CN113913442A - 一种布鲁氏菌蛋白抗原及其原核表达方法和应用 - Google Patents
一种布鲁氏菌蛋白抗原及其原核表达方法和应用 Download PDFInfo
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- CN113913442A CN113913442A CN202010661025.1A CN202010661025A CN113913442A CN 113913442 A CN113913442 A CN 113913442A CN 202010661025 A CN202010661025 A CN 202010661025A CN 113913442 A CN113913442 A CN 113913442A
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Abstract
本发明属于基因工程技术领域，特别是涉及一种布鲁氏菌蛋白及其原核表达方法和应用。本发明采用原核表达载体构建包含抗原蛋白BAB的编码基因的重组载体，实现了抗原蛋白BAB的原核表达，方法简单易行，获得的表达产物在纯化后不经任何处理即具有明显的免疫效果，为布鲁氏菌诊断方法及iELISA诊断试剂盒的建立提供基础。
Description
技术领域
本发明属于基因工程技术领域，特别是涉及一种布鲁氏菌蛋白抗原及其原核表达方法和应用。
背景技术
布鲁菌病(布病)是由布鲁氏菌引起的以流产和发热为特征的人兽共患病，严重地威胁着人和多种动物的生命健康，对农业生产和公共卫生造成了巨大的威胁。布鲁氏菌具有内寄生的特性，因此被感染的人或动物一般需要接受长时间的抗生素治疗，而且往往会留下严重后遗症。
反刍动物和猪对布鲁氏菌高度易感。布鲁氏菌特征性地侵害其生殖器官并导致母畜流产和不孕。当患病母畜分娩时，胎盘组织中布鲁氏菌达到极高数量，每克组织中细菌数可高达1014个，因此母畜流产是该病的传染性病症。在世界卫生组织(WHO)实验室生物安全手册中，布鲁氏菌被列为Ⅲ类危险级。布鲁氏菌病易传播给人，引起急性发热性疫病(波浪热)，可进一步发展成慢性型，引起肌肉-骨骼系统、心血管系统和中枢神经系统的严重并发症。本病流行区域应采取预防措施，防止人感染。职业性接触是常见的感染原因，口腔、呼吸道和结膜是基本感染途径。
布鲁菌是一种兼性胞内寄生菌，感染人畜后可在宿主体内建立慢性感染且难以被彻底清除，布鲁菌被认为缺乏病原菌所具有的经典毒力因子，因此对毒力基因的鉴定和研究对阐述其致病机制有着重要的意义。
鉴于此，特提出本发明。
发明内容
本发明的首要发明目的在于提供一种编码布鲁氏菌蛋白抗原的基因序列。
本发明的第二发明目的在于提供一种布鲁氏菌蛋白抗原的重组载体。
本发明的第三发明目的在于提供布鲁氏菌蛋白抗原的原核表达的方法。
本发明的第四发明目的在于提供该布鲁氏菌蛋白抗原的应用。
为了实现本发明的发明目的，采用的技术方案为：
本发明涉及一种编码布鲁氏菌蛋白抗原的基因序列，所述基因序列的核苷酸序列如SEQ ID No.1所示。
本发明还涉及一种布鲁氏菌蛋白抗原的重组载体，所述重组载体中包含如SEQ IDNo.1所示的基因序列。
本发明还涉及一种布鲁氏菌蛋白抗原的原核表达的方法，至少包括以下步骤：
(1)扩增编码布鲁氏菌蛋白抗原的基因序列，所述基因序列的核苷酸序列如SEQID No.1所示，
(2)构建重组载体：将含有SEQ ID No.1所示核苷酸序列的PCR扩增产物连接到pMD19-T克隆载体中，构建得到重组质粒pMD19-BAB；分别酶切重组质粒pMD19-BAB和原核表达载体pET-30a(+)，构建得到包含BAB的编码基因的重组载体pET30-BAB；
(3)表达重组载体pET30-BAB：将重组载体pET30-BAB转化至BL21菌株中，利用IPTG诱导后收集包含BAB的细菌，经离心、纯化得到所述布鲁氏菌蛋白抗原。
可选的，在步骤(1)中，扩增编码布鲁氏菌蛋白抗原的基因序列的引物如SEQ IDNo.2、SEQ ID No.3所示。
可选的，在步骤(3)中，采用含尿素和咪唑的PBS重悬液处理离心破碎后的细胞菌液，离心并收集沉淀，获得沉淀重悬液；采用梯度离心法纯化所述沉淀重悬液，获得纯化后的抗原蛋白。
本发明还涉及一种由本发明的基因序列编码、或由本发明所述的方法制备得到布鲁氏菌蛋白抗原。
具体的，所述布鲁氏菌蛋白抗原的氨基酸序列如SEQ ID No.4所示。
本发明还涉及布鲁氏菌蛋白抗原在制备用于检测布鲁氏菌的试剂或试剂盒中的应用。
本发明至少具有以下有益的效果：
本发明采用原核表达载体构建包含抗原蛋白BAB的编码基因的重组载体，实现了抗原蛋白BAB的原核表达，方法简单易行，获得的表达产物在纯化后不经任何处理即具有明显的免疫效果，为布鲁氏菌诊断方法及iELISA诊断试剂盒的建立提供基础。
附图说明
图1为PCR扩增或胶回收产物凝胶电泳结果；
图2为双酶切PCR回收产物和pET30a载体凝胶电泳结果；
图3为SDS-PAGE检测蛋白表达的电泳结果；
图4为SDS-PAGE电泳分析蛋白在沉淀中表达的电泳结果；
图5为目的蛋白纯化后SDS-PAGE电泳结果；
图6为目的蛋白纯化后Western-Blot电泳结果；
图7为高免血清的鉴定Western-B Blot电泳结果。
保藏说明
重组质粒pET-30a-BAB保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.19806，分类命名：大肠埃希氏菌Escherichia coli，保藏日期为2020年5月11日。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本申请而不用于限制本发明的范围。
具体实施方式
本发明实施例从布鲁氏菌Rev.I株基因组中扩增BAB-22915基因片段。布鲁菌的BAB22915基因为编码裂解酶基因，突变株表现出在RAW264.7细胞和小鼠体内存活率下降；突变株BAB22915在宿主细胞内不与溶酶体融合，不引起明显的病理损伤。本发明实施例构建重组质粒pET-30a-BAB进行原核表达、纯化后经SDS-PAGE鉴定，得到较纯蛋白，并经实验发明该蛋白具有良好的反应原性。
具体的，编码该布鲁氏菌蛋白抗原的基因序列如SEQ ID No.1所示。SEQ ID No.1所示的核苷酸序列具体为：
ggatccatgaagattatcgcctcagttctggctgcggccttgtttgcctccacggctctatcgggagccggggcagcgacaaccccggctcccgcagccgcgacagataatgccgtcaacatgccgaagccggaatgtgaaactgacttcggtcaatggatggaaaatctgaccagggaagcccgtgaagccggcgtcggtgaaaaaggcattgccgaattgcacaaggcttccatcgaccagaaagttctgggccgcgaccgcaagcagaccgttttcaatctcactttcaccgaattttccaagcgcctcatttccgaggcacgcctgaagaaaggccaggaaaatctcgtcaaatatgccgatgtcttcaagaaagttgaagatgcttatggcgttccgggtccggttcttgccgctttctgggggcttgaaaccgactatggcgcgattcagggcgatttcgacacgctcaatgcgctcgtcacgctctcctacgactgccgccgcccggacctgttccgtccgcagctcatcgccctgctcaagctcttcgatcagggcgtggtcgatgcaaatacgaccggcgcctgggcaggcgaaatcggcatgatgcagcttctgccgaaagactatctggaacgcggcgtcgatggcgacggcgatggcaaggtcgatctgaagaacagcgtgcccgatgccatgatgaccgcagggcgcatgctttcggaactgggctggaagcgcggccagccatggcttgaagaagtgcgcctcaccaaggacctcccctgggaagaggcgatccgcaccaaccgcaagccgcattcatggtgggctgagcatggcgtgacgggcctcaatggcccactcggccctgatgatggcgatgcatcccttctgctgccgcttggccgcaagggaccggctttcctgtcctatctgaattttgacatcttcgtcgaatggaacaaatccatagtctatgcgacgactgccgcctattttgcaacccgccttgcaggtgcccctgctttcgatccgggcaccccggttccggggctgacgcaggatcaactcaaggaattgcagaccaagcttcaggcgcgcggttatgaaatgggcaagatcgatggtgttttcggtgtcgcgacccgcgatgcggtacgctccgaacagttgcgccttggcatgcccgccgattcctggccgacacaggaacttctcgacaggcttctcgag
本发明实施例的基因序列所编码的布鲁氏菌蛋白抗原是一种新型的免疫抗原，由414个氨基酸构成。
本发明实施例还涉及一种布鲁氏菌蛋白抗原的重组载体，重组载体中包含如SEQID No.1所示的基因序列。
本发明实施例还涉及一种布鲁氏菌蛋白抗原的原核表达的方法，该原核表达方法获得的抗原蛋白在纯化后不经任何处理即具有明显的免疫原性。
具体的，至少包括以下步骤：
(1)扩增编码布鲁氏菌蛋白抗原的基因序列，基因序列的核苷酸序列如SEQ IDNo.1所示，
(2)构建重组载体：将含有SEQ ID No.1所示核苷酸序列的PCR扩增产物连接到pMD19-T克隆载体中，构建得到重组质粒pMD19-BAB；分别酶切重组质粒pMD19-BAB和原核表达载体pET-30a(+)，构建得到包含BAB的编码基因的重组载体pET30-BAB；
(3)表达重组载体pET30-BAB：将重组载体pET30-BAB转化至BL21菌株中，利用IPTG诱导后收集包含BAB的细菌，经离心、纯化得到所述布鲁氏菌蛋白抗原。
可选的，在步骤(1)中，扩增编码布鲁氏菌蛋白抗原的基因序列的引物如SEQ IDNo.2、SEQ ID No.3所示。
具体的，SEQ ID No.2、SEQ ID No.3的核苷酸序列如表1所示：
表1
| 核苷酸编号 | 名称 | 核苷酸序列 |
| SEQ ID No.2 | 正向引物 | cgcg<u>gatcc</u>gcgatgaagattatcgcctc |
| SEQ ID No.3 | 反向引物 | ccg<u>ctcgag</u>cggtcaaagcctgtcgagaa |
可选的，在步骤(3)中，采用含尿素和咪唑的PBS重悬液处理离心破碎后的细胞菌液，离心并收集沉淀，获得沉淀重悬液；采用梯度离心法纯化沉淀重悬液，获得纯化后的抗原蛋白。
本发明实施例还涉及一种由本发明的基因序列编码、或由本发明所述的方法制备得到布鲁氏菌蛋白抗原。具体的，该蛋白抗原的氨基酸序列如SEQ ID No.4所示，经实验证实该蛋白具有良好的反应原性。
具体的，SEQ ID No.4的氨基酸序列为：
GSMKIIASVLAAALFASTALSGAGAATTPAPAAATDNAVNMPKPECETDFGQWMENLTREAREAGVGEKGIAELHKASIDQKVLGRDRKQTVFNLTFTEFSKRLISEARLKKGQENLVKYADVFKKVEDAYGVPGPVLAAFWGLETDYGAIQGDFDTLNALVTLSYDCRRPDLFRPQLIALLKLFDQGVVDANTTGAWAGEIGMMQLLPKDYLERGVDGDGDGKVDLKNSVPDAMMTAGRMLSELGWKRGQPWLEEVRLTKDLPWEEAIRTNRKPHSWWAEHGVTGLNGPLGPDDGDASLLLPLGRKGPAFLSYLNFDIFVEWNKSIVYATTAAYFATRLAGAPAFDPGTPVPGLTQDQLKELQTKLQARGYEMGKIDGVFGVATRDAVRSEQLRLGMPADSWPTQELLDRLLE
本发明实施例还涉及布鲁氏菌蛋白抗原在制备用于检测布鲁氏菌的试剂或试剂盒中的应用。
下面通过具体实施例对本发明的实验步骤、过程和结果做具体说明。
实施例1
一、采用的材料和来源如下：
1、菌种与质粒
羊种布鲁氏菌Rev.I核酸、大肠杆菌(Escherichia coli)DH5α菌株、BL21(DE3)菌株及pET-30a载体。
2、试剂
DNA Marker：购自大连宝生物工程有限公司；T4连接酶：购自NEB公司；限制性内切酶(EcoRI/XhoI)、2×Es TaqMasterMix、细菌质粒小提取试剂盒以及琼脂糖凝胶回收试剂盒：购自thermo fisher scientific公司；HRP标记的兔抗羊IgG抗体：购自abcom公司；蛋白Marker：购自北京全式金公司；显色剂：购自碧云天生物公司。
3、主要仪器
超净工作台；PCR仪；制冰机；超速离心机；恒温培养箱；恒温振荡器；高速冷冻离心机；超声波细胞粉碎机；水浴锅；聚丙烯酰胺凝胶电泳仪；蛋白纯化仪等。
二、原核表达方法如下
1、目的基因扩增
将羊种布鲁氏菌Rev.I株灭活菌液(水浴锅中85℃灭活1h)，按细菌DNA提取试剂盒操作步骤进行菌体DNA提取。
根据GenBank中登录的布鲁氏菌BAB22915基因序列中BAB基因设计引物，具体如表1所示，由北京华大基因有限公司合成。
目的基因PCR扩增采用25μL反应体系：
Mix 12.5μL、ddH2O 9.5μL、上游引物1μL、下游引物1μL、DNA模板1μL。
反应条件为：95℃变性3min；94℃变性45s，35个循环，55℃退火45s，72℃延伸2min，最后72℃延伸10min。
所得PCR产物用2％琼脂糖凝胶电泳进行检测，得到凝胶电泳结果如图1所示，其中，M为maker，1～3为PCR扩增BAB产物。回收目的基因片段，将其与pMD19-T载体在16℃条件下连接，构建重组质粒pMD19-T-BAB。
重组质粒转化至大肠杆菌DH5α感受态细胞，并在氨苄抗性的LB固体培养基上进行阳性筛选，然后进行菌液PCR并送往北京华大有限公司进行测序鉴定，将测序结果在GenBank中进行比对。与羊种布鲁氏菌Rev.1株同源性为99％。测序正确。
2、原核表达载体构建
将送公司测序正确的质粒pMD19-T-BAB和pET-30a载体用限制性内切酶BamHI和XhoI进行双酶切，经琼脂糖凝胶回收试剂盒回收目的片段和酶切后的线性pET-30a载体，得到凝胶电泳结果如图2所示，其中，M为maker，1为双酶切PCR回收产物BAB，2为双酶切载体pET-30a。将回收的产物按一定摩尔比在16℃金属浴中过夜连接，后转化至大肠杆菌BL21(DE3)感受态细胞，涂布于卡那抗性平板培养12～16h，对阳性克隆进行摇菌提取质粒，并进行菌液RCR检测及质粒双酶切鉴定。鉴定结果如图2所示，条带大小与目的片段一致，重组质粒pET-30a-BAB构建成功，将该重组质粒进行保藏，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCCNo.19806，分类命名：大肠埃希氏菌Escherichia coli，保藏日期为2020年5月11日。
3、重组蛋白SDS-PAGE电泳检测
从转化的平板上挑单克隆到15mL含相应抗性的LB液体培养基中，37℃，200r/min培养至OD＝0.6～0.8，用0.5mmol/L的IPTG，37℃，200r/min诱导培养2h。取1mL诱导的菌液，12 000r/min，离心1min，弃上清；将沉淀用10mmol/L Tris-HCl pH8.0溶液吹散，将等体积的缓冲液及2×loading buffer加入到1.5mL离心管中，100℃煮10min，SDS-PAGE电泳检测后，按照1:50比例接菌到卡那抗性的LB液体培养基中进行诱导表达。得到的实验结果如图3和图4所示。在图3中，M为maker，1～2为IPTG诱导沉淀(BL21)，3为未诱导对照(BL21)、12％SDS-PAGE；在图4中，M为maker，1为超声后上清，2为超声后、12％SDS-PAGE。
4重组蛋白纯化
用20～30mL10mM Tris-HCL(pH8.0)溶液重悬超声离心得到的沉淀，静置10min；将静置溶液12 000r/min，离心10min，上清转入另一管中保存；用20～30mL10mM Tris-HCL(pH8.0)溶液重悬沉淀，静置10min；12 000r/min，离心10min，弃上清；重复此步骤一次；加入5～10mL10mM Tris-HCL(pH8.0)溶液重悬沉淀，再加5～10mL含8M尿素的10mM Tris-HCL(pH8.0)溶液溶解蛋白。12000rpm，离心10min，收集上清，取50μL电泳。得到的实验结果如图5所示，其中，M为make，1为纯化蛋白稀释5倍、12％SDS-PAGE。
5重组蛋白Western-Blot检测
对纯化的蛋白进行SDS-PAGE后转移到PVDF膜上，14V电压电转50min；将膜放入预先用PBST缓冲液洗涤后的平皿，用1％的脱脂乳4℃过夜封闭，用PBST缓冲液洗涤PVDF膜3次，每次15min；加入1:50比例稀释的经本实验室用虎红平板凝集试验鉴定的羊种布鲁氏菌阳性血清，37℃孵育4h；用PBST缓冲液洗涤PVDF膜3次，每次15min，然后加入1:5 000比例稀释的辣根过氧化氢酶标记的兔抗羊IgG抗体，37℃孵育2h；PBST缓冲液洗涤PVDF膜3次，每次15min，后用显色剂显色后拍照。得到的实验结果如图6所示，其中，M为make，1为纯化蛋白、12％SDS-PAGE。
实施例2免疫原性实验方法和结果
1.1实验动物
BALB/c小鼠6只，分为2组，一组为实施例1制备得到的免疫纯化后BAB蛋白。一组为对照组，免疫PBS。
1.2方法
1.2.1高纯度抗原的制备
取实施例1制备得到的纯化后的BAB蛋白，BCA试剂盒测稀释液浓度，根据公式C1V1＝C2V2将终浓度稀释至2mg/mL。将1mL的弗氏佐剂(首免为完全佐剂，二免、三免为不完全佐剂)和抗原溶液分别吸入两个注射器内，两注射器之间以一细胶管相连，注意排净空气，然后交替推动针管，直至形成粘稠的乳剂为止。将乳化剂滴入冷水中，若保持完整不分散，成滴状浮于水面，即乳化完全。
1.2.2.高免疫血清的制备
1.2.2.1动物免疫
采用皮下多点注射对6只小鼠进行免疫。首先将6只小鼠分为两组，第一组为实验组，皮下注射高纯度抗原。第二组为对照组，皮下注射等剂量PBS。首免7天后进行二免，21天后进行第三次加强免疫。
1.2.2.2血清中抗体的检测和抗血清采集
小鼠免后10～14天试血，WB鉴定抗体是否为阳性，如WB条带明显，第21天三免后，心脏采血或颈动脉放血，按常规的方法分离、收集血清。分装小管，每管1～2mL。
1.2.3高免血清的鉴定
对纯化的蛋白进行SDS-PAGE后转移到PVDF膜上，14V电压电转50min；将膜放入预先用PBST缓冲液洗涤后的平皿，用1％的脱脂乳4℃过夜封闭，用PBST缓冲液洗涤PVDF膜3次，每次15min；加入1:50比例稀释的高免血清，37℃孵育4h；用PBST缓冲液洗涤PVDF膜3次，每次15min，然后加入1:5000比例稀释的辣根过氧化氢酶标记的兔抗鼠IgG抗体，37℃孵育2h；PBST缓冲液洗涤PVDF膜3次，每次15min，后用显色剂显色后拍照。得到的实验结果如图7所示，其中，M为make，1为目的条带、12％SDS-PAGE。
本申请虽然以较佳实施例公开如上，但并不是用来限定权利要求，任何本领域技术人员在不脱离本申请构思的前提下，都可以做出若干可能的变动和修改，因此本申请的保护范围应当以本申请权利要求所界定的范围为准。
序列表
<110> 内蒙古农业大学
<120> 一种布鲁氏菌蛋白抗原及其原核表达方法和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1242
<212> DNA
<213> 羊属布鲁氏菌(Brucella melitensis)
<400> 1
ggatccatga agattatcgc ctcagttctg gctgcggcct tgtttgcctc cacggctcta 60
tcgggagccg gggcagcgac aaccccggct cccgcagccg cgacagataa tgccgtcaac 120
atgccgaagc cggaatgtga aactgacttc ggtcaatgga tggaaaatct gaccagggaa 180
gcccgtgaag ccggcgtcgg tgaaaaaggc attgccgaat tgcacaaggc ttccatcgac 240
cagaaagttc tgggccgcga ccgcaagcag accgttttca atctcacttt caccgaattt 300
tccaagcgcc tcatttccga ggcacgcctg aagaaaggcc aggaaaatct cgtcaaatat 360
gccgatgtct tcaagaaagt tgaagatgct tatggcgttc cgggtccggt tcttgccgct 420
ttctgggggc ttgaaaccga ctatggcgcg attcagggcg atttcgacac gctcaatgcg 480
ctcgtcacgc tctcctacga ctgccgccgc ccggacctgt tccgtccgca gctcatcgcc 540
ctgctcaagc tcttcgatca gggcgtggtc gatgcaaata cgaccggcgc ctgggcaggc 600
gaaatcggca tgatgcagct tctgccgaaa gactatctgg aacgcggcgt cgatggcgac 660
ggcgatggca aggtcgatct gaagaacagc gtgcccgatg ccatgatgac cgcagggcgc 720
atgctttcgg aactgggctg gaagcgcggc cagccatggc ttgaagaagt gcgcctcacc 780
aaggacctcc cctgggaaga ggcgatccgc accaaccgca agccgcattc atggtgggct 840
gagcatggcg tgacgggcct caatggccca ctcggccctg atgatggcga tgcatccctt 900
ctgctgccgc ttggccgcaa gggaccggct ttcctgtcct atctgaattt tgacatcttc 960
gtcgaatgga acaaatccat agtctatgcg acgactgccg cctattttgc aacccgcctt 1020
gcaggtgccc ctgctttcga tccgggcacc ccggttccgg ggctgacgca ggatcaactc 1080
aaggaattgc agaccaagct tcaggcgcgc ggttatgaaa tgggcaagat cgatggtgtt 1140
ttcggtgtcg cgacccgcga tgcggtacgc tccgaacagt tgcgccttgg catgcccgcc 1200
gattcctggc cgacacagga acttctcgac aggcttctcg ag 1242
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cgcggatccg cgatgaagat tatcgcctc 29
<210> 3
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccgctcgagc ggtcaaagcc tgtcgagaa 29
<210> 4
<211> 414
<212> PRT
<213> 羊属布鲁氏菌(Brucella melitensis)
<400> 4
Gly Ser Met Lys Ile Ile Ala Ser Val Leu Ala Ala Ala Leu Phe Ala
1 5 10 15
Ser Thr Ala Leu Ser Gly Ala Gly Ala Ala Thr Thr Pro Ala Pro Ala
20 25 30
Ala Ala Thr Asp Asn Ala Val Asn Met Pro Lys Pro Glu Cys Glu Thr
35 40 45
Asp Phe Gly Gln Trp Met Glu Asn Leu Thr Arg Glu Ala Arg Glu Ala
50 55 60
Gly Val Gly Glu Lys Gly Ile Ala Glu Leu His Lys Ala Ser Ile Asp
65 70 75 80
Gln Lys Val Leu Gly Arg Asp Arg Lys Gln Thr Val Phe Asn Leu Thr
85 90 95
Phe Thr Glu Phe Ser Lys Arg Leu Ile Ser Glu Ala Arg Leu Lys Lys
100 105 110
Gly Gln Glu Asn Leu Val Lys Tyr Ala Asp Val Phe Lys Lys Val Glu
115 120 125
Asp Ala Tyr Gly Val Pro Gly Pro Val Leu Ala Ala Phe Trp Gly Leu
130 135 140
Glu Thr Asp Tyr Gly Ala Ile Gln Gly Asp Phe Asp Thr Leu Asn Ala
145 150 155 160
Leu Val Thr Leu Ser Tyr Asp Cys Arg Arg Pro Asp Leu Phe Arg Pro
165 170 175
Gln Leu Ile Ala Leu Leu Lys Leu Phe Asp Gln Gly Val Val Asp Ala
180 185 190
Asn Thr Thr Gly Ala Trp Ala Gly Glu Ile Gly Met Met Gln Leu Leu
195 200 205
Pro Lys Asp Tyr Leu Glu Arg Gly Val Asp Gly Asp Gly Asp Gly Lys
210 215 220
Val Asp Leu Lys Asn Ser Val Pro Asp Ala Met Met Thr Ala Gly Arg
225 230 235 240
Met Leu Ser Glu Leu Gly Trp Lys Arg Gly Gln Pro Trp Leu Glu Glu
245 250 255
Val Arg Leu Thr Lys Asp Leu Pro Trp Glu Glu Ala Ile Arg Thr Asn
260 265 270
Arg Lys Pro His Ser Trp Trp Ala Glu His Gly Val Thr Gly Leu Asn
275 280 285
Gly Pro Leu Gly Pro Asp Asp Gly Asp Ala Ser Leu Leu Leu Pro Leu
290 295 300
Gly Arg Lys Gly Pro Ala Phe Leu Ser Tyr Leu Asn Phe Asp Ile Phe
305 310 315 320
Val Glu Trp Asn Lys Ser Ile Val Tyr Ala Thr Thr Ala Ala Tyr Phe
325 330 335
Ala Thr Arg Leu Ala Gly Ala Pro Ala Phe Asp Pro Gly Thr Pro Val
340 345 350
Pro Gly Leu Thr Gln Asp Gln Leu Lys Glu Leu Gln Thr Lys Leu Gln
355 360 365
Ala Arg Gly Tyr Glu Met Gly Lys Ile Asp Gly Val Phe Gly Val Ala
370 375 380
Thr Arg Asp Ala Val Arg Ser Glu Gln Leu Arg Leu Gly Met Pro Ala
385 390 395 400
Asp Ser Trp Pro Thr Gln Glu Leu Leu Asp Arg Leu Leu Glu
405 410
Claims (8)
1.一种编码布鲁氏菌蛋白抗原的基因序列，其特征在于，所述基因序列的核苷酸序列如SEQ ID No.1所示。
2.一种布鲁氏菌蛋白抗原的重组载体，其特征在于，所述重组载体中包含如SEQ IDNo.1所示的基因序列。
3.一种布鲁氏菌蛋白抗原的原核表达的方法，其特征在于，至少包括以下步骤：
(1)扩增编码布鲁氏菌蛋白抗原的基因序列，所述基因序列的核苷酸序列如SEQ IDNo.1所示，
(2)构建重组载体：将含有SEQ ID No.1所示核苷酸序列的PCR扩增产物连接到pMD19-T克隆载体中，构建得到重组质粒pMD19-BAB；分别酶切重组质粒pMD19-BAB和原核表达载体pET-30a(+)，构建得到包含BAB的编码基因的重组载体pET30-BAB；
(3)表达重组载体pET30-BAB：将重组载体pET30-BAB转化至BL21菌株中，利用IPTG诱导后收集包含BAB的细菌，经离心、纯化得到所述布鲁氏菌蛋白抗原。
4.根据权利要3所述的原核表达的方法，其特征在于，在步骤(1)中，扩增编码布鲁氏菌蛋白抗原的基因序列的引物如SEQ ID No.2、SEQ ID No.3所示。
5.根据权利要3所述的原核表达的方法，其特征在于，在步骤(3)中，采用含尿素和咪唑的PBS重悬液处理离心破碎后的细胞菌液，离心并收集沉淀，获得沉淀重悬液；采用梯度离心法纯化所述沉淀重悬液，获得纯化后的抗原蛋白。
6.如权利要求1所述的基因序列编码、或由权利要求3～5任一项所述的方法制备得到布鲁氏菌蛋白抗原。
7.根据权利要求3所述的布鲁氏菌蛋白抗原，其特征在于，所述布鲁氏菌蛋白抗原的氨基酸序列如SEQ ID No.4所示。
8.如权利要求6所述的布鲁氏菌蛋白抗原在制备用于检测布鲁氏菌的试剂或试剂盒中的应用。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20110117134A1 (en) * | 2009-11-16 | 2011-05-19 | Ding Xuan Z | Gene library of Brucella suis surface antigens |
| CN102532271A (zh) * | 2011-12-14 | 2012-07-04 | 南方医科大学 | 一种布鲁氏菌b细胞表位及其单克隆抗体和应用 |
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| US20110117134A1 (en) * | 2009-11-16 | 2011-05-19 | Ding Xuan Z | Gene library of Brucella suis surface antigens |
| CN102532271A (zh) * | 2011-12-14 | 2012-07-04 | 南方医科大学 | 一种布鲁氏菌b细胞表位及其单克隆抗体和应用 |
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| BAO YANQING 等: "Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate", 《VETERINARY RESEARCH》, vol. 48, no. 17, 31 December 2017 (2017-12-31), pages 1 - 13 * |
| SETUBAL, J.C. 等: "GenBank: EEP63355.1", 《GENBANK》, 19 May 2009 (2009-05-19) * |
| 杭天宇 等: "羊种布鲁氏菌Rev.1株VjbR基因的克隆、原核表达及生物信息学分析", 《中国动物检疫》, vol. 36, no. 12, 6 December 2019 (2019-12-06), pages 68 - 75 * |
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