CN113354742A - 一种布鲁氏杆菌基因工程亚单位疫苗及其制备方法和应用 - Google Patents
一种布鲁氏杆菌基因工程亚单位疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种布鲁氏杆菌基因工程亚单位疫苗及其制备方法和应用,具体为一种原核表达的布鲁氏杆菌外膜蛋白的串联表位蛋白,包括牛种布鲁氏杆菌(B.abortus)、羊种布鲁氏杆菌(B.melitensis)、猪种布鲁氏杆菌(B.suis)、犬种布鲁氏杆菌(B.canis)的第二组外膜蛋白OMP2蛋白以及第三组外膜蛋白中的OMP25、OMP31、OMP28、OMP22蛋白的抗原表位。该疫苗具备良好的安全性与有效性,牛、羊经过肌肉或皮下注射后,可激发良好的抗体水平。
Description
技术领域
本发明涉及生物制药技术领域,涉及一种布鲁氏杆菌基因工程亚单位疫苗及其制备方法和应用。
背景技术
布鲁氏杆菌是一种革兰氏阴性的胞内寄生菌,目前已经确认的布鲁氏杆菌有9个种,其中宿主为陆生动物的有7个种,分别为牛种(B.abortus)、羊种(B.melitensis)、猪种(B.suis)、绵羊睾丸种(B.ovis)、犬种(B.canis)、沙林鼠种(B.neotomae)与田鼠种(B.microti)。宿主为海洋动物的有2个种,分别为鲸鱼种(B.ceti)与海豹种(B.pinnipedialis)。其中羊种、牛种、猪种、犬种布鲁氏杆菌是一种传播性极强、宿主广泛的病原菌,尤其以羊种布鲁氏杆菌的致病性最强,可通过皮肤、口腔、粘膜、交配进入动物体内感染致病,且患病个体可通过乳液、***、脓液、***分泌物等多种途径向体外传播。
布鲁氏杆菌病属于重大人畜共患传染病,对人和动物的危害都极大,与狂犬病、非典型肺炎、高致病性禽流感等同属于乙类传染病。现有的牛种布鲁氏菌S19株活疫苗、猪种布鲁氏菌S2株活疫苗及羊种布鲁氏菌M5株活疫苗在布鲁氏菌病防控工作中起到了重要的作用。但这三种疫苗的应用均存在不足:(1)安全性差,不能接种于孕畜、种畜、奶畜;(2)疫苗接种动物与野毒感染动物无法鉴别;(3)疫苗对人与动物存在感染性,危害公共卫生安全。上述不足使得疫苗使用范围受到了一定限制,而且不能对种畜与奶畜使用。这导致大部分地区只能通过监测与捕杀来控制布氏杆菌病的流行,继续一种安全有效的布鲁氏杆菌疫苗。
外膜蛋白成为了布鲁氏杆菌的研究热点,并已发现外膜蛋白与布鲁氏杆菌在宿主内体鉄摄取等重要繁殖生长过程有关,且具备重要免疫原性与抗原保护作用。目前已经发现了7种外膜蛋白,并根据分子量大小可以分为三组外模蛋白。其中第一组外膜蛋白包括OMP10、OMP19等,与细菌结构稳定性方面有关;第二组外膜蛋白为OMP2,参与细菌营养物质运输;第三组外模蛋白包括OMP25、OMP31等,参与维持细菌的外模功能性结构的稳定。其中OMP25在布鲁氏杆菌感染、致病过程中发挥重要作用、OMP31与布鲁氏杆菌体在内寄生过程中摄取铁元素密切相关。因此,基于布鲁氏杆菌外膜蛋白研制一种安全有效的新型疫苗,对布鲁氏杆菌并的防控与净化以及公共卫生安全具有重要意义。
发明内容
本发明为了解决上述问题,提供了一种布鲁氏杆菌外膜蛋白串联表位蛋白,利用其制备得到的疫苗用于牛、羊等动物,可安全有效的预防布鲁氏杆菌感染。
本发明目的之一在于提供一种布鲁氏杆菌外膜蛋白串联表位蛋白,对牛种布鲁氏杆菌(B.abortus)、羊种布鲁氏杆菌(B.melitensis)、猪种布鲁氏杆菌(B.suis)、犬种布鲁氏杆菌(B.canis)的第二组外膜蛋白OMP2、第三组外膜蛋白中的OMP25、OMP31、OMP28、OMP22蛋白进行生物信息学分析,得到外膜蛋白的抗原表位。通过人工合成,制备包含所述外膜蛋白抗原表位的串联表位DNA片段,将所述DNA片段连接到pET28a原核表达载体,构建串联表位蛋白重组表达载体(pET28a-MutiE),具体技术方案如下:
一种布鲁氏杆菌外膜蛋白串联表位蛋白,所述布鲁氏杆菌外膜蛋白串联表位蛋白的氨基酸序列由羊种Omp2a蛋白、羊种Omp2b蛋白、牛种Omp2b蛋白、猪种Omp2b蛋白、牛种Omp25蛋白、犬种Omp25蛋白、猪种Omp25蛋白、绵羊睾丸种Omp25蛋白、羊种Omp31蛋白、犬种Omp31蛋白、绵羊睾丸种Omp31蛋白、猪种Omp31蛋白、绵羊睾丸种Omp22蛋白、牛种Omp22蛋白、羊种Omp28蛋白和牛种Omp28蛋白的氨基酸序列组成。
具体的,所述布鲁氏杆菌外膜蛋白串联表位蛋白的氨基酸序列由羊种Omp2a蛋白的22-51位、144-158位;羊种Omp2b蛋白的233-245位、314-336;牛种Omp2b蛋白的4-29位;猪种Omp2b蛋白的41-53位;牛种Omp25蛋白的4-46位、187-197位、143-154位;犬种Omp25蛋白的87-94位;猪种Omp25蛋白的120-137位;绵羊睾丸种Omp25蛋白的10-27位;羊种Omp31蛋白的19-27位;犬种Omp31蛋白的4-14位;绵羊睾丸种Omp31蛋白的95-102位、224-237位;猪种Omp31蛋白的72-89位;绵羊睾丸种Omp22蛋白的157-177位;牛种Omp22蛋白的74-80位;羊种Omp28蛋白的240-247位和牛种Omp28蛋白的103-111位氨基酸序列组成。
具体的,所述布鲁氏杆菌外膜蛋白串联表位蛋白的氨基酸序列如SEQ ID NO.2所示。
本发明目的之二在于提供过来一种布鲁氏杆菌外膜蛋白串联表位蛋白,具体的,所述布鲁氏杆菌外膜蛋白串联表位蛋白的核苷酸序列如SEQ ID NO.1所示。
本发明目的之三在于提供了一种表达布鲁氏杆菌外膜蛋白串联表位蛋白的重组质粒,具体技术方案如下:
一种表达布鲁氏杆菌外膜蛋白串联表位蛋白的重组质粒,所述重组质粒包含上述技术方案中的布鲁氏杆菌外膜蛋白串联表位蛋白的核苷酸序列。
本发明目的之四在于请求保护一种利用上述技术方案中的重组质粒制备得到的基因工程菌株。
具体方法为将上述方案中的重组质粒转化宿主菌构建基因工程菌株,所述基因工程菌株经鉴定后制备布鲁氏杆菌外膜蛋白串联表位蛋白基因工程菌株种子株,所述宿主菌为大肠杆菌BL 21菌株。
进一步地,所述基因工程菌株的鉴定方法为将所述基因工程表达菌株用LB培养基在37℃条件下震荡培养至对数期,然后在LB培养基内添加终浓度为1mM的IPTG并继续培养2-3小时。离心收集菌体沉淀,用冰冷的生理盐水按10:1(W/V)比例重悬菌体后,再离心收集菌体沉淀,重复2-3次,去除培养基成分。菌体通过SDS-PAGE电泳检测,与为未转化的大肠杆菌BL21宿主菌相比,在34kD处有明显条带。
进一步地,所述基因工程菌株的制备方法为,将经过鉴定的基因工程菌株用LB培养基在37℃条件下震荡培养至对数期,在洁净条件下收集菌液,按1:1比例加入冰冷的冻干保护剂并震荡混匀,分装后冷冻干燥。
本发明目的之五在于请求保护利用上述的基因工程菌株制备得到的布鲁氏杆菌亚单位疫苗。
本发明目的之六在于提供一种利用上述技术方案中的基因工程菌株制备布鲁氏杆菌亚单位疫苗的方法,具体技术方案如下:
利用上述技术方案中的基因工程菌株制备布鲁氏杆菌亚单位疫苗的方法,包括如下步骤:
(1)发酵培养所述基因工程菌;
(2)加入疫苗配制缓冲液重悬、破碎、离心得到上清液;
(3)将所述上清液纯化过滤得到疫苗配制用抗原溶液;
(4)添加冻干保护剂冷冻干燥得到布鲁氏杆菌亚单位疫苗。
进一步地,步骤(1)的发酵培养具体为将所述基因工程菌按1:1000的比例接种于发酵培养基中进行发酵培养。
具体的,所述发酵温度为35℃-38℃,发酵至OD值大于20时,添加IPTG至终浓度为1mM,继续发酵2-3小时。
进一步地,所述发酵培养基由蛋白胨5g/L、酵母提取物2.5g/L、甘油8g/L、KH2PO410.5g/L、(NH4)2HPO4 6g/L、柠檬酸1.7g/L、MgSO41.68g/L、ZnSO4·7H2O 5.25g/L、MnSO4·4H2O 0.5g/L、CaCl2 2.0g/L、FeSO4·7H2O10 g/L、Na2B4O7·10H2O 0.23g/L、(NH4)6Mo7O240.1g/L组成。
进一步地,步骤(3)所述的疫苗配制用抗原溶液用疫苗配制缓冲液稀释至目标蛋白含量为10mg/mL之后,再按1:1添加冻干保护剂进行步骤(4)的冷冻干燥。
具体的,所述疫苗配制缓冲液由Tris 6.057g/L、EDTA 0.146g/L、NaCl 2.922g/L、DTT 0.77g/L、尿素180.18g/L和水组成。
进一步地,所述冻干保护剂由Tris 6.057g/L、EDTA 0.146g/L、NaCl 2.922g/L、蔗糖100g/L、脱脂奶粉200g/L和水组成。
本发明目的之七在于请求保护上述技术方案中所述的布鲁氏杆菌亚单位疫苗在制备抗布鲁氏杆菌抗体中的应用。
本发明的有益之处在于:本发明的布鲁氏杆菌基因工程亚单位疫苗,主要成分为原核表达的布鲁氏杆菌外膜蛋白串联表位蛋白,经免疫动物后,可以激发良好的细胞免疫与体液免疫,用于牛、羊等动物,可安全有效的预防布鲁氏杆菌感染。
并且本发明进行了安全性与免疫原性实验,通过皮下或肌肉注射免疫牛、羊,没有发现免疫损伤或由疫苗接种而导致的发病迹象,具有安全性。在0天、21天皮下或肌肉免疫牛、羊两次,在第二次免疫后14天时采外周血检测抗体滴度可达1:51200,具备良好的免疫原性。
附图说明
图1为MutiE蛋白DNA的PCR检测
图2为MutiE蛋白SDS-PAGE电泳检测
图3为本发明的布鲁氏杆菌基因工程亚单位疫苗免疫羊后ELISA抗体水平
图4为本发明的布鲁氏杆菌基因工程亚单位疫苗免疫牛后ELISA抗体水平
具体实施方式
下面通过实施例对本发明进行进一步详细说明,应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明,本领域技术人员应该理解的是,在不偏离本发明的结构思路、使用范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。
以下实施例中未注明具体条件的实验方法,通常按照常规条件,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著)中所述的条件,或按照制造厂商所建议的条件实施。
实施例1重组表达载体的构建
对来自Genebank的牛种、羊种、绵羊睾丸种、猪种、犬种布鲁氏杆菌的外膜蛋白OMP2、OMP25、OMP31、OMP28、OMP22蛋白的氨基酸序列进行生物信息学分析,从上述布鲁氏杆菌外膜蛋白中筛选得到如下抗原表位:羊种Omp2a蛋白(GenBank:AMM72580.1)的22-51位、144-158位;羊种Omp2b蛋白(GenBank:AMM72579.1)的233-245位、314-336;牛种Omp2b蛋白(GenBank:AAX94577.1)的4-29位;猪种Omp2b蛋白(GenBank:AAX94574.1)的41-53位;牛种Omp25蛋白(GenBank:AFJ79953.1)的4-46位、187-197位、143-154位;犬种Omp25蛋白(GenBank:CDL76104.1)的87-94位;猪种Omp25蛋白(GenBank:AHN46339.1)的120-137位;绵羊睾丸种Omp25蛋白(GenBank:ABU93464.1)的10-27位羊种Omp31蛋白(GenBank:ACS50328.1)的19-27位;犬种Omp31蛋白(GenBank:AAL27296.1)的4-14位;绵羊睾丸种Omp31蛋白(GenBank:AAL27294.1)的95-102位、224-237位;猪种Omp31蛋白(GenBank:AAL27286.1)的72-89位;绵羊睾丸种Omp22蛋白(GenBank:AAS84602.1)的157-177位;牛种Omp22蛋白(GenBank:AAS84601.1)的74-80位;羊种Omp28蛋白(GenBank:AAB17713.1)的240-247位;牛种Omp28蛋白(GenBank:AAX74798.1)的103-111位。所筛选表位蛋白的氨基酸序列如表1所示。
表1.表位蛋白氨基酸序列统计表
将所述表位蛋白的氨基酸序列经一定顺序的排序后,得到布鲁氏杆菌串联表位蛋白MutiE的氨基酸序列,如SEQ:ID:2所示。所述氨基酸序列经生物信息学软件翻译与密码子优化,得到布鲁氏杆菌串联表位蛋白MutiE的核苷酸序列,如SEQ:ID:1所示。人工合成所述MutiE蛋白的DNA片段,在DNA片段5’端引入Xba I酶切位点,在DNA片段的3’端引入Xho I酶切位点。将所述DNA片段连接到pET28a原核表达载体的多克隆位点中,构建重组表达载体pET28a-MutiE,如图1所示(“M”为DNA Marker、“1”为pET28a-MutiE载体样品、“2”为工程菌株原始种子样品、“3”为工程菌种生产种子样品、“NC”为未转化表达载体的空白大肠杆菌BL21样品)。
实施例2质粒稳定性鉴定
选用大肠杆菌Bl21细胞为宿主细菌,采用热激法将实施例1构建的重组pET28a-MutiE表达载体转化大肠杆菌Bl21感受态细胞,然后筛选稳定高表达MutiE蛋白的基因工程菌株。具体步骤如下:①在冰上解冻制备好的感受态大肠杆菌Bl21细胞100μL,然后加入制备好的pET28a-MutiE载体1.5μg;②混匀后,冰上放置30min,45℃水浴90s;③冰上放置3min,添加700μL LB培养基,37℃活化1h;④将活化好的菌液涂布于卡那霉素浓度为100μg/mL的LB固体平板中,于37℃倒置培养过夜;⑤随机挑选饱满的单菌落,编号后接种于卡那霉素浓度为100μg/mL的5mL LB液体培养基中,37℃震荡培养,6h后加IPTG至终浓度为1mM诱导表达2-3h。然后将表达MutiE蛋白的菌株划线传代培养40代,经鉴定,第10代、20代、30代与40代的菌种均可稳定表达MutiE蛋白,菌种生长情况与蛋白表达情况无差异。
实施例3种子库建立
原始种子库建立:取实施例2制备的菌种按接着量为1%接入卡那霉素浓度为100μg/mL的1.5mL LB培养基中,于37℃进行震荡培养至OD600达到0.8左右。然后将培养液用划线法接种于卡那霉素浓度为100μg/mL的LB固体平板中,于37℃倒置培养过夜。随机挑选饱满的单菌落,编号后接种于5mL卡那霉素浓度为100μg/mL的LB培养基中,37℃震荡培养。将培养至对数期的菌液按1:1比例与冻干保护剂混合,之后在无菌条件下取3mL于无菌10mL西林瓶中,冷冻干燥,命名为布鲁氏杆菌基因工程亚单位疫苗原始种子,PCR鉴定结果如图1所示。
生产种子库建立:取用LB培养基将原始种子复溶后,用划线法筛选单菌落,随机挑选饱满的单菌落扩大培养。将培养至对数期的菌液按1:1比例与冻干保护剂混合,之后在无菌条件下取3mL于无菌10mL西林瓶中,冷冻干燥,命名为布鲁氏杆菌基因工程亚单位疫苗生产种子。为保证生产批次间稳定,生产时每次取一只生产种子,直接活化后用于生产,PCR鉴定结果如图1所示。
实施例4工程菌株的发酵
取冻干保存的生产种子,按接种量为0.1%接种于卡那霉素浓度为100μg/mL的LB培养基中扩大培养,37℃震荡培养至对数期时。在发酵罐中提前加入发酵培养基并在线霉菌,待培养基温度降低至35-38℃时,将扩大培养的生产种子按1:1000比例加入发酵罐,37℃通气搅拌发酵。每小时记录一次OD值,待OD值大于20时,加入终浓度为1mM的IPTG诱导外源蛋白表达,继续发酵2-3小时。SDS-PAGE结果如图2(“M”为蛋白质Marker、“NC”为未转化表达载体的空白大肠杆菌BL21样品、“发酵”为工程菌种发酵后样品、“纯化”为疫苗制备用抗原样品)所示。
实施例5疫苗抗原溶液的制备
离心收集发酵后菌体,用冰冷的疫苗配制缓冲液按1:10(W/V)比例重悬,之后再次离心收集菌体,重复2-3次,去除培养基成分。将菌体用冰冷的疫苗配制缓冲液按1:10(W/V)重悬,用高压均质机破碎菌体,离心收集破碎后上清。上清用0.45μm孔径的中空纤维柱超滤去除杂质。收集滤出液,并用10kD截留分子量的中空纤维柱超滤浓缩,浓缩至原体积的20-30%。收集浓缩后的回流液,经BSA法蛋白质定量检测,总蛋白质含量应不低于30mg/mL,经SDS-PAGE电泳检测,如图2所示,MutiE蛋白质条带占比应不低于80%。回流液经除菌过滤后,即为疫苗配制用抗原溶液。所述抗原溶液的外观为淡黄色澄清液体,pH值为7.0-7.2,通过血平板进行杂菌鉴定结果为阴性,通过半流体和肉汤培养基培养进行支原体鉴定结果为阴性,采用Reed&Muench法计算抗血清中和效价高于1:2800。
实施例6布鲁氏杆菌基因工程亚单位疫苗的配制与应用
将实施例5中疫苗配制用抗原溶液用疫苗配制缓冲液稀释,确保MutiE蛋白质含量不小于10mg/mL,之后按1:1添加冻干保护剂混合均匀。按3mL/瓶,分装与无菌西林瓶后冷冻干燥并封口包装。最终成品为淡黄色疏松固体,即为15头份/瓶(绵羊及山羊用)或3头份/瓶(牛用)的布鲁氏杆菌基因工程亚单位疫苗,放2-8℃保存。所述疫苗使用15mL无菌生理盐水或氢氧化铝溶液稀释,则为含MutiE抗原1mg/mL的绵羊及山羊用布鲁氏杆菌基因工程亚单位疫苗,每只绵羊或山羊在0天、21天分别通过肌肉或皮下注射1mL可激发良好的免疫反应。所述疫苗使用3mL无菌生理盐水或氢氧化铝溶液稀释,则为含MutiE抗原5mg/mL的牛用布鲁氏杆菌基因工程亚单位疫苗,每头牛在0天、21天分别通过肌肉或皮下注射1mL可激发良好的免疫反应。
实验例7布鲁氏杆菌基因工程亚单位疫苗对实验动物的免疫应答实验
将实施例6制备的疫苗分别用无菌生理盐水与氢氧化铝溶液稀释,作为为实验组,同时用无菌生理盐水与氢氧化铝溶液为对照组,分别在0天、21天通过肌肉或皮下免疫4-6月龄的羔羊与6月龄的犊牛。末次免疫后14天采集各组实验羊与试验牛的外周血、分离血清。通过ELISA法测定血清抗体效价在1:12800以上,本发明疫苗不管是加入佐剂与没有佐剂均都能产生良好的抗体水平。实验羊与试验牛抗体水平检测结果如图3(V0为第一次免疫前、V1为第一次免疫后14天、V2为第二次免疫后14天)、图4(V0为第一次免疫前、V1为第一次免疫后14天、V2为第二次免疫后14天)所示。
实验例8布鲁氏杆菌基因工程亚单位疫苗的安全性实验
(1)无菌、支原体试验:将实施例6制备的疫苗分别接种硫乙醇酸盐培养基、营养琼脂斜面培养基和改良马丁培养基培养14d,并以无菌生理盐水做阴性对照,培养温度为25℃、35℃,结果应无细菌生长。分别将疫苗用半流体培养基和肉汤培养基,在37℃初代培养21天,次代培养21天,无菌生理盐水做阴性对照,结果应无显无支原体生长。
(2)溶血性试验:选取体重为350g左右的豚鼠,采集新鲜豚鼠血1mL,用PBS洗涤3次,再将血细胞体积恢复并稀释10倍。用生理盐水将实施例6所制备的疫苗分别稀释2倍、4倍、8倍,将豚鼠血细胞加入到稀释的待检佐剂中,8小时后,评价血球溶解以目测或者上清浓度检测为准,并在570nm处检测吸光度。结果应无血球破裂,无溶血现象。
(3)急性毒性试验:将实施例6制备的疫苗加1.5mL无菌生理盐水稀释,通过腹腔注射体重为12~18g Balb/C小鼠1.5mL,每组10只,同时设生理盐水对照组,连续14天内,实验小鼠应全部存活,没有出现竖毛、精神萎靡、行动迟缓等不良症状,而体重呈现增加。14天后处死进行解剖检查,应未见脏器有病理改变。说明在10倍羊的剂量、1.5倍牛的剂量下,所述疫苗对小鼠安全。
(5)兔热源质试验:取预检合格的体重为2~3kg家兔3只固定,30分钟后测量体温,共测2次,间隔30分钟,要求2次温差不大于0.2℃,各家兔2次平均温度在38.6-39.5℃。将实施例6制备的疫苗加1.5mL无菌生理盐水稀释,并预热至38℃,于第2次测温后15分钟内,自家兔耳边静脉分别缓缓注入0.5mL/只。注射后每隔30分钟测量体温1次,连测6次。结果应未引起家兔发热反应,家兔个体升温均应未超过0.2℃。
实验例9布鲁氏杆菌基因工程亚单位疫苗稳定性实验
将实施例6制备的疫苗分别在2-8℃、室温(20-25℃)、37℃条件下,放置1周、2周、1个月、3个月、6个月、12个月、15个月。取样观察外观、pH值、无菌、安全性均应表现正常。在室温放置6个月、在37℃放置1个月后免疫动物,抗体水平应达到1:12800。在2-8℃放置15个月免疫动物,抗体水平应达到1:12800。说明本发明疫苗有效期不低于12个月。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等均应包含在本发明的保护范围之内。
序列表
<110> 重庆市畜牧科学院
<120> 一种布鲁氏杆菌基因工程亚单位疫苗及其制备方法和应用
<130> 2021
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1005
<212> DNA
<213> Artificial Sequence
<400> 1
gcggcggatg cgattgtggc gccggaaccg gaagcggtgg aatatgtgcg cgtgtgcgat 60
gcgtatggcg cgggctattt ttatattccg ctgcagtttg cgtatattac cctgggcggc 120
tttaaagtgg gcattattgc gggcgtggtg gcgtatgata gcgtgattga agaagcggtg 180
accgcgaacg tggcgtatga actggtgccg ggctttaccg tgaccccgga agtgagctat 240
accaccagcg cgctggtgcc ggcgattgcg gtgggcaccg gcgtgaacgt gattgcggcg 300
ctgtatgtgg tgaccgatgt gaacgtggaa gtggcgagcg cggtgagcgt ggcgaacgcg 360
aaaaacctgc tgggcgcgag cctggtggcg gtgattacca gcaccagcgc gtatgcggcg 420
gatgcgattg tggcgcagga accggcgccg attgcgattg cgccgagctt tagctgggcg 480
ggcgcgtata ccgataacgt gctgctgcgc ctggaatatc gctttatgcc gtatattgcg 540
ggcggcgtgg cgtttggcga tcagattgtg tatggcgtgg aacgcgcgcg cgtgggctat 600
gatctgaacc cggtgatgcc gtatctgacc gcgggccgcg cgcgcgtggg ctatgatctg 660
aacccggtga tgccgtatct gaccgcgggc gcggcggatg tggtggtgag cgaaccggtg 720
attctggcga gcattgcggc gatgtttgcg gataacggcg tggtgctggg cgcggaaagc 780
aaagtgaact ttcataccgt gcgcgtgggc ctgaacagcg gcagcctgga tgtgaccgcg 840
ggcggctttg tgggcggcgt gcaggcgggc ggcgtgctga ttggcgcggg cgtggaacag 900
gcgctgagcg gcccgctgag cgtgaaagcg gaaggcggca ttgtggtggg caaaaacgtg 960
agcgtgaacg tggtgtttat tcagccgatt tatgtgtatc cggat 1005
<210> 2
<211> 335
<212> PRT
<213> Artificial Sequence
<400> 2
Ala Ala Asp Ala Ile Val Ala Pro Glu Pro Glu Ala Val Glu Tyr Val
1 5 10 15
Arg Val Cys Asp Ala Tyr Gly Ala Gly Tyr Phe Tyr Ile Pro Leu Gln
20 25 30
Phe Ala Tyr Ile Thr Leu Gly Gly Phe Lys Val Gly Ile Ile Ala Gly
35 40 45
Val Val Ala Tyr Asp Ser Val Ile Glu Glu Ala Val Thr Ala Asn Val
50 55 60
Ala Tyr Glu Leu Val Pro Gly Phe Thr Val Thr Pro Glu Val Ser Tyr
65 70 75 80
Thr Thr Ser Ala Leu Val Pro Ala Ile Ala Val Gly Thr Gly Val Asn
85 90 95
Val Ile Ala Ala Leu Tyr Val Val Thr Asp Val Asn Val Glu Val Ala
100 105 110
Ser Ala Val Ser Val Ala Asn Ala Lys Asn Leu Leu Gly Ala Ser Leu
115 120 125
Val Ala Val Ile Thr Ser Thr Ser Ala Tyr Ala Ala Asp Ala Ile Val
130 135 140
Ala Gln Glu Pro Ala Pro Ile Ala Ile Ala Pro Ser Phe Ser Trp Ala
145 150 155 160
Gly Ala Tyr Thr Asp Asn Val Leu Leu Arg Leu Glu Tyr Arg Phe Met
165 170 175
Pro Tyr Ile Ala Gly Gly Val Ala Phe Gly Asp Gln Ile Val Tyr Gly
180 185 190
Val Glu Arg Ala Arg Val Gly Tyr Asp Leu Asn Pro Val Met Pro Tyr
195 200 205
Leu Thr Ala Gly Arg Ala Arg Val Gly Tyr Asp Leu Asn Pro Val Met
210 215 220
Pro Tyr Leu Thr Ala Gly Ala Ala Asp Val Val Val Ser Glu Pro Val
225 230 235 240
Ile Leu Ala Ser Ile Ala Ala Met Phe Ala Asp Asn Gly Val Val Leu
245 250 255
Gly Ala Glu Ser Lys Val Asn Phe His Thr Val Arg Val Gly Leu Asn
260 265 270
Ser Gly Ser Leu Asp Val Thr Ala Gly Gly Phe Val Gly Gly Val Gln
275 280 285
Ala Gly Gly Val Leu Ile Gly Ala Gly Val Glu Gln Ala Leu Ser Gly
290 295 300
Pro Leu Ser Val Lys Ala Glu Gly Gly Ile Val Val Gly Lys Asn Val
305 310 315 320
Ser Val Asn Val Val Phe Ile Gln Pro Ile Tyr Val Tyr Pro Asp
325 330 335
Claims (10)
1.一种布鲁氏杆菌外膜蛋白串联表位蛋白,其特征在于,所述布鲁氏杆菌外膜蛋白串联表位蛋白的氨基酸序列由羊种Omp2a蛋白、羊种Omp2b蛋白、牛种Omp2b蛋白、猪种Omp2b蛋白、牛种Omp25蛋白、犬种Omp25蛋白、猪种Omp25蛋白、绵羊睾丸种Omp25蛋白、羊种Omp31蛋白、犬种Omp31蛋白、绵羊睾丸种Omp31蛋白、猪种Omp31蛋白、绵羊睾丸种Omp22蛋白、牛种Omp22蛋白、羊种Omp28蛋白、和牛种Omp28蛋白的氨基酸序列组成。
2.根据权利要求1所述的布鲁氏杆菌外膜蛋白串联表位蛋白,其特征在于,所述布鲁氏杆菌外膜蛋白串联表位蛋白的氨基酸序列由羊种Omp2a蛋白的22-51位、144-158位;羊种Omp2b蛋白的233-245位、314-336;牛种Omp2b蛋白的4-29位;猪种Omp2b蛋白的41-53位;牛种Omp25蛋白的4-46位、187-197位、143-154位;犬种Omp25蛋白的87-94位;猪种Omp25蛋白的120-137位;绵羊睾丸种Omp25蛋白的10-27位;羊种Omp31蛋白的19-27位;犬种Omp31蛋白的4-14位;绵羊睾丸种Omp31蛋白的95-102位、224-237位;猪种Omp31蛋白的72-89位;绵羊睾丸种Omp22蛋白的157-177位;牛种Omp22蛋白的74-80位;羊种Omp28蛋白的240-247位和牛种Omp28蛋白的103-111位氨基酸序列组成。
3.根据权利要求1所述的布鲁氏杆菌外膜蛋白串联表位蛋白,其特征在于,所述布鲁氏杆菌外膜蛋白串联表位蛋白的氨基酸序列如SEQ ID NO.2所示。
4.一种布鲁氏杆菌外膜蛋白串联表位蛋白,其特征在于,所述布鲁氏杆菌外膜蛋白串联表位蛋白的核苷酸序列如SEQ ID NO.1所示。
5.一种表达布鲁氏杆菌外膜蛋白串联表位蛋白的重组质粒,其特征在于,所述重组质粒包含权利要求4所述的布鲁氏杆菌外膜蛋白串联表位蛋白的核苷酸序列。
6.一种利用权利要求5所述的重组质粒制备得到的基因工程菌株。
7.利用权利要求6所述的基因工程菌株制备得到的布鲁氏杆菌亚单位疫苗。
8.权利要求7所述的布鲁氏杆菌亚单位疫苗的制备方法,其特征在于,包括如下步骤:
(1)发酵培养所述基因工程菌株;
(2)加入疫苗配制缓冲液重悬、破碎、离心得到上清液;
(3)将所述上清液纯化过滤得到疫苗配制用抗原溶液;
(4)添加冻干保护剂冷冻干燥得到布鲁氏杆菌亚单位疫苗。
9.根据权利要求8所述的方法,其特征在于,所述冻干保护剂由Tris 6.057g/L、EDTA0.146g/L、NaCl 2.922g/L、蔗糖100g/L、脱脂奶粉200g/L和水组成。
10.权利要求7所述的布鲁氏杆菌亚单位疫苗在制备抗布鲁氏杆菌抗体中的应用。
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