073ΔaroA株的菌数即使达到1.0×108CFU也没有引起鸡的发病,而对照组的攻毒量为1.0×106CFU时引起鸡发病,表明本发明所筛选的菌株R073ΔaroA具有很好的生物安全性。
实施例5:使用肠炎沙门氏菌R073ΔaroA株制备疫苗
1)制备活疫苗:
把肠炎沙门氏菌R073ΔaroA株划线于改良马丁平板上,37℃静置培养24h,取出备用。从培养好的平板菌种中,挑取3个菌落于试管液体培养基中,倾斜45°固定在摇床中,37℃,200rpm震荡培养过夜,取出备用。按2%的比例,将上培养的菌液接种于三角瓶培养基中(挡板三角瓶,300ml/瓶),各培养1瓶。37℃,200rpm震荡培养5h,取出菌液。按菌液:保护剂为9:1的比例加入冻干保护剂,冻干保存菌种,对冻干菌种进行活菌计数,用改良马丁肉汤稀释后,1ml/羽。其中佐剂和保护剂都可选用常用的试剂。
2)疫苗的免疫效果
将1日龄的SPF鸡随机分成四组,每组10只;其中实验组共30只,对照组10只。分别用R073ΔaroA株口服免疫30只SPF鸡,对照组肌肉注射生理盐水,隔离器中饲养观察14天,然后分别对实验组和对照组用野生型R073进行攻毒实验。
隔离器中饲养观察14天,记录各组试验鸡的发病和死亡情况。实验结果如下表6所示。
表6:使用R073ΔaroA疫苗株免疫效果数据表
上述实验结果表明,本发明的R073ΔaroA株制备的疫苗能对野生R073株的感染提供完全的保护,该组实验动物未表现任何临床症状,也无死亡现象。
为了检测R073ΔaroA疫苗株对不同血清型的肠炎沙门氏菌交叉免疫效力,分别把SPF鸡用R073ΔaroA疫苗株制备的疫苗免疫后分别用鸡白痢沙门氏菌SP04、鼠伤寒沙门氏菌ST032、肠炎沙门氏菌960等血清型的沙门氏菌进行攻毒保护实验,隔离器中饲养观察14天,记录各组试验鸡的发病和死亡情况。实验结果如下表7所示。
表7:使用R073ΔaroA疫苗株交叉免疫效果数据表
上述实验结果表明,本发明的R073ΔaroA株制备的疫苗能对市场上分离到的鸡白痢沙门氏菌、鼠伤寒沙门氏菌、肠炎沙门氏菌960等血清型的沙门氏菌菌株的感染提供较好的交叉保护。
为了检测R073ΔaroA疫苗株接种方式是否影响免疫效果,分别选择肌肉注射、喷雾、饮水三种接种方式进行疫苗免疫,14天后用野生株R073进行攻毒实验,隔离器中饲养观察14天,记录各组试验鸡的发病和死亡情况。实验结果如下表8所示。
表8:使用R073ΔaroA疫苗株不同接种方式的免疫效果数据表
上述实验结果表明,本发明的R073ΔaroA株制备的疫苗株可以使用肌肉注射、喷雾和饮水三种接种方式,均能对野生R073株的感染提供完全的保护,该组实验动物未表现任何临床症状,也无死亡现象。
为了评价本发明的R073ΔaroA疫苗株与国内外市场现售的鸡肠炎沙门氏菌疫苗免疫效果,以礼来公司的商品化肠炎沙门氏菌活疫苗Salmonella vac E为对照组进行比较。分别用R073ΔaroA疫苗株和/>Salmonella vac E对SPF鸡进行免疫,14天后用野生株R073进行攻毒实验,隔离器中饲养观察14天,记录各组试验鸡的发病和死亡情况。实验结果如下表9所示。
表9:使用R073ΔaroA疫苗株与Salmonella vac E免疫效果数据表
结果表明本发明的肠炎沙门氏菌R073ΔaroA株制备的疫苗株和市售的肠炎沙门氏菌疫苗Salmonella vac E均能对野生肠炎沙门氏菌R073株的感染提供保护,但疫苗/>Salmonella vac E无法提供完全的保护(9/10),而本发明的肠炎沙门氏菌R073ΔaroA制备的疫苗株能够提供完全的保护(10/10),并且该组实验动物未表现任何临床症状,也无死亡现象。
综上所述,本发明的肠炎沙门氏菌R073ΔaroA株是利用Red/ET同源重组技术删除了肠炎沙门氏菌R073基因组中的aroA基因,保持了良好的免疫原性,通过动物实验表明该疫苗对鸡只安全,能够诱导产生较高的中和抗体,并且能够保护当前市场上流行的鸡白痢沙门氏菌、鼠伤寒沙门氏菌、肠炎沙门氏菌等血清型强毒株的攻击,保护效果良好,具有广阔的应用前景。
序列表
<110> 青岛易邦生物工程有限公司
<120> 一种肠炎沙门氏菌活疫苗
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1539
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aattgaagag tttgatcatg gctcagattg aacgctggcg gcaggcctaa cacatgcaag 60
tcgaacggta acaggaagca gcttgcnctt cgctnnnnag tggcggacgg gtgagtaatg 120
tctgggaaac tgcctgatgg agggggataa ctactggaaa cggtggctaa taccgcataa 180
cgtcgcaaga ccaaagaggg ggaccttcgg gcctcttgcc atcagatgtg cccagatggg 240
attagctagt tggtgaggta acggctcacc aaggcgacga tccctagctg gtctgagagg 300
atgaccagcc acactggaac tgagacacgg tccagactcc tacgggaggc agcagtgggg 360
aatattgcac aatgggcgca agcctgatgc agccatgccg cgtgtatgaa gaaggccttc 420
gggttgtaaa gtactttcag cggggaggaa ggtgttgtgg ttaataaccg cagcaattga 480
cgttacccgc agaagaagca ccggctaact ccgtgccagc agccgcggta atacggaggg 540
tgcaagcgtt aatcggaatt actgggcgta aagcgcacgc aggcggtctg tcaagtcgga 600
tgtgaaatcc ccgggctcaa cctgggaact gcattcgaaa ctggcaggct tgagtcttgt 660
agaggggggt agaattccag gtgtagcggt gaaatgcgta gagatctgga ggaataccgg 720
tggcgaaggc ggccccctgg acaaagactg acgctcaggt gcgaaagcgt ggggagcaaa 780
caggattaga taccctggta gtccacgccg taaacgatgt ctacttggag gttgtgccct 840
tgaggcgtgg cttccggacg taacgcgtta agtagaccgc ctggggagta cggccgcaag 900
gttaaaactc aaatgaattg acgggggccg cacaagcggt ggagcatgtg gtttaattcg 960
atgcaacgcg aagaacctta cctggtcttg acatccacag aagaatccag agatggattg 1020
gtgccttcgg gaactgtgag acaggtgctg catggctgtc gtcagctcgt gttgtgaaat 1080
gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg attaggtcgg 1140
gaactcaaag gagactgcca gtgataaact ggaggaaggt ggggatgacg tcaagtcatc 1200
atggccctta cgaccagggc tacacacgtg ctacaatggc gcatacaaag agaagcgacc 1260
tcgcgagagc aagcggacct cataaagtgc gtcgtagtcc ggattggagt ctgcaactcg 1320
actccatgaa gtcggaatcg ctagtaatcg tggatcagaa tgccacggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgcaaaaga agtaggtagc 1440
ttaaccttcg ggagggcgct taccactttg tgattcatga ctggggtgaa gtcgtaacaa 1500
ggtaaccgta gggnaacctg cggttggatc acctcctta 1539
Claims (4)
1.一种肠炎沙门氏菌弱毒株,其特征在于,所述的肠炎沙门氏菌弱毒株的保藏编号为CCTCC M 2022376。
2.权利要求1所述的肠炎沙门氏菌弱毒株在制备肠炎沙门氏菌弱毒株活疫苗中的应用。
3.一种肠炎沙门氏菌活疫苗,其特征在于,所述的活疫苗包含有抗原和佐剂,其中抗原是权利要求1所述的肠炎沙门氏菌弱毒株。
4.如权利要求3所述的肠炎沙门氏菌活疫苗,其特征在于,所述的佐剂为水性疫苗佐剂。
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鸡沙门氏菌疫苗研究进展;韩春来等;《家禽科学》(第11期);第40-42页 * |
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