CN113136356A - 一种重组链球菌及其制备方法和应用 - Google Patents
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Abstract
本发明涉及基因工程技术领域,尤其涉及一种重组链球菌及其制备方法和应用。所述重组链球菌为:通过将猪圆环病毒2型的Cap蛋白的编码序列插入链球菌中Szp基因序列中得到。本发明通过构建包含有链球菌中Szp基因上下同源臂片段和猪圆环病毒2型的Cap蛋白的编码序列的重组载体,进而同源重组得到一种重组链球菌ST171‑Cap,其对于猪圆环病毒2型和链球菌都具有较好的保护力,且相较于单独的猪圆环病毒2型的Cap蛋白亚单位疫苗而言,具备更高的免疫原性。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及一种重组链球菌及其制备方法和应用。
背景技术
猪圆环病毒Ⅱ型(Porcinecircovirus2,PCV2)是圆环病毒科圆环病毒属的一种无囊膜、共价闭环单股负链DNA病毒(ssDNA),是目前为止发现的所有动物病毒中最小的之一。猪对PCV2均易感,但是其危害主要为感染断奶仔猪并引起多系统衰竭综合征(PMWS),该病对养猪业的危害相当严重,是影响猪业发展的一种重要疾病。1991年,加拿大首次发现猪圆环病毒病的流行,之后在全球范围迅速流行,严重影响全球养猪业健康发展。PCV2基因大小约为1.7kb,含有11个开放阅读框(ORF),ORF1和ORF2是其中两个最主要的开放阅读框,分别编码病毒复制相关蛋白(Rep)和233~235个氨基酸大小的病毒衣壳结构蛋白(Cap)。其中Cap蛋白是病毒的免疫保护性抗原,宿主细胞含有与其结合的受体,与病毒的感染密切相关,含有病毒诱导机体产生特异性免疫应答的主要抗原表位。因此,该蛋白是研制PCV2相关疫苗的焦点和检测PCV2特异性免疫反应的血清学诊断方法的靶抗原。
现已证实,PCV2感染主要侵袭猪只的免疫系统,并引起猪只多个系统进行性功能衰竭,导致免疫抵抗力下降和免疫抑制,猪只机体难以产生和维持对其他病原体的免疫抗体,从而继发其他疾病。研究揭示了多种与PCV2感染猪群相关的并发感染病原体,猪链球菌就是其中之一。因此,PCV2的危害性不能孤立而言。此外,PCV2对外界理化因子的抵抗力相当强,可于外界环境存在很长一段时间,即使是酸性环境和氯仿溶液中也可存活较长时间,在72℃温度作用下也能存活一段时间。PCV2于养殖场中很难净化,免疫接种是预防猪圆环病毒病以及猪链球菌病的重要措施。目前的商品化疫苗种类繁多,包括弱毒苗、灭活苗、亚单位疫苗、口服疫苗和病毒(细菌)载体疫苗等,临床应用免疫效果良好。但是由于PCV2的致病机理仍未十分确切,其至今仍是影响养猪业发展的重要疾病之一。现有的商业化疫苗免疫成本高,且因为PCV2基因型变异等因素注存在免疫失败的风险。目前PCV2仍在猪群中流行,这促使高效廉价的疫苗研发成为必要,增强免疫效果和降低经济损失。
免疫预防是防制传染病的有效手段之一,目前市面上的商品化猪链球菌疫苗种类繁多,均是各种不同菌株的弱毒疫苗以及灭活疫苗。目前用于预防PCV2感染的商品化疫苗,诸如灭活苗、亚单位疫苗、口服疫苗和病毒载体疫苗等价格普遍较高,给养猪业增加了生产成本。
Wang等将截断型的PCV2Cap基因克隆于乳酸链球菌穿梭载体得到重组载体pSEC:LEISS-dCap。将其电转化并构建了重组乳酸菌LEISS-dCap口服疫苗株。同样,Kim等电转化将PCV2Cap基因克隆入aroA基因缺失的支气管炎博德特氏菌,获得表达Cap蛋白的重组菌BBS-MCP。该两种重组菌通过免疫小鼠或猪只均能诱导高水平抗Cap蛋白特异性抗体,且能有效保护猪群。
发明内容
为了解决现有技术存在的问题,本发明提供一种重组链球菌及其制备方法和应用。
第一方面,本发明提供一种重组链球菌,所述重组链球菌为:
通过将猪圆环病毒2型的Cap蛋白的编码序列插入链球菌中Szp基因序列中得到。
进一步地,所述链球菌为猪链球菌ST171弱毒株。
猪链球菌ST171弱毒株的基因序列已公开,GenBank登录号为CP002904。
本发明通过同源重组筛选发现,将猪圆环病毒2型的Cap蛋白的编码序列插入猪链球菌ST171弱毒株中Szp基因序列后,既可以使得猪圆环病毒2型的Cap蛋白顺利表达,同时不会影响猪链球菌ST171弱毒株本身的免疫原性。
同源重组技术指将同源重组载体通过电穿孔技术转化到细菌体内,利用同源重组载体的特性,使载体上的基因片段与细菌基因组片段发生交换,从而将外源片段整合到细菌基因组中,使细菌能表达外源片段对应的蛋白。但由于同源重组双交换具有随机性,且重组菌株缺少与亲本菌株不一致的抗性基因,导致重组菌株的筛选工作变得十分困难,往往需要进行大量的筛选才能得到正确的重组菌株(同时,由于不带有和亲本菌株不一致的抗性基因,本发明提供的重组菌株相较于现有技术其他类型同时整合了抗性基因的重组菌株而言具备更高的安全性,适用范围更广)。另外,本发明所构建的重组链球菌ST171-Cap中Cap蛋白的插入位点为猪链球菌ST171弱毒株的膜蛋白,相较于其他位点,在该位点插入的外源蛋白有着更高的表达量。
进一步地,所述猪圆环病毒2型的Cap蛋白的编码序列包括如SEQ ID NO:1所示核苷酸序列。
第二方面,本发明进一步提供所述重组链球菌的制备方法,包括:
构建包含有所述重组链球菌中Szp基因上下同源臂片段和所述猪圆环病毒2型的Cap蛋白的编码序列的重组载体;
将所述重组载体转化至所述链球菌中进行同源重组。
进一步地,所述重组载体中,所述猪圆环病毒2型的Cap蛋白的编码序列位于所述链球菌中Szp基因的上同源臂片段和下同源臂片段之间。
本发明进一步提供一种用于鉴定所述重组链球菌的引物对,包括:
M1:5’-TCTAGGACTGATAGGTCCAT-3’,
M2:5’-CTAATGCTACTTCGGTCT-3’。
本发明进步一步提供一种疫苗,所述疫苗包括所述重组链球菌和佐剂。
进一步地,所述佐剂为MONTANIDETMGEL 01佐剂。
本发明进一步提供所述重组链球菌在制备免疫猪圆环病毒2型和/或链球菌的药物中的应用。
本发明具备如下有益效果:
本发明以猪链球菌ST171弱毒株作为细菌活载体,把PCV2的Cap基因插入ST171的Szp基因中制成重组活载体疫苗。该疫苗不仅生产成本较低,而且能同时预防PCV2和链球菌的感染。此外,该疫苗相较于PCV2 Cap蛋白而言具备更高的免疫原性。
附图说明
图1为本发明实施例1提供的构建重组链球菌的流程示意图。
图2为本发明实施例1提供的同源重组载体pG+host5-SzP-Cap的构建结果;其中a为重组载体的构建策略,b为重组载体的酶切鉴定结果。
图3为本发明实施例1提供的重组菌株ST171-Cap的构建策略示意图。
图4为本发明实施例1提供的重组菌株ST171-Cap的鉴定结果;其中,左图为单交换鉴定结果,右图为双交换鉴定结果。
图5为本发明实施例1提供的荧光定量PCR检测基因转录水平的结果。
图6为本发明实施例1提供的PCV2特异性抗体检测结果。
图7为本发明实施例1提供的小鼠存活曲线示意图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
本发明提供一种构建重组链球菌的方法,构建思路如图1所示,具体包括如下流程:
1、引物设计与合成
本试验所用引物(表1)均使用Primer 5设计,并于上海生工生物工程股份有限公司合成。具体引物信息如下:根据NCBI网站所公布的SEZ ATCC 35246株基因序列(GenBank登录号:CP002904)设计引物S1、S2用于扩增Szp同源臂上臂片段;S3、S4用于扩增Szp同源臂下臂片段;设计M1、M2引物用于鉴定重组菌株。根据PCV2 VRL13-AUG-2018株基因序列(GenBank登录号:NC_005148.1)设计引物S-PCV-1、S-PCV-2用于扩增Cap片段,设计引物PCV-S-1、PCV-S-2用于PCR鉴定Cap基因。
表1引物序列
2、同源重组载体pG+host5-SzP-Cap的构建
从猪圆环2型阳性猪的脾脏中提取DNA,使用S-PCV-1、S-PCV-2引物扩增获得Cap片段;从ST171中提取DNA,分别使用S1、S2与S3、S4引物扩增获得Szp上下同源臂片段。使用以上片段酶切位点对应的酶将Szp上下同源臂片段与Cap片段酶切连接至链球菌温敏自杀型穿梭载体pG+host5中,获得同源重组载体pG+host5-SzP-Cap。
图2为同源重组载体pG+host5-SzP-Cap的构建结果,其中a为重组载体的构建策略,b为重组载体的酶切鉴定结果。
3、重组菌株ST171-Cap的构建
将构建的同源重组载体pG+host5-SzP-Cap电转入链球菌感受态ST171中,使用5mm电转杯,电转参数为:电压2.5kV,电容25uFD,脉冲电阻500Ω。将电转后的产物转移至1.5mLEP管中,28℃摇床培养2h后,涂布于含有红霉素的TSA平板上,28℃培养36h至单菌落出现。挑取单菌落在37℃进行第一次同源重组。之后再选取单菌落连续传代4代后筛选出红霉素敏感菌株。将筛选出的红霉素敏感菌株提取DNA进行PCR验证,并送往生工测序。
图3为重组菌株ST171-Cap的构建策略示意图。图4为重组菌株ST171-Cap的鉴定结果;其中,左图为单交换鉴定结果,右图为双交换鉴定结果。
4、qRT-PCR检验重组菌株Cap基因转录水平
使用传统法抽提重组菌株ST171-Cap和亲本菌株ST171的RNA。按照promega公司的DNase I试剂盒与反转录试剂盒对此次提取的细菌RNA进行处理。使用Takara公司的荧光定量试剂与罗氏LightCycler480荧光定量PCR仪对突变菌株的Cap基因与亲本菌株的Szp基因转录水平进行荧光定量PCR分析。定量PCR体系为:TB Green,10μL;上下游引物,各1μL;cDNA,2μL;ddH2O,6μL;反应程序为95℃预变性30s;95℃变性5s、60℃退火30s、72℃延伸45s,40个循环。
图5为荧光定量PCR检测基因转录水平的结果,可以看出,Szp和Cap都在重组菌株ST171-Cap中顺利表达。
5、小鼠半数致死量测定
分别挑取亲本菌株和突变菌株的单菌落,接种于含5%犊牛血清TSB培养基中震荡培养12h。细菌计数后,PBS洗涤一遍,分别做倍比稀释至10-9CFU/mL、10-8CFU/mL、10-7CFU/mL、10-6CFU/mL,小鼠腹腔分别接种500μL,观察小鼠临床变化以测定突变菌株和亲本菌株的半数致死量。半数致死量的计算分析采用改良寇式法(Karber氏法)进行,计算公式为LD50=log-1[Xm-i(Σp-0.5)]。
6、ELISA检验重组菌株诱导的抗体水平
将6周龄的雌BALB/c小鼠随机分为4组,每组10只。将突变菌株用无菌PBS调整为1×1010CFU/mL,使用4%福尔马林灭活,并使用MONTANIDETMGEL 01进行乳化,通过腹腔注射免疫第一组小鼠,14天后相同步骤加强免疫,每次免疫剂量为0.5mL。第二组小鼠接种某商品化PCV2全病毒灭活疫苗作为阳性对照(Positive),第三组小鼠接种乳化后的亲本菌株作为阴性对照(Negative),第四组接种无菌PBS作为空白对照,免疫程序同第一组。分别对二免后14天的所有小鼠进行断尾采血并分离血清,使用猪圆环病毒Ⅱ型抗体检测试剂盒检测PCV2 Cap抗原特异性抗体水平。
图6为PCV2特异性抗体检测结果,由图中可以看出重组菌株ST171-Cap在接种后产生的PCV2特异性抗体水平要显著高于阳性对照(某商品化的PCV2全病毒灭活疫苗)。
7、链球菌感染小鼠试验
将6周龄的雌性BALB/c小鼠随机分为4组,每组10只。将重组菌株ST171-Cap和亲本菌株ST171的浓度用无菌PBS调整为1×1010CFU/mL,经过4%的福尔马林灭活后,加入终浓度为10%-20%的佐剂MONTANIDETMGEL 01并乳化,通过腹腔注射分别免疫第一组和第二组小鼠,14d后用相同的步骤加强免疫一次,每次免疫剂量为0.5mL。第三组小鼠接种PBS,注射剂量为0.5ml,14d后以相同步骤再注射一次。第四组小鼠接种乳化后的PBS,步骤同第三组。二次免疫14d后接种1×105CFU的马链球菌兽疫亚种野毒株C55138,观察小鼠死亡状况。
图7为小鼠存活曲线示意图,从图中可以看出,重组菌株ST171-Cap可以对小鼠产生有效保护。
表2为猪链球菌ST171弱毒株和重组菌株ST171-Cap的半数致死量测定结果。
表2 ST171与ST171-Cap感染小鼠半数致死量测定
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 佛山科学技术学院
广东永顺生物制药股份有限公司
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gggaccaaca aaatctctat accctttgaa tactacagaa taagaaaggt taaggttgaa 180
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ctagatgaca actttgtaat aaaggccaca gcccaaacct atgaccccta tgtaaactac 300
tcctcccgcc atacaatccc ccaacccttc tcctaccact cccgttactt cacacccaaa 360
cctgttcttg attccactat tgattacttc caaccaaata acaaaaggaa tcagctgtgg 420
atgagactac aaaccagtag aaatgtggac cacgtaggcc tcggcactgc gttcgaaaac 480
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Claims (9)
1.一种重组链球菌,其特征在于,所述重组链球菌为:
通过将猪圆环病毒2型的Cap蛋白的编码序列插入链球菌中Szp基因序列中得到。
2.根据权利要求1所述的重组链球菌,其特征在于,所述链球菌为猪链球菌ST171弱毒株。
3.根据权利要求1所述的重组链球菌,其特征在于,所述猪圆环病毒2型的Cap蛋白的编码序列包括如SEQ ID NO:1所示核苷酸序列。
4.权利要求1-3任一项所述重组链球菌的制备方法,其特征在于,包括:
构建包含有所述链球菌中Szp基因上下同源臂片段和所述猪圆环病毒2型的Cap蛋白的编码序列的重组载体;
将所述重组载体转化至所述链球菌中进行同源重组。
5.根据权利要求4所述的制备方法,其特征在于,所述重组载体中,所述猪圆环病毒2型的Cap蛋白的编码序列位于所述链球菌中Szp基因的上同源臂片段和下同源臂片段之间。
6.一种引物对,其特征在于,所述引物对用于鉴定权利要求1-3任一项所述重组链球菌;
所述引物对包括:
M1:5’-TCTAGGACTGATAGGTCCAT-3’,
M2:5’-CTAATGCTACTTCGGTCT-3’。
7.一种疫苗,其特征在于,所述疫苗包括权利要求1-3任一项所述重组链球菌和佐剂。
8.根据权利要求7所述疫苗,其特征在于,所述佐剂为MONTANIDETMGEL 01佐剂。
9.权利要求1-3任一项所述重组链球菌在制备免疫猪圆环病毒2型和/或链球菌的药物中的应用。
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