CN116574160A - 一种猪链球菌抗原蛋白及其应用 - Google Patents
一种猪链球菌抗原蛋白及其应用 Download PDFInfo
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Abstract
本发明公开了一种猪链球菌高免疫原性抗原蛋白,该蛋白质的氨基酸序列如SEQ ID NO.2所示,编码基因的核苷酸序列如SEQ ID NO.1所示,本发明还公开了该蛋白质的制备方法及其用途,属于分子生物学领域。该蛋白的基因存在于猪链球菌的核心基因组中,在不同的血清型中都高度保守,具有很高的免疫原性,能诱导机体产生高水平的Th1和Th2型免疫反应,该蛋白可用于猪链球菌的临床检测和制备亚单位疫苗,具有很好的应用前景。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种猪链球菌的高免疫原性抗原蛋白,本发明还涉及该蛋白的制备以及在病原检测和疫苗制备中的应用。
背景技术
猪链球菌(Streptococcus suis,S.suis)是一种危害严重并能引起人畜共患的病原体,对全世界的养猪业造成了重大经济损失。感染猪链球菌的主要临床症状主要包括脑膜炎,心内膜炎,关节炎,败血病甚至是引起猪的突然死亡等。由于猪链球菌具有多种血清型,并且猪链球菌还可以利用毒力因子来逃避宿主的免疫系统,这些特征为开发高效的猪链球菌疫苗带来了巨大的挑战。
目前针对猪链球菌病的预防主要依赖于疫苗,虽然市场上已有一些预防猪链球菌的灭活疫苗,但是大部分疫苗主要预防特定菌株或者某些血清型。亚单位疫苗是一类以病原体主要抗原为靶标的新型疫苗,与灭活疫苗相比,重组蛋白没有感染性、成本低廉、安全性高,是当前疫苗研发的重点。亚单位疫苗不仅可以预防猪链球菌的传播,还可以减少抗生素的耐药风险,是当前预防猪链球菌的迫切需求,而寻找新型有效的免疫原性抗原是猪链球菌疫苗研发和临床诊断的关键。
发明内容
本发明的目的是提供一种猪链球菌高免疫原性抗原蛋白及其制备方法和应用,旨在为猪链球菌亚单位疫苗和诊断制剂的研发提供新的靶标。
申请人前期以噬菌体展示文库、免疫共沉淀、二代高通量测序等多种技术为手段,建立了一种在细菌全基因组水平上无偏见、高通量筛选免疫原性抗原蛋白的方法,该方法已于本专利申请日前申请了另一项中国发明专利,专利申请号为202310239675.0,名称为“一种细菌全基因组水平高通量筛选免疫原性抗原蛋白的方法”。利用上述方法,申请人从猪链球菌的基因组中筛选出多个免疫原性抗原蛋白。
其中一种蛋白质的氨基酸序列如SEQ ID NO.2所示,编码基因的核苷酸序列如SEQID NO.1所示。
申请人使用PCR方法,从猪链球菌的基因组DNA中克隆上述目的基因,构建重组表达载体,通过原核表达系统转化宿主菌并获得所述重组蛋白。该蛋白存在于猪链球菌的核心基因组中,在不同的血清型中序列高度保守。经验证,该蛋白具有很高的免疫原性,能诱导机体产生高水平的Th1和Th2型免疫反应,利用该蛋白,不仅能对抗体血清进行间接ELISA检测,而且还能免疫动物产生保护力,抵御猪链球菌对机体的攻击。该蛋白及其功能是申请人首次从猪链球菌中获得并表征,在此之前未有任何文献记载。
本发明还提供了包含所述基因的表达载体,以及包含所述蛋白质的宿主菌。
本发明还提供了一种制备所述蛋白质的方法,该方法包括以下步骤:
1)以猪链球菌的基因组DNA为模板,设计PCR引物扩增编码所述蛋白质的目的基因;
2)将扩增产物克隆至pET32a载体,获得连接有所述目的基因的重组表达质粒;
3)将重组表达质粒转化至感受态BL21(DE3)细胞进行诱导表达,获得包含所述蛋白质的宿主菌;
4)使用His标签的蛋白纯化预装柱从宿主菌中分离纯化所述蛋白质。
优选地,所述PCR引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
本发明进一步提供了所述的蛋白质在制备猪链球菌检测试剂盒中的应用。
说明书中提供了一个利用所述蛋白对猪链球菌抗体血清进行间接ELISA检测的实施例,当然,本发明的检测应用并不局限于该实施例,根据该蛋白的功能特性,本领域技术人员也能在不经创造性劳动的基础上将该蛋白用于其它检测,例如,利用该蛋白制备抗体并对抗原进行直接ELISA检测,这些不经过创造性劳动的改进方案也都在本发明的保护范围内。
本发明再进一步提供了所述的蛋白质在制备猪链球菌亚单位疫苗中的应用。
在本发明的一个具体实施例中,将重组蛋白与赛彼科ISA 201佐剂混合并免疫小鼠,猪链球菌SC19攻毒后统计小鼠的发病及死亡情况,结果表明,免疫小鼠的临床症状明显减轻,死亡率显著下降。
本蛋白的名称已被注释,为FMN-binding protein,NCBI登录号为WP_012027664。申请人将本发明中的该蛋白的基因命名为8455。
本发明的有益效果是:
本发明为猪链球菌的病原检测和预防提供了另一种候选抗原,在猪链球菌的亚单位疫苗和诊断试剂研发方面有很高的应用前景。
附图说明
图1:8455基因的PCR扩增条带。
图2:8455蛋白表达质粒酶切鉴定的电泳结果。
图3:SDS-PAGE以及Western-blot验证表达的8455蛋白。
图4:ELISA检测免疫8455蛋白的小鼠产生特异性IgG抗体。
图5:ELISA检测免疫8455蛋白的小鼠产生特异性IgG1和IgG2a抗体。
图6:通过攻毒实验验证8455蛋白对小鼠的保护作用。
具体实施方式
下面结合实例对本发明作进一步的详细说明。下列实施例仅用于说明本发明,而不应视为限定本发明的范围。
遗传资源来源说明:猪链球菌SC19株是申请人华中农业大学构建的一株猪链球菌2型血清型弱毒株,该毒株已保藏于中国典型培养物保藏中心(CCTCC),保藏编号为:CCTCCNO:M 2016584,且已在CN 108410784A的专利文献中公开。
实施例1:构建8455蛋白的表达质粒并纯化蛋白
1.构建蛋白的表达质粒
1)PCR扩增目的基因
首先我们根据8455的基因序列设计引物,引物包括保护性碱基、酶切位点以及与模板结合的核苷酸序列,酶切位点选择了EcoRⅠ和HindⅢ,将设计好的引物送至生工生物工程(上海)股份有限公司合成。挑取菌株SC19的单菌落至含有10%的新生牛血清的TSB液体培养基中,37℃培养过夜,按照说明书提取猪链球菌的基因组。以SC19的基因组DNA为模板,通过PCR扩增8455的基因片段,扩增产物的长度为495bp,扩增结果如图1所示,目的条带明显。基因扩增后通过琼脂糖凝胶电泳,采用OMEGA凝胶回收试剂盒回收该片段的DNA并测量DNA浓度,用QuickCutTM HindⅢ/QuickCutTM EcoRⅠ(Takara)将片段的DNA进行双酶切,并经过琼脂糖凝胶电泳切胶回收DNA,分光光度计测量浓度后保存备用。PCR扩增体系、酶切体系以及反应条件如下:
8455基因片段的PCR扩增体系:
引物序列(下划线的为酶切位点):
8455F:5’-CCGGAATTCATGAAAACAACTAAAGTTGT-3’(SEQ ID NO.3)
8455B:5’-GGCAAGCTTGTCAAGTTTTACAGTTTCTGT-3’(SEQ ID NO.4)
8455基因片段的PCR扩增条件:
95℃5min,95℃15sec,60℃15sec,72℃20sec,进行30个循环,72℃延伸5min。
8455基因片段的DNA酶切体系:
反应条件:
酶切产物置于37℃恒温水浴锅孵育1h,经琼脂糖凝胶电泳后切胶回收备用。
2)构建8455的表达载体
将实验室已经提取好的pET32a质粒载体用QuickCutTM HindⅢ/QuickCutTM EcoRⅠ双酶切后进行琼脂糖凝胶电泳,切胶后采用OMEGA凝胶回收试剂盒回收DNA。回收后采用赛默飞的T4连接酶(货号EL0012),将酶切好的8455的DNA片段与pET32a质粒载体体外连接。连接后将连接产物加入DH5α感受态中,置于冰上冰浴30分钟,42℃热激90秒,热激后将感受态置于冰上再次冰浴2分钟,在感受态中加入500μl的LB液体培养基,放入37℃摇床180rpm孵育45分钟,孵育后5000rpm离心3分钟,弃去500μl上清,剩余100μl上清重悬沉淀后将菌液涂至含有100μg/ml氨苄青霉素的LB固体平板上,将平板置于37℃的恒温培养箱中培养过夜。次日挑取单菌落至10ml含有100μg/ml氨苄青霉素的LB液体培养基中,培养12小时后提取质粒并酶切鉴定,鉴定结果如图2所示,三个质粒经酶切,均有两条目的条带,一条为酶切后的载体,另一条为酶切后的8455基因片段,证明表达质粒连接成功。鉴定后挑取阳性克隆送往测序公司测序。酶切体系、连接体系及反应条件如下:
pET32a质粒酶切体系如下:
反应条件:
酶切产物置于37℃恒温水浴锅孵育1h,经琼脂糖凝胶电泳后切胶回收备用。
表达质粒的连接体系:
反应条件:
将上述混合物置于PCR仪16℃过夜孵育。
8455质粒酶切体系如下:
反应条件:
酶切产物置于37℃恒温水浴锅孵育1h,经琼脂糖凝胶电泳鉴定酶切产物。
2.蛋白的表达及纯化:
将测序正确的质粒用移液器吸取10ng加入到表达感受态BL21(DE3)中,置于冰上冰浴30分钟,42℃热激45秒,热激后将感受态置于冰上再次冰浴2分钟,在感受态中加入500μl的LB液体培养基,放入37℃摇床180rpm孵育45分钟,孵育后5000rpm离心3分钟,弃去500μl上清,剩余100μl上清重悬沉淀后将菌液涂至含有100μg/ml氨苄青霉素和34μg/mL氯霉素的固体LB平板上,37℃恒温培养箱培养12小时,挑取单菌落至含有10mL培养基的细菌瓶中,并添加氨苄青霉素和氯霉素并使其终浓度为100μg/ml和34μg/mL。次日按照1:100转接至1L的LB培养基中,放入37℃摇床中200rpm摇菌至OD值约为0.6左右,将摇床转速调至170rpm,并加入1ml的IPTG使其终浓度为1mmol/L,诱导表达5小时。5小时后将培养的细菌倒入离心瓶中,6000rpm离心10min收集沉淀,用PBS将沉淀洗涤两遍。150ml的结合缓冲液(300mMNaCl,20mM Tris-HCl,浓盐酸调至pH为8.0,10mM咪唑)重悬细菌沉淀,高压破碎仪将蛋白低温破碎约半个小时,待液体澄清后收集破碎后的蛋白,30000g离心20min收集蛋白上清,并用0.22μm的滤器过滤上清,将过滤好的蛋白上清通过蠕动泵用His标签的蛋白纯化预装柱纯化蛋白。待蛋白液流尽之后更换洗涤缓冲液(300mM NaCl,20mM Tris-HCl,浓盐酸调至pH为8.0,20mM咪唑)洗涤杂蛋白,洗涤30分钟后将缓冲液更换为洗脱缓冲液(300mM NaCl,20mM Tris-HCl,浓盐酸调至pH为8.0,400mM咪唑)洗脱与His柱结合的目的蛋白。将洗脱后的目的蛋白用10kDa的超滤管对蛋白进行超滤浓缩,先用超纯水将超滤管洗涤两遍,将蛋白加至超滤管中,4℃4000rpm离心,离心时间根据蛋白而定。每次离心后,加入一定体积的GEbuffer(20mM Tris,100mM NaCl,用浓盐酸调至pH为8.0),用来置换蛋白中高浓度的盐和咪唑。大约离心置换约4-5次后,离心后的体积约为置换前体积的十分之一,从超滤管中吸出蛋白至1.5mL离心管中混匀后将蛋白分装至PCR小管中,每管50μL,分装后放至-80℃冰箱保存备用。
3.SDS PAGE和Western blot验证纯化的蛋白:
将纯化后的蛋白取出10μL加入含有30μL PBS的1.5ml EP管中,再加入10μL 5×蛋白上样缓冲液,混匀后100℃煮10min。取出两块已制备好的SDS-聚丙烯酰胺凝胶,一块用于SDS PAGE验证所纯化的蛋白,另一块用于Western blot验证。先80V电压下跑完浓缩胶后切换电压至120V跑完分离胶。蛋白胶跑完之后一块直接放入含有2.5g/L的考马斯亮蓝染液中染色2h,之后转至脱色液(10%冰醋酸,5%乙醇)中进行脱色。而另一块蛋白胶则用于Western blot验证。
切下蛋白凝胶的胶块,在65V的电压下转40分钟将蛋白转至PVDF膜上。TBST洗膜3遍,每次间隔5min,5%脱脂奶粉室温封闭2h,TBST洗膜3遍,每次间隔5min,鼠抗His-Tag的单克隆抗体作为一抗室温孵育2h(公司:Abclonal货号:AE003,抗体用TBST以1:4000倍稀释),TBST洗膜5遍,每次间隔5min,HRP标记的山羊抗小鼠IgG作为二抗孵育45min(公司:Abbkine货号:A21010,用TBST以1:8000倍稀释),TBST洗膜5遍,每次间隔5min,ECL发光显色液A:B等体积混合后避光使用化学发光仪显色(公司:Biosharp货号:BL520A),所纯化的蛋白结果如图3所示,结果发现所纯化的8455蛋白约37kDa,与预测结果一致。
实施例2:动物实验验证8455蛋白对小鼠是否具有保护效果
1.动物免疫实验
随机取10只六周龄BALB/c雌鼠分成两组,每组5只,一组为实验组,另外一组为对照组。实验组的每只小鼠免疫30μg纯化的蛋白,将蛋白用PBS稀释至100μL并与赛彼科ISA201佐剂等体积混合并完全乳化,每只小鼠200μL进行腹腔免疫。对照组则将PBS与佐剂等体积混合后每只小鼠腹腔注射200μL。一共免疫两次,每次间隔14天。在第二次免疫前一天对小鼠进行尾静脉采血,二免后10天对小鼠进行二次采血并分离血清;
2.间接ELISA检测小鼠的血清抗体
在第二次采血后,检测小鼠的血清抗体。将纯化好的蛋白用包被液(0.05M碳酸盐缓冲液,pH9.6)稀释并按照每孔100ng/100μL包被酶标板,放置4℃过夜。次日弃去包被液后用PBST洗涤5次,每孔加200μL 3%的BSA于37℃封闭1h。封闭结束后弃去封闭液,PBST洗涤5遍后除第一孔加入200μL的PBST,其余孔加上100μL的PBST,将蛋白二次免疫后的血清从第一个孔以1:5000开始按照2倍比依次往后稀释,37℃孵育1h。弃去血清,PBST洗涤5遍,每孔加100μL HRP标记的羊抗鼠的二抗(公司:Abbkine,货号:A21010)37℃孵育50min。弃去二抗,PBST洗涤5遍,每孔加100μL TMB显色液,置于避光处,室温显色至不变色后每孔加100μL2M的浓硫酸终止显色。最后通过酶标仪测定OD450处的吸光度。结果如图4所示,免疫蛋白的小鼠抗体水平明显高于对照组的小鼠。
为了对IgG亚型进行分析,将8455蛋白为包被抗原,通过间接ELISA检测IgG1和IgG2a的抗体滴度。一抗为蛋白二次免疫后的血清,从第一个孔以1:1000开始按照2倍比依次往后稀释,二抗将IgG换成HRP标记的羊抗鼠IgG1和IgG2a(1:3,000稀释),其余步骤同上。结果如图5所示,与对照组相比,8455蛋白与佐剂混合后诱导的IgG1抗体水平和IgG2a抗体水平高于对照组小鼠。IgG1抗体与Th2型反应相关,IgG2a抗体与Th1型反应相关,证明了8455蛋白免疫小鼠,可以诱导小鼠的Th1和Th2型免疫反应。
3.动物攻毒实验
在第二次采血后,通过动物攻毒实验来验证筛选的蛋白对动物是否具有保护效果。挑取新鲜的SC19单菌落至10ml含有10%的新生牛血清的TSB培养基中培养过夜,次日按照1:100转接至50ml含有10%的新生牛血清的TSB培养基中培养9小时,将细菌倒入离心管中8000g离心5min,弃去上清,加入10ml PBS洗涤两遍。最后将细菌沉淀用2ml的PBS重悬,并且按照10倍比稀释法对细菌进行稀释,稀释后将细菌涂至含有10%新生牛血清的TSA平板上,37℃恒温培养箱培养过夜,次日通过平板计数法对细菌进行计数。计数后用PBS将菌液浓度调整至3.0×1010cfu/ml,每只小鼠的腹腔攻毒200μL的细菌,剂量为6×109cfu,攻毒后对小鼠观察一周,统计小鼠的发病及死亡情况。结果如图6所示,免疫蛋白的小鼠虽然在攻毒后的24小时内精神较差,毛发粗糙,但是与对照组相比,状态明显好于对照组,且在24小时后,对照组小鼠全部死亡,而实验组小鼠全部存活并逐渐恢复精神状态。证明8455蛋白免疫小鼠后,可对小鼠产生免疫保护,有望成为研发猪链球菌疫苗的重要候选靶标。
Claims (10)
1.一种蛋白质,该蛋白质的氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述蛋白质的基因,该基因的核苷酸序列如SEQ ID NO.1所示。
3.包含权利要求2所述基因的表达载体。
4.包含权利要求1所述蛋白质的宿主菌。
5.一种制备权利要求1所述蛋白质的方法,其特征在于包括以下步骤:
1)以猪链球菌的基因组DNA为模板,设计PCR引物扩增编码所述蛋白质的目的基因;
2)将扩增产物克隆至pET32a载体,获得连接有所述目的基因的重组表达质粒;
3)将重组表达质粒转化至感受态BL21(DE3)细胞进行诱导表达,获得包含所述蛋白质的宿主菌;
4)使用His标签的蛋白纯化预装柱从宿主菌中分离纯化所述蛋白质。
6.如权利要求5所述蛋白质的制备方法,其特征在于:所述PCR引物的核苷酸序列如SEQID NO.3和SEQ ID NO.4所示。
7.权利要求1所述的蛋白质,或权利要求2所述的基因,或权利要求3所述的表达载体,或权利要求4所述的宿主菌在制备猪链球菌检测试剂盒中的应用。
8.一种猪链球菌检测试剂盒,该试剂盒含有权利要求1所述的蛋白质。
9.权利要求1所述的蛋白质,或权利要求2所述的基因,或权利要求3所述的表达载体,或权利要求4所述的宿主菌在制备猪链球菌亚单位疫苗中的应用。
10.一种猪链球菌亚单位疫苗,该疫苗含有权利要求1所述的蛋白质。
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