CN107129527A - 一种马链球菌兽疫亚种保护性抗原hp0623及其制备方法 - Google Patents
一种马链球菌兽疫亚种保护性抗原hp0623及其制备方法 Download PDFInfo
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- CN107129527A CN107129527A CN201710395446.2A CN201710395446A CN107129527A CN 107129527 A CN107129527 A CN 107129527A CN 201710395446 A CN201710395446 A CN 201710395446A CN 107129527 A CN107129527 A CN 107129527A
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Abstract
本发明涉及一种马链球菌兽疫亚种保护性抗原HP0623及其制备方法，所述马链球菌兽疫亚种保护性抗原HP0623为SEZ HP0623重组蛋白，由429个氨基酸残基组成，分子量为47.04kDa；所述制备方法包括基因克隆、酶切酶连、诱导表达、蛋白纯化；所述HP0623可用于疫苗的制备。本发明所述马链球菌兽疫亚种保护性抗原HP0623在小鼠实验中表现出良好的免疫反应原性，产生的抗体能提供较强保护力，诱导高水平的杀菌能力，马链球菌兽疫亚种保护性抗原HP0623位于SEZ表面，rHP0623可以抑制SEZ对Hep‑2细胞的粘附。
Description
技术领域
本发明涉及链球菌保护性抗原，具体为一种马链球菌兽疫亚种保护性抗原HP0623及其制备方法。
背景技术
链球菌病是一种极为常见的动物致病菌，根据群特异性抗原可以将其分为20个群，从A～V，缺I和J。动物病原菌以B群链球菌(Group B Streptococci,GBS)和C群链球菌(Group C Streptococci,GCS)较多，而人的致病菌则多为A群链球菌(GroupAStreptococci,GAS)。
马链球菌兽疫亚种(Streptococcus zooepidemicus,SEZ)属于兰氏分群的C群链球菌，无宿主专嗜性，易发生变异，来源不同的菌株抗原性可能不同。该菌可以经消化道、手术、外伤、注射疫苗和外伤等途径感染动物，从而引起动物的败血症、关节炎、乳房炎、脑膜炎和心内膜炎等动物疫病。人则可能通过与患病动物密切接触或食用该菌污染的食物而引发该病的感染。青霉素和阿莫西林等抗生素一直被用于预防和治疗此病，但由于抗生素的大量使用，导致该菌耐药性的产生和动物制品中抗生素的残留等问题。因此，采用疫苗来防治该病才能够从根本上解决这些问题。但是由于灭活的全菌疫苗常会引起免疫综合症等副作用，因此需要研发新型的亚单位蛋白疫苗来预防该类疾病的发生。细胞表面锚定的糖结合蛋白(HP0623)是马链球菌兽疫亚种细胞表面的一种新发现的保护性抗原，其主要作用于宿主细胞，因此有望被开发为防治SEZ的亚单位疫苗。
发明内容
为了解决上述问题，本发明提供了一种马链球菌兽疫亚种保护性抗原HP0623及其制备方法，所述一种马链球菌兽疫亚种保护性抗原HP0623能有效用于防治马链球菌兽疫亚种。
本发明的技术方案为：
一种马链球菌兽疫亚种保护性抗原HP0623，所述HP0623重组蛋白为SEZ HP0623重组蛋白(rHP0623)，由sez_0623基因编码，由429个氨基酸残基组成，分子量为47.04kDa，氨基酸序列如SEQ ID NO.2所示。
一种马链球菌兽疫亚种保护性抗原HP0623的制备方法，包含以下步骤：
1)PCR扩增：使用马链球菌兽疫亚种SEZ C55138株基因组为模板，用引物进行PCR扩增；
2)与载体连接：PCR产物经限制性内切酶酶切后与经相同酶酶切的pET-32a载体连接；所述的限制性内切酶为BamH I和Hind III；
3)转化和诱导：将上述连接好的载体转化进入大肠杆菌(E.coli BL21)后，置于37℃摇床培养，待其OD值为0.6～1.0时，加入IPTG诱导3-5h；
4)纯化：离心收集菌体，经PBS重悬后，高压破碎后离心收集上清，将上清液的目的蛋白经过Ni-NTA层析柱进行纯化获得纯化的HP0623重组蛋白。
所述引物包括正向引物和反向引物，所述正向引物和反向引物各有1个酶切位点。
进一步地，所述引物为：
正向引物(SEQ ID NO.3)：5’-CCCGGATCCGCTTGCCTGCTAGTG-3’，划线部分为BamH I酶切位点；
反向引物(SEQ ID NO.4)：5’-CTCAAGCTTGAGGGGAAGATCGTATC-3’，划线部分为HindIII酶切位点；
马链球菌兽疫亚种保护性抗原HP0623在制备马链球菌兽疫亚种疫苗中的应用。
应用所述马链球菌兽疫亚种保护性抗原HP0623制备马链球菌兽疫亚种疫苗的方法为：将成功表达并经过纯化处理的重组蛋白，即马链球菌兽疫亚种保护性抗原HP0623，与Montanide Gel 01PR(SEPPIC，France)佐剂乳化后作为疫苗，疫苗中所述重组蛋白浓度为100μg/mL，所述浓度为疫苗中重组蛋白的最低有效浓度。
本发明的技术效果为：
与现有技术相比，本发明通过基因克隆、表达、纯化等步骤得到纯化的马链球菌兽疫亚种保护性抗原HP0623重组蛋白。本发明首先通过PCR技术获取目的基因，通过酶切酶连的方法获得重组质粒，将其转入大肠杆菌中进行诱导表达HP0623重组蛋白，经蛋白免疫印记试验，表明该重组蛋白具有良好的免疫原性。HP0623重组蛋白经佐剂乳化后免疫小鼠可以为其提供较高的保护效力，二免小鼠血清可以为小鼠提供较好的被动免疫保护。经rHP0623重组蛋白免疫后的小鼠血清经ELISA测定其抗体滴度，表明重组蛋白可诱发高水平的抗体滴度，且免疫反应类型以Th2为主。HP0623重组蛋白抗体可以诱导较强的杀菌能力，当SEZ感染小鼠后，在其脾脏组织中所提取的SEZ的sez_0623基因的转录水平要高于体外培养的转录水平，rHP0623可以抑制SEZ对Hep-2细胞的粘附和侵染。
附图说明
图1.表示HP0623蛋白的SDS-PAGE和免疫印记图。
图1中，泳道1～3分别为重组大肠杆菌诱导前、诱导后和纯化后的HP0623蛋白；泳道M为蛋白预染Marker。
泳道4为rHP0623与SEZ感染康复猪血清的Westernblot检测结果。
图2、图3为重组蛋白免疫小鼠后在小鼠体内引起的免疫反应ELISA检测结果图。
图2中为免疫组和阴性对照组的抗体水平比较；图3中为HP0623诱导的抗体反应类型比较。
图4表示免疫组、阴性对照组和阳性对照组的小鼠，经SEZ攻毒后，小鼠的存活率对比图。
图5表示rHP0623二免血清(实验组)、SEZ灭活疫苗二免血清(阳性对照组)和正常血清(阴性对照组)为小鼠提供被动免疫保护的情况对比图。
图6.显示了定量PCR的分析结果图。
图6中从左至右分别为三只小鼠各自脾脏内细菌基因表达情况。
图7、图8为.流式细胞术分析HP0623在SEZ的表面分布图。
图9.表示小鼠全血致死使用结果对比图。
图9中，1为实验组、2为阳性对照组和3为阴性对照组。
图10.SEZ对Hep-2细胞的粘附检测结果图。
具体实施方式
为了更好的理解本发明，下面将结合实施例和附图来做进一步说明。
本发明实施例中采用常规实验方法和条件，未在本发明中条件是本领域的技术人员所熟知的，本领域的技术人员可以结合现有技术实现本发明。
实施例一、菌株与生长条件
菌株为SEZ C55138菌株；培养条件为加入5％胎牛血清的TSB培养基或TSA培养基；在37℃震荡培养约8h，或OD600达到0.6～1.0。载体为大肠杆菌pET-32a，感受态细胞为大肠杆菌BL21。
所述SEZ C55138菌株，大肠杆菌pET-32a、胎牛血清和大肠杆菌BL21感受态细胞均为市售产品。
实施例二、制备方法
HP0623蛋白由sez_0623基因编码，所述HP0623基因正向引物有1个BamH I酶切位点，反向引物有1个Hind III酶切位点。所述正向和反向引物由SEZ基因组序列设计，由引物合成公司合成。
马链球菌兽疫亚种保护性抗原HP0623的制备方法，包含以下步骤：
1)PCR扩增：使用C55138菌株的基因组作为模板，使用下述引物进行PCR扩增：
正向引物(SEQ ID NO.3)：5’-CCCGGATCCGCTTGCCTGCTAGTG-3’，划线部分为BamH I酶切位点；
反向引物(SEQ ID NO.4)：5’-CTCAAGCTTGAGGGGAAGATCGTATC-3’，划线部分为HindIII酶切位点；
2)与载体连接：PCR产物使用限制性内切酶酶切后与经相同酶酶切的pET-32a载体连接；
3)转化和诱导：将上述连接好的载体转化大肠杆菌感受态细胞BL21后，在37℃恒温摇床中待其到指数生长期，加入终浓度为1mM的IPTG，诱导3h；同时做非诱导对照试验。
4)纯化：离心收集诱导后菌体，用无菌PBS重悬后经高压破碎，离心收集重组蛋白上清，将上清液中的目的蛋白经过Ni-NTA层析柱纯化后获得的纯化的重组蛋白。
免疫印记分析：纯化后的重组蛋白，使用感染过SEZ的康复猪血清作为一抗，山羊抗猪IgG(H+L)-HRP作为二抗进行免疫印迹分析。
所述的C55138菌株的基因组由常规方法从C55138菌株中提取得到。
引物由武汉金开瑞生物工程有限公司合成，所述限制性内切酶为BamH I和HindIII。
IPTG即异丙基-β-D-硫代半乳糖苷，是β-半乳糖苷酶的活性诱导物质。基于这个特性，带有lacZ基因载体DNA以lacZ缺失细胞为宿主进行转化时，它可以作为具有lac或tac等启动子的表达载体的表达诱导物使用。
实验结果
PCR得到sez_0623的基因序列(SEQ ID NO.1)，将SEZ C55138菌株sez_0623基因克隆后，表达出了429个氨基酸组成的蛋白质(SEQ ID NO.2)，分子量为47.04kDa。比较诱导和非诱导培养的细菌，可以证明成功表达了目的蛋白，且目的蛋白的条带大小与预测的大小一致。通过Ni-NTA亲和层析的方式获得纯化的重组蛋白，通过免疫分析实验表明，该蛋白与感染SEZ的康复期猪血清表现出良好的免疫反应性，如图1所示，HP0623蛋白大小与预测的大小一致，且其具有良好的免疫反应原性。
实施例三、抗体滴度测定
通过酶联免疫吸附试验测定小鼠血清抗体IgG，将纯化的重组蛋白HP0623于4℃过夜包被后，将二免免疫10天后的小鼠采血分离血清，梯度倍比稀释后，所有稀释度均取100μL加入酶联板，37℃反应后加入辣根酶标记的山羊抗鼠IgG，待显色完成后终止反应，用酶标仪读取OD值，取OD值大于对照血清平均OD值+3倍方差SD(标准差)的血清最大稀释倍数作为血清抗体。为了推断免疫类型，进一步检测IgG1和IgG2a以确定Th2和Th1的免疫反应。
实验结果：
免疫组的IgG滴度的水平显著高于阴性对照组的抗体水平，如图2所示免疫组的抗体滴度明显高于阴性对照组。
结果表明HP0623可以引起显著的Th1/Th2免疫反应，与产生的IgG2抗体相比，IgG1抗体滴度占主导地位，如图3所示在HP0623诱导的抗体反应类型中，Th2反应占主导地位。
实施例四、小鼠免疫接种与攻毒试验
1、取30只6～8周龄KM雌鼠，随机分为3组，每组10只；
2、实验组：50μg纯化的rHP0623用Montanide Gel 01PR(SEPPIC，France)佐剂乳化后，腹腔注射免疫第一组小鼠，间隔14天后，用同样的方式和剂量对小鼠进行二次免疫；
3、阳性对照组：将SEZ C55138株灭活，经Montanide Gel 01PR(SEPPIC，France)佐剂乳化后，以1×109CFU/mL免疫第二组小鼠，采用腹腔注射的方式，间隔14日后，同样的方式进行二次免疫；
4、阴性对照组：第三组10只小鼠均腹腔注射经Montanide Gel 01PR(SEPPIC，France)佐剂乳化的PBS做对照；
5、所有小鼠二次免疫10天后，尾静脉采血离心收集血清，然后所有小鼠均攻毒SEZC55138菌株(2×105CFU/mL)；
6、观察并记录所有小鼠的生长情况。
实验结果：
1)实验组的10只小鼠，攻毒后第三天开始出现死亡，存活的小鼠表现出轻微的临床症状，如精神状态较差，消瘦，在6天内即全部恢复正常，最终有4只小鼠死亡；
2)阳性对照组的小鼠总体均未表现出明显的临床症状，整个攻毒期间只有1只小鼠出现死亡；
3)阴性对照组的小鼠在攻毒后第二天即开始死亡，存活的小鼠表现出明显的临床症状，如被毛零乱，对外界刺激反应迟钝，不好动等，并在攻毒后6天内逐渐死亡，最终只有1只存活。
如图4所示，阳性对照组和免疫组均能为小鼠提供较高的免疫保护，且阳性对照组要高于实验组。结果表明重组rHP0623蛋白可以为小鼠提供免疫保护，抵御SEZ强毒株的感染。
实施例五、被动保护试验分析
为了证实保护是归功于HP0623特殊的刺激免疫反应，用rHP0623二免血清被动免疫小鼠，然后再进行攻毒试验。
1、30只6周龄KM雌鼠，随机分为3组，每组10只；
2、阳性对照组：用SEZ灭活疫苗二免血清静脉注射免疫第一组小鼠；
3、阴性对照组：用正常小鼠血清静脉注射免疫第二组小鼠；
4、实验组：用rHP0623二免血清静脉注射免疫第三组小鼠；
5、免疫24h后，所有小鼠均用SEZ C55138菌株进行攻毒试验(2×105CFU/mL)；
6、观察并记录所有小鼠的生长情况。
实验结果：
1)阳性对照组小鼠在攻毒试验中均未表现出临床症状，整个攻毒期间有1只小鼠最终死亡；
2)阴性对照组小鼠在攻毒后主要表现为精神萎靡，对外界刺激反应迟缓、被毛粗乱等症状，且在3天内均陆续死亡；
3)实验组小鼠在攻毒后主要表现为精神萎靡，对外界刺激反应迟缓、被毛粗乱等症状，但3天后基本恢复正常，小鼠的最终的存活率为70％。
如图5所示，结果表明，实验组和阳性对照组对小鼠的被动保护能力明显强于阴性对照组。
实施例六、荧光定量PCR分析
为了评估SEZ sez_0623基因在小鼠体内外的表达水平，通过荧光定量PCR技术定量检测sez_0623基因体内外的转录水平。
1、从感染SEZ的小鼠脾脏内获得体内增殖细菌，无菌收集脾脏组织，碾磨后于4℃，850×g离心5min，取上层清液于4℃，15500×g离心5min，沉淀即为脾脏组织内的细菌；体外培养的细菌待其生长到指数中期后离心收集沉淀即可；
2、用酸酚法提取细菌总RNA；
3、将RNA逆转录为cDNAs，以cDNA样品为模板进行定量PCR反应，所有反应进行三个重复；
在实时荧光定量PCR中，使用下述引物对sez_0623基因进行定量分析：
正向引物(SEQ ID NO.5)：5’-CGGTATAGTATAGCCAAGC-3’；
反向引物(SEQ ID NO.6)：5’-GCATTTCTCCCAACGA-3’；
在实时荧光定量PCR中，使用下述引物对16S rRNA基因进行定量分析：
正向引物(SEQ ID NO.5)：5’-ATCCGAACTGAGATTGGC-3’；
反向引物(SEQ ID NO.6)：5’-CCCTTATGACCTGGGCTA-3’；
实验结果：
如图6所示，结果表明小鼠脾脏内提取的SEZ的sez_0623基因的转录水平要明显高于体外培养的SEZ的转录水平。
实施例七、流式细胞术分析
1、用PBS/BSA(含1％BSA的PBS)重悬细菌，取菌液200μL；
2、实验组加入经rHP0623免疫的小鼠血清100μL，对照组加入经PBS免疫的小鼠血清100μL，4℃孵育15min，然后室温孵育30min；
3、5000rpm离心3min，收集细菌；
4、用PBS/BSA洗3次，加入FITC-山羊抗鼠二抗，室温下孵育45min；
5、用PBS/BSA洗3次，用500μL的PBS重悬，加入到流式细胞仪中进行检测。
实验结果：
如图7、图8所示，相对于空白对照组，SEZ HP0623抗体能在SEZ表面引起更加强烈的荧光信号，对照组细菌平均荧光强度较弱，而实验组细菌的平均荧光强度有较大的提升，表明HP0623蛋白分布于SEZ的表面。
实施例八、全血致死量分析
为测定HP0623抗体的杀菌活性，吧小鼠血液肝素化后，使用rHP0623二免血清或对照血清与血液中存活的SEZ孵育后进行评估。
1、待SEZ C55138菌株生长至指数中期，离心收集菌体，用无菌PBS将细菌浓度调整为104CFU/mL；
2、阳性对照组：取900μL健康小鼠经肝素抗凝全血，与100μLSEZ灭活疫苗二免血清混合；
3、阴性对照组：取900μL健康小鼠经肝素抗凝全血，与100μL正常小鼠血清混合；
4、实验组：取900μL健康小鼠经肝素抗凝全血，与100μL重组rHP0623二免血清混合；
5、分别向混合液中加入100μL SEZ菌液，置于37℃恒温摇床震荡培养90min；
6、每组分别取100μL样品，涂布于TSA平板中，进行3个重复，计算孵化前后存活的细菌量；
7、存活率计算为剩下细菌量相对于开始细菌量的百分比。
实验结果：
阴性对照组杀菌率为9.21±1.29％；阳性对照组杀菌率为91.6±1.35％；实验组杀菌率为61.08±1.60％。
如图9所示，结果表明anti-rHP0623抗体可以诱导高水平的杀菌能力，anti-HP0623抗体能够对SEZ具有较强的杀伤能力。
实施例九、粘附抑制试验
为检测HP0623对SEZ粘附Hep-2细胞的影响，通过Hep-2细胞的粘附试验进行检测，主要步骤如下：
1、在24孔板中加入含10％胎牛血清的DMEM培养Hep-2细胞，将其置于37℃细胞培养箱中培养，待其长至80％左右；
2、使用预冷的PBS/BSA洗涤细胞3次；
3、每孔加入200μg/ml的纯化的rSec_205孵育2h，相同的细胞加入200μg/ml的BSA孵育作为对照组；
4、孵育完成后，用PBS/BSA洗涤3次，然后向各孔中加入1ml 5×107CFU/ml的SEZC55138株在37℃，5％CO2条件下孵育2h；
5、用PBS/BSA洗去未粘附的细菌，然后用1ml PBS(含0.2％的Triton X-100)将细胞吹散，稀释后平板计数；
6、HP0623对SEZ粘附Hep-2细胞的抑制率通过如下公式计数：
抑制率＝[1-(HP0623处理的细胞的细菌数/BSA处理的细胞的细菌数)]×100％。
实验结果：rHP0623能够抑制SEZ对Hep-2细胞的粘附，如图10所示，相较于对照组，抑制率为14.9％。
从上述实施例可以看出，本发明所述马链球菌兽疫亚种保护性抗原HP0623的制备方法，包括目的基因克隆、酶切酶连、诱导表达、蛋白纯化。重组HP0623蛋白经蛋白免疫印记分析，表明其具有良好的免疫反应原性。重组蛋白免疫后的小鼠血清中抗体滴度明显高于阴性对照组小鼠，且免疫小鼠产生的抗体类型中，IgG1滴度要高于IgG2a。rHP0623免疫小鼠后可以为小鼠提供较强的保护效力，anti-rHP0623小鼠二免血清对小鼠有明显的被动保护力，anti-rHP0623抗体可以诱导高水平的杀菌能力。sez_0623基因在SEZ感染的小鼠体内的转录水平要高于体外培养的转录水平，流式细胞术检测结果表明，HP0623位于SEZ表面，rHP0623可以抑制SEZ对Hep-2细胞的粘附，抑制率为14.9％。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为本发明专利范围的限制。对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变性和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
SEQUENCE LISTING
<110> 湖北大学
<120> 一种马链球菌兽疫亚种保护性抗原HP0623及其制备方法
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Claims (7)
1.一种马链球菌兽疫亚种保护性抗原HP0623，其特征在于：为SEZ HP0623重组蛋白，由sez_0623基因编码，由429个氨基酸残基组成，分子量为47.04kDa，氨基酸序列如SEQ IDNO.2所示。
2.一种马链球菌兽疫亚种保护性抗原HP0623的制备方法，分为以下步骤：
1)PCR扩增：使用马链球菌兽疫亚种SEZ C55138基因组为模板，用引物进行PCR扩增；
2)载体的连接：PCR产物经限制性内切酶酶切后与PET-32a载体连接；
3)转化和诱导：将步骤2)连接好的载体转化至大肠杆菌后，培养至OD值到0.6～1.0，加入IPTG诱导培养3-5h；
4)纯化：离心收集步骤3)中培养好的菌体，用无菌PBS重悬后经破碎后，离心收集蛋白上清，取上清液经Ni-NTA层析柱纯化，获得纯化的重组蛋白，即为马链球菌兽疫亚种保护性抗原HP0623。
3.根据权利要求2所述的马链球菌兽疫亚种保护性抗原HP0623的制备方法，其特征在于：所述引物为：
正向引物：5’-CCCGGATCCGCTTGCCTGCTAGTG-3’；
反向引物：5’-CTCAAGCTTGAGGGGAAGATCGTATC-3’。
4.根据权利要求3所述的马链球菌兽疫亚种保护性抗原HP0623的制备方法，其特征在于：所述正向引物和反向引物分别有1个酶切位点。
5.根据权利要求4所述的马链球菌兽疫亚种保护性抗原HP0623的制备方法，其特征在于：所述正向引物的酶切位点为BamH I酶切位点；反向引物酶切位点为Hind III酶切位点。
6.根据权利要求1所述马链球菌兽疫亚种保护性抗原HP0623在制备马链球菌兽疫亚种疫苗中的应用。
7.一种马链球菌兽疫亚种保护性抗原HP0623制备马链球菌兽疫亚种疫苗的方法，其特征在于：将权利要求2-5中任一权利要求所述步骤4)纯化处理得到的重组蛋白与MontanideGel 01 PR佐剂乳化后即为马链球菌兽疫亚种疫苗，所述重组蛋白的浓度为100μg/mL。
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