CN112501096B - 一组肠道外致病大肠杆菌糖蛋白结合疫苗的基因工程大肠杆菌的构建及应用 - Google Patents
一组肠道外致病大肠杆菌糖蛋白结合疫苗的基因工程大肠杆菌的构建及应用 Download PDFInfo
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Abstract
本发明公开了一组肠道外致病大肠杆菌糖蛋白结合疫苗的基因工程大肠杆菌的构建及应用。基于CRISPR‑cas9删除E.coli K‑12 MG1655菌株中zwf、pfkB、lacZ、manA和pykA基因,构建能够协同利用葡萄糖和甘油的菌株,分别用于合成O抗原多糖和维持细菌生理代谢;进一步删除waaL、肠杆科共有抗原(ECA)和O16基因簇,构建底盘细胞为OSLA。分别导入O5和O7抗原合成基因簇,同时导入表达糖基转移酶以及载体蛋白,合成各血清型糖蛋白结合疫苗。纯化的糖蛋白经过动物免疫实验证实能够刺激小鼠产生具有保护性作用的抗体,保护率可以达到90%以上。本发明构建的重组大肠杆菌糖蛋白结合疫苗为抗肠道外致病大肠杆菌感染的免疫治疗提供了新的选择。
Description
技术领域
本发明属于合成生物学技术领域,设计一组肠道外致病大肠杆菌O5和O7糖蛋白结合疫苗的基因工程大肠杆菌的构建及应用。
背景技术
肠道外致病大肠杆菌(extraintestinal pathogenic Escherichia coli,ExPEC)是人畜共患病的病原体,它们通常在人类(和其他动物)的肠道微生物群中占据特定生态位,然后从肠道这个“蓄水池”迁移到其他位点,引起肠道外的感染。ExPEC具有复杂的进化谱系,含有众多的毒力因子,基因组可塑性也较大。这些致病菌株不仅感染泌尿系系统,还会引起菌血症或败血症。根据感染位点的差异,可以简单地将ExPEC分为禽类致病大肠杆菌(avian pathogenic Escherichia coli, APEC)、尿路致病大肠杆菌(uropathogenicEscherichia coli, UPEC)和致新生儿脑膜炎大肠杆菌(neonatal meningitis-causingEscherichia coli, NMEC)。这三个簇之间相互有交叉,如,大肠杆菌O2血清型,既能引起禽类感染,也能引起人来的尿路感染;O18血清型引起禽类肠败血症,也会导致新生儿脑膜炎;引起尿路感染的UPEC菌株,通常也能引起严重的败血症。
直到20世纪90年代末,ExPEC对最广泛使用的抗菌药物高度敏感,如氨苄西林和甲氧苄啶磺胺甲恶唑。然而,在过去十年中,ExPEC已成为一线抗菌药物(包括头孢菌素类、氟喹诺酮类,氨苄西林和甲氧苄啶磺胺甲恶唑)的重要耐药库。更让人担忧的是,ExPEC能够对所有主要抗生素的形成耐药性。在早产儿中,由多重耐药ExPEC引起的早发性新生儿败血症发病率呈上升趋势。某些ExPEC菌株能够耐受M型头孢克肟,另一些菌株能够分泌超广谱β内酰胺酶,成为名副其实的多重耐药菌。新生儿败血症和脑膜炎感染的抢救窗口期很短,这类病原体的感染显著增加了治疗难度,延长病患住院时间,并且死亡率致残率也更高。
通常情况下,细菌表面多糖(O抗原、K抗原)可作为细菌的保护性抗原,免疫后可以刺激成熟的B淋巴细胞,激发免疫系统产生强烈的抗体,可以保护机体抵御随后病菌的侵袭。但是,在众多的医学实践中,人们逐渐发现多糖结合疫苗有个重大缺陷——既不能诱导易受细菌感染人群,特别是老人和不超过两岁的孩子形成保护性抗体和记忆效应。其免疫过程没有T细胞的参与,诱导免疫系统产生的抗体主要为IgM,亲和力低,而且存在时间较短。细菌表面多糖可以被化学试剂活化并与载体蛋白共价偶联,形成多糖蛋白结合疫苗。该类疫苗能够诱发机体免疫系统产生更理想的免疫应答,如长效免疫记忆以及在多糖疫苗中不存在的高亲和力的IgG抗体,有效弥补了多糖疫苗的缺陷,是目前为止人类最成功的疫苗,可以给予儿童以抵抗细菌感染并且也可以给成年人提供长时间持续的免疫应答。
目前,合成糖蛋白疫苗的方法主要是化学法。化学法合成糖蛋白结合疫苗包含多个步骤:1)培养致病菌和蛋白生产菌株;2)纯化细菌表面多糖和载体蛋白;3)脱除内毒素并用化学试剂活化,同时也活化载体蛋白;4)化学偶联活化的多糖分子和载体蛋白;5)精细纯化得到目的产品。在每个步骤中都会发生相当大的所耗,并且由于化学偶联的随机性,最终产品偶联的位置不明确,影响批次间的稳定性。该过程耗时,成本高昂,并且通常需要大规模培养病原菌以进行多糖生物合成。基于此,亟待建立和开发新的糖蛋白疫苗生产方法和工艺,从而大幅降低生产成本,普及其商业化应用。
发明内容
本发明进一步公开了合成肠道外致病大肠杆菌糖蛋白结合疫苗的大肠杆菌基因工程菌的构建方法,其特征在于,步骤如下:
一组合成肠道外致病大肠杆菌糖蛋白结合疫苗的大肠杆菌底盘细胞,其特征在于:基于CRISPR-Cas9的方法删除zwf、pfkB、lacZ、manA和pykA基因得菌株OS12,该菌株细胞能够协同利用葡萄糖和甘油;OS12中O抗原连接酶WaaL和肠杆科共有抗原(enterobacterial common antigen,ECA)被删除,同时原始已失活的O16抗原基因簇被全部删除,构建底盘细胞为OSLA;含有肠道外致病大肠杆菌(extraintestinal pathogenicEscherichia coli, ExPEC)O5和O7抗原合成基因簇分别导入上述底盘细胞OSLA,命名为OSLA-O5和OSLA-O7。所述含有ExPEC血清型O5和O7抗原合成基因簇指的是:通过对实验室保存的从婴幼儿脑脊液中分离的致病大肠杆菌基因组进行测序并定位出其抗原合成基因簇区域,将其转入OSLA菌株。
所述含有糖基化系统是指,通过将来自脑膜炎奈瑟球菌(Campylobacter jejuni)并经过大肠杆菌密码子优化的O-糖基转移酶编码基因(undecaprenyl-diphosphooligosaccharide--protein glycotransferase,GI:905417)与合成的P15启动子和BBa_B1002终止子共同导入OSLA-O5、OSLA-O7中,其中P15启动子序列为组成型表达的中等强度启动子。
本发明进一步公开了所述合成肠道外致病大肠杆菌糖蛋白结合疫苗的大肠杆菌细胞底盘的构建方法,其特征在于按如下的步骤进行:
1)将质粒pCas转化到宿主菌E.coli MG1655 K-12中,获得携带该质粒的宿主菌;
2)以基因组为模板,设计待删除基因的sgRNA以及同源臂,通过Gibson组装的方法体外构建pTarget质粒,并转化感受态细胞DH5α;
3) 提取测序正确的pTarget质粒;
4) 向步骤1所得的携带质粒pCas的宿主菌中转化pTarget质粒,电击复苏后涂布于LB+壮观霉素(50 ug/ml)+L-(+)-arabinose(10 mM)的平板,获得目的基因被删除的重组菌;
5) 以步骤4所得的删除一个基因的重组菌为宿主菌,重复步骤2)3)4)的操作,获得删除zwf、pfkB、lacZ、manA、pykA、waaL、ECA和O16基因簇的重组菌;
6) 所述构建zwf、pfkB、lacZ、manA、pykA、waaL、ECA和O16删除引物和鉴定引物的核苷酸序列如SEQ ID NO:1-8所示。
本发明同时也公开了所述融合O-糖基化一致序列和DsbA信号肽序列的霍乱毒素亚基B基因ctxB和寡糖转移酶pglL导入菌株中,其特征在于步骤如下:
1)将质粒pCas分别导入转化到宿主菌OSLA-O5、OSLA-O7中,获得携带该质粒的宿主菌;
2)以基因组为模板,设计sgRNA、霍乱毒素亚基B基因ctxB和寡糖转移酶基因片段pglL和相应的P15启动子和BBa_B1004终止子以及同源臂,通过Gibson组装的方法体外构建pTarget质粒,并转化感受态细胞DH5α;
3)提取测序正确的pTarget质粒;
4)向步骤1所得的携带质粒pCas的宿主菌中转化pTarget质粒,电击复苏后涂布于LB+壮观霉素(50ug/ml)+L-(+)-arabinose(10mM)的平板,获得目的基因插入正确的重组菌;
5)所述构建融合O-糖基化一致序列和DsbA信号肽序列的霍乱毒素亚基B基因ctxB和寡糖转移酶pglL的扩增引物和鉴定引物的核苷酸序列如SEQ ID NO:1-8所示。
本发明还公开了一组采用重组菌进行糖蛋白疫苗细胞发酵合成肠道外致病大肠杆菌血清型糖蛋白结合疫苗的方法,其特征在于按如下的步骤进行:
(1)培养基和发酵方法:
NBSYT培养基(1L):Tryptone(胰蛋白胨):1-10g,Yeast Extract(酵母提取物):1-5g,(NH4)2HPO4(磷酸二氢钾):1-3.5g,K2HPO4:1-3.5g,KH2PO4•3H2O:1-6.5g。若配置固体培养基,则再加入10-15g Agar(琼脂),将所得细胞工厂在20 ml含有葡萄糖 20-100 g/L,甘油20-100 g/L的NBSYT培养基中37℃,220 rpm过夜活化,活化菌转入1 L 含葡萄糖 20-100g/L,甘油20-100 g/L的NBSYT培养基,37℃,220rpm培养至OD 600≈0.6,并转至30℃,180 rpm培养,约72h后,离心收集菌体,超高压破碎仪破碎细胞,借助于AKTA Primeplus蛋白纯化工作站大量纯化糖蛋白;
(2)将活化的ExPEC O5:K1、O7:K1、O18:K1菌株培养至OD 600≈0.6,经巴氏杀菌灭活后,经三次皮下注射于兔子后取适量的血,离心得粗血清,粗血清经过毛细管沉淀纯化得到高纯度血清,上述血清可分别与E. coli OSLA-O5、OSLA-O7菌株发生凝集反应,且经过western-blotting鉴定条带特异。
本发明进一步公开了一组合成肠道外致病大肠杆菌糖蛋白结合疫苗的大肠杆菌底盘细胞在合成不同血清型的肠道外致病大肠杆菌糖蛋白结合疫苗在制备治疗肠道外致病大肠杆菌感染中的应用。糖蛋白结合疫苗诱导小鼠产生特异性抗体,并保护小鼠免于ExPEC感染,保护率达到70%以上。实验结果显示,糖蛋白结合疫苗诱导小鼠产生特异性抗体,并保护小鼠免于ExPEC感染,具体包括:
糖蛋白疫苗免疫小鼠的方法与攻毒实验,其步骤如下:
纯化后的糖蛋白疫苗与弗氏完全佐剂混合,经三次皮下注射5周龄CD1小鼠,免疫后的小鼠进行攻毒实验:尾静脉注射致死量的ExPEC O5:K1、O7:K1,观察疫苗的对免疫小鼠的保护率。结果显示经过糖蛋白结合疫苗注射过的小鼠成活率较高,达到了70%(见图7)。
糖蛋白结合疫苗诱导小鼠产生特异性抗体实验,其步骤如下:
第三次免疫后的小鼠,尾静脉取血,4℃,1000 × g离心30 min,取上清制备成血清。通过酶联免疫法测定血液中特异性的IgG抗体,该蛋白结合疫苗能够刺激机体产生具有保护性的抗体IgG(见图8)。
本发明构建的基因工程菌经酚水法提取脂多糖,凝胶电泳和银染显示E.coli K-12 MG1655和OS12能够合成ExPEC O5/O7血清型的LPS,而且western-blotting结果显示有ladder状条带,证明合成了正确的糖蛋白。
本发明更加详细的描述如下:
本发明的第一个目的在于提供一株发酵葡萄糖合成ExPEC O5/O7血清型糖蛋白结合疫苗的细胞工厂。该大肠杆菌工程菌从E.coli K-12 MG1655出发,删除了O抗原链接酶基因(O-antigen ligase,waaL)和ECA,同时删除了删除zwf、pfkB、lacZ、manA、pykAF、waaL、和O16基因使其能够同时利用葡萄糖和甘油;同时该菌含有组成型表达的糖基化质粒pCDF-P15-pglL-P15-CTB和表达O5/O7抗原的穿梭质粒pCRG16-O5/O7。pCDF-P15-pglL-P15-CTB改造自pCDFDuet-1,插入合成的两对P15启动子和BBa_B1002终止子替换原始的T7启动子和终止子,其中ctboh为来自于霍乱弧菌(Vibrio cholerae)经过大肠杆菌密码子优化,且添加了O糖基化识别位点和组氨酸标签的的霍乱毒素B基因(cholerae toxin B,GI:877850);pglL为来自脑膜炎奈瑟球菌(Campylobacter jejuni)并经过大肠杆菌密码子优化的O-糖基转移酶编码基因(undecaprenyl-diphosphooligosaccharide--proteinglycotransferase,GI:905417)pCRG16- O5/O7为含有肠道外致病大肠杆菌O5/O7抗原基因簇表达质粒,同时含有合成O5/O7重复单元的基因,以及将重复单元聚合起来的聚合酶和翻转酶。
本发明的第二个目的是提供一种所述发酵葡萄糖合成ExPEC O5/O7血清型糖蛋白结合疫苗的构建方法,该方法的步骤如下:
1.删除E.coli K-12 MG1655菌株中的zwf、pfkB、lacZ、manA、pykA、waaL、ECA和O16基因簇。
1.1 E.coli K-12 MG1655/pCas菌株的构建
1.1.1 提取pCas质粒
用生工柱式质粒小量抽提试剂盒提取pCas质粒,具体步骤见试剂盒说明书。
1.1.2制备E.coli K-12 MG1655感受态细胞
1.1.3 E.coli K-12 MG1655/pCas菌株的获得
将提取的pCas质粒通过电穿孔仪转化至E.coli K-12 MG1655感受态细胞中,然后转至复苏培养基中复苏半小时左右,涂布卡那霉素抗性2YT平板,于30℃培养箱过夜培养。次日可以看到平板上长出的单菌落即为含pCas质粒的E.coli K-12 MG1655菌株。
1.2 pTarget质粒的构建,以waaL基因为例
1.2.1设计gRNA原则:打开http://www.rgenome.net/cas-designer/ PAM Type选择SpCas9 from Streptococcus pyogenes: 5'-NGG-3'(因Cas9蛋白来自于Streptococcuspyogenes),Organism Type中选择others,genome选择Escherichia coli (K-12,MG1655),取waaL的CDS序列约900bp(不超过1000bp)黏贴至Target sequence,提交,选择评分高的spacer,spacer中最后三个碱基为PAM序列,不要复制,spacer+cas9BIRE+spy-tem构成gRNA,根据gRNA序列,设计hanging over引物,上游引物的spacer为非结合区,下游引物5’含有25-30bp碱基,该碱基序列与上游同源臂左端序列一致,以pTarget为模板扩增,高保真引物扩增,推荐在扩增时加入0.5 μL-1μL的DMSO,利于长引物的次级结构的解开。
1.2.2 扩增上游同源臂和下游同源臂,原则:下游同源臂左端与上游同源臂右端25-30个碱基序列一致,可通过overlap extension pcr连接上下游同源臂得到,也可以不进行overlap连接。
1.2.3 反向扩增pTarget质粒除gRNA以外的序列,左端含有25-30个同源碱基,与gRNA的左端一致。
1.2.4 按照NEBuilder® HiFi DNA Assembly的说明,构建pTarget-GwaaL-HwaaL。
1.2.5 大肠杆菌中导入pCas质粒,卡那霉素平板筛选正确的转化子
1.2.6 制备含有pCas质粒的感受态(于30℃培养)(该感受态可用10mM-100mM L-(+)-arabinose诱导,也可以不诱导,同源序列位于质粒上,则可以不诱导;同源序列以片段的形式导入,则需要诱导),转化pTarget-GwaaL-HwaaL质粒,复苏于SOC培养基,30℃,~2h。
1.2.7 复苏完的菌液全部接种至20ml LB+Kan+SpeC+10mM L-(+)-arabinose,30℃培养~48 h,菌液划线于LB无抗性平板,37℃培养,也可以划线于LB+Kan+SpeC+L-(+)-arabinose平板。Kan和SpeC工作浓度可以在这一步减半。
1.2.8 挑单克隆PCR验证,测序。电泳图见图3
2.pCDF-pglL-CTBOH载体的构建
2.1 pCDFDuet-1质粒的改造
合成含有两对tac启动子和BBa_B1002终止子的片段,通过酶切连接替换原始的T7启动子和T7终止子
2.2 pCDF-pglL-CTB
合成密码子优化的pglL和CTB基因,PCR扩增CTB基因,通过酶切连接至PCDF载体上。
3. pCRG16-O质粒的构建、鉴定与测序
3.1 酵母感受态细胞的制备
3.2 pCRG16线性克隆载体的获得
(1)用质粒小量抽提试剂盒提取pCRG16质粒。
(2)NotI限制性内切酶消化pCRG16
3.3 DNA Assembler片段的获得
以ExPEC O5:K1为例,提取ExPEC相应血清型的基因组,通过BSPdb数据库(http://bspdb.nankai.edu.cn/index.html)定位相应的O抗原合成基因簇,将整个O抗原合成基因簇成5个部分扩增,每片段约5K。第一个片段在5’含有pCRG16线性克隆载体NotI酶切位点处同源序列40mer碱基,第二个片段含有第一个片段3’同源序列约100-200mer,依次类推,直至最后一个片段的3’含有pCRG16线性克隆载体另一个NotI酶切位点同源序列40mer碱基。详细的原理如图6所示
3.4酿酒酵母in vivo重组构建O抗原表达质粒
将上述扩增得到的片段与pCRG16线性克隆载体共电转化至CRY1-2酿酒酵母,复苏于1 ml SD尿嘧啶缺陷液体培养基,取复苏的培养基100μL涂布于SD 尿嘧啶缺陷平板,30℃培养48-72h,随机挑取3-5个单菌落,用索莱宝公司的酵母质粒提取试剂盒提取酵母质粒,PCR方法鉴定质粒,通过大小判断是否重组成功。从酵母中提取的重组质粒转化至NEB-10β,提取NEB-10β中的质粒,送测序。
4.脂多糖提取,凝胶电泳与银染
将测序正确的O抗原表达载体先转化至至JM109感受态细胞中,涂布氯霉素抗性2YT平板,经鉴定正确的单克隆用20 ml液体2YT培养基培养至OD600≈0.6时提取LPS。配置12% SDS-PAGE电泳胶,将提取的LPS进行凝胶电泳,然后银染。
5.ELISA法测定免疫小鼠血清的抗体滴度
(1)用包被液稀释的LPS包被96孔板,每孔100 μL,含LPS 10 μg,4℃过夜。设置空白对照和阴性对照。
(2)包被结束后PBST洗3次,每次3分钟。洗完后甩干96孔板,加入200 μL封闭液37℃封闭2小时。
(3)甩干96孔板,加入保温液倍比稀释的小鼠血清,每孔100 μL。37℃孵育1小时。
(4)PBST洗3次,每次3分钟。洗完后甩干96孔板,加入保温液稀释的二抗,每孔100μL,37℃孵育1小时。
(5)二抗孵育结束后,再用PBST洗5次,每次3分钟。洗完后甩干96孔板,加入100 μLOPD-H2O2显色液,避光反应15分钟。
(6)加入50μL终止液,终止5分钟后,用酶标仪读取490 nm吸光值。
本发明提供了一种具体应用,所述大肠杆菌工程菌合成糖蛋白结合疫苗的方法和相应的纯化步骤和检测方法具体步骤如下:
6.糖蛋白结合疫苗的发酵合成、纯化与检测:
将pCRG16-O5/O7和pCDF-pglB-CTB共转入JM109∆waaL∆ECA感受态细胞中,复苏1-2h,涂布氯霉素和链霉素双抗平板,37℃培养48-60h。对长出的单克隆进行煮菌PCR鉴定。鉴定正确的克隆进行保菌。
糖蛋白结合疫苗合成与纯化:
(1)将上述菌株接种于20 mL的LB培养基中并加入链霉素(50 μg/mL)和氯霉素(10μg/mL),37℃,180 rpm过夜培养。
(2)次日早晨,将过夜培养的菌液按照1:100的比例转接至1L的NBSYT培养基中,37℃,200 rpm培养10-12 h。
(3)加入IPTG诱导蛋白表达(终浓度为1 mM),30℃,180 rpm诱导约10-12 h。
(4)用高速离心机12000 rpm,10 min收菌,并将菌体转至50 mL离心管中。
(5)菌体用0.9%NaCl洗涤一次,然后再加入Binding buffer重悬菌体至OD600≈200。
(6)加入蛋白酶抑制剂(终浓度为1 mM)和溶菌酶(终浓度为1 mg/mL)。冰浴30min。
(7)将离心管放在冰上,用超声破碎仪破碎菌体细胞。超3S,停2S,累计超声时间约40 min,直至液体呈棕褐色且液体流动性变好。所述的蛋白酶抑制剂购买于thermo fisherscientific公司。
(8)加入少量的DNA酶和RNA酶(终浓度为5 μg/mL),冰浴15 min。
(9)4℃,8000 rpm离心15 min,将上清转至另一50 mL离心管备用(可置于-80℃保存2-3天)。
用AKTA Primeplus系统进行蛋白纯化,步骤如下:
(1)配制IMAC buffer A、IMAC buffer B、去内毒素buffer、AEC bufferA、AECbufferB以及20%的无水乙醇。以上试剂均需过水膜处理并超声30 min。
(2)打开仪器,在load模式下清洗机器管路,待显示屏上曲线都平稳后装上镍离子鳌合层析柱。
(3)换用IMAC buffer A再次进行清洗,直至曲线再次平稳。
(4)将超声破碎后的上清液过0.22 μm滤膜,并进样至上样环中。此时如果曲线有所波动继续在此模式下用IMAC buffer A冲洗至平稳。
(5)将仪器调至inject模式,使上样环中的样品与镍柱结合。直至上样环中的样品完全与镍柱结合。
(6)重新调至load模式,先用去内毒素buffer冲洗40个柱体积再用IMAC buffer A继续冲洗,直至曲线再次平稳。
(7)调至梯度洗脱模式,使IMAC buffer B浓度在6个柱体积内从0%上升至100%进行梯度洗脱目的蛋白。当显示屏上指示UV的曲线出现明显的吸收峰时,收集出峰时所对应的收样管中的样品即为目的蛋白。
(8)样品放入透析袋并在透析液中置于4℃冰箱透析2h以除去咪唑。
(9)用镍离子亲和层析纯化完的蛋白用离子交换色谱柱再次进行纯化。将镍离子鳌合层析柱换成离子交换色谱柱,用AEC buffer A冲洗管路。
(10)将蛋白上样至上样环,待曲线平稳后调至inject模式使蛋白与离子交换色谱柱充分结合。
(11)用AEC buffer A冲洗10个柱体积。
(12)用AEC buffer A和AEC buffer B进行梯度洗脱目的蛋白。当显示屏上指示UV的曲线出现明显的吸收峰时,收集出峰时所对应的收样管中的样品即为我们的目的蛋白(如SEQ ID NO:9所示),加10%的甘油-80℃保存。
糖蛋白结合疫苗检测——western blot检测:
(1)样品经过SDS-PAGE分离后,把凝胶放入1×转膜Buffer 中,置于水平摇床上摇15min。
(2)准备合适大小的PVDF膜和滤纸,PVDF膜在使用前需在无水甲醇中浸泡5min,滤纸需在1×转膜Buffer中浸润。将海绵、滤纸、凝胶、PVDF膜、滤纸、海绵按此顺序依次夹好,放入转膜仪中。4℃,恒压70V,转1h。
(3)转膜完成后,把PVDF膜放入孵育容器中,在水平摇床上用脱脂奶粉溶液室温封闭1h。
(4)PVDF膜用配好的TBST溶液洗涤三次,每次10 min,加入稀释好的一抗,室温水平摇床上孵育1h(或者4℃过夜孵育)。
(5)用TBST洗涤三次,每次10 min,加入稀释好的二抗,室温水平摇床上孵育1h。
(6)再用TBST洗涤三次,每次10 min,用TBS洗涤一次。使用高灵敏ECL发光试剂盒于AI600超灵敏多功能成像仪曝光成像。检测结果显示:阴性对照没有72KD处条带,实验组在72KD处有明显的ladder状条带,证明合成了正确了糖蛋白结合疫苗。
该糖蛋白结合疫苗能够刺激机体免疫系统产生T细胞依赖的免疫应答,极大地提高了多糖的免疫原性,同时具有很强的特异性。在整个免疫过程中,糖蛋白并不像多糖一样需要与抗体广泛交联,因此较短的糖链依然可以产生有效的免疫反应。糖蛋白引起的免疫机制与多糖类完全不同,它能引起T、B细胞的联合识别,明显提高了免疫效果。糖蛋白还可产生免疫记忆,因此糖蛋白结合疫苗被认为是人类最成功的疫苗。糖蛋白结合疫苗在临床使用过程中对疾病的预防产生了非常显著的效果。这类疫苗目前已成功应用于临床的有三种,分别是针对B型流感嗜血杆菌、肺炎链球菌以及脑膜炎奈瑟菌的糖蛋白结合疫苗。此外,还有许多疫苗正处于临床试验中,如金黄色葡萄球菌、宋内及福氏志贺氏菌等。
附图说明
图1 质粒pCRG16-O5/O7;用于表达致脑膜炎大肠杆菌O5/O7抗原合成基因簇;
图2 质粒pCDFm的图谱,用来表达糖基化系统;
图3 waaL基因被删除的电泳图;
(图中,M:Marker;泳道1-2分别为:对照,waaL删除菌株);
图4 pCRG16-O5/O7质粒的构建方法;
图5 免疫和攻毒实验示意图;
图6 血清型O5/O7最小致死菌量,A、B分别对应O5、O7血清型;
图7 免疫小鼠攻毒后的生存曲线图,A、B分别对应O5、O7血清型;
图8 免疫小鼠血清中的抗体滴度,A、B分别对应O5、O7血清型。
具体实施方式
下面结合具体实施例对本发明做进一步的说明,但本发明不受实施例的限制;
以下实施例中所用的材料、试剂、仪器和方法未经特殊说明均为本领域中的常规材料、试剂、仪器和方法,均可通过商业渠道获得;
本发明中质粒提取采用生工生物工程(上海)有限公司的SanPrep柱式质粒DNA小量抽提试剂盒(Catalog NO.:B518191),切胶回收是采用生工生物工程(上海)有限公司的SanPrep柱式胶回收试剂盒(Catalog NO.:B518131),DNA片段的连接是使用fermentas公司的T4 DNA Ligase(Catalog NO.:EL0014),DNA片段的扩增使用fermentas公司的pfu DNApolymerase(Catalog NO.:EP0571),PCR质粒模板的消化使用fermentas公司的FastDigest KpnI(Catalog NO.:FD0524),BamHI(Catalog NO.:FD0054) NcoI(Catalog NO.:FD0574)BglII(Catalog NO.:FD0083),NotI(Catalog NO.:FD0593)
E.coli电击转化实验使用Bio-Rad的电转仪(Catalog NO.:165-2100)。细菌基因组提取使用北京康为世纪生化科技有限公司的细菌基因组提取试剂盒(Catalog NO.:CW0552S)。
表1删除waaL所用引物序列
表2本发明用到的菌株与原料的来源
实施例1
基因的获取
本实施例中,获取来源于霍乱弧菌(Vibbrio cholerae)经过大肠杆菌密码子优化的霍乱毒素B(CTB,GI:877850);和来自脑膜炎奈瑟球菌(Campylobacter jejuni)并经过大肠杆菌密码子优化的O-糖基转移酶编码基因(undecaprenyl-diphosphooligosaccharide--protein glycotransferase,GI:905417)。
实施例2
基因删除引物的设计
本实施例使用CRISPR-cas9 λRed重组系统删除E.coli K-12 MG1655的基因下面以waaL基因为例,详细阐述基因敲除的步骤,其他基因删除引物设计原则与此相同。
查找E.coli K-12 MG1655 waaL核苷酸序列,设计waaL的删除引物和鉴定引物。waaL的删除引物为GwaaL-F,HwaaL-GwaaL-R,waaL-up-F,waaL-up-Rm,waaL-down-Fm,waaL-down-R,HwaaL-pTarget-backbone-F,GwaaL-pTarget-backbone-R鉴定引物为S2-waaL-F/R。核苷酸序列如表1-8所示。
实施例3
3.1 E.coli K-12 MG1655 ΔwaaL的构建
(1) 提取pCas质粒
用生工柱式质粒小量抽提试剂盒提取pCas质粒,具体步骤见试剂盒说明书。
(2)制备E.coli K-12 MG1655感受态细胞
(3)E.coli K-12 MG1655/pCas菌株的获得
将提取的pCas质粒通过电穿孔仪转化至E.coli K-12 MG1655感受态细胞中,然后转至复苏培养基中复苏半小时左右,涂布卡那霉素抗性2YT平板,于30℃培养箱过夜培养。次日可以看到平板上长出的单菌落即为含pCas质粒的E.coli K-12 MG1655菌株。
3.2 pTarget-waaL质粒的构建
(1)设计gRNA原则:打开http://www.rgenome.net/cas-designer/ PAM Type选择SpCas9 from Streptococcus pyogenes: 5'-NGG-3'(因Cas9蛋白来自于Streptococcuspyogenes),Organism Type中选择others,gemone选择Escherichia coli (K-12,MG1655),取waaL的CDS序列约900bp(不超过1000bp)黏贴至Target sequence,提交,选择评分高的spacer,spacer中最后三个碱基为PAM序列,不要复制,spacer+cas9BIRE+spy-tem构成gRNA,根据gRNA序列,设计hanging over引物,上游引物的spacer为非结合区,下游引物5’含有25-30bp碱基,该碱基序列与上游同源臂左端序列一致,以pTarget为模板扩增,高保真引物扩增,推荐在扩增时加入0.5 μL-1μL的DMSO,利于长引物的次级结构的解开。
(2)扩增上游同源臂和下游同源臂,原则:下游同源臂左端与上游同源臂右端25-30个碱基序列一致,可通过overlap pcr连接上下游同源臂得到,也可以不进行overlap连接。overlap连接好的利于下一步组装。
(3)反向扩增pTarget质粒除gRNA以外的序列,左端含有25-30个同源碱基,与gRNA的左端一致。
(4)按照NEBuilder® HiFi DNA Assembly的说明,构建pTarget-GwaaL-HwaaL。
(5)大肠杆菌中导入pCas质粒,卡那霉素平板筛选正确的转化子。
(6)制备含有pCas质粒的感受态(于30℃培养)(该感受态可用10 mM-100 mM L-(+)-arabinose诱导,也可以不诱导),转化pTarget-GwaaL-HwaaL质粒,复苏于SOC培养基,30℃,~2 h。
(7)复苏完的菌液全部接种至20 ml LB+Kan+SpeC+10mM L-(+)-arabinose,30℃培养~48 h,菌液划线于 LB无抗性平板,37℃培养,也可以划线于LB+Kan+SpeC+L-(+)-arabinose平板。Kan和SpeC工作浓度可以在这一步减半。
(8)挑单克隆 PCR验证,测序。电泳图见图3。
实施例4
4.1糖蛋白结合疫苗的合成
(1)将上述菌株接种于20 mL的LB培养基中并加入链霉素(50 μg/mL)和氯霉素(10μg/mL),37℃,180 rpm过夜培养。
(2)次日早晨,将过夜培养的菌液按照1:100的比例转接至1L的NBSYT培养基中,37℃,200 rpm培养10-12 h。
(3)加入IPTG诱导蛋白表达(终浓度为1 mM),30℃,180 rpm诱导约10-12 h。
4.2 糖蛋白结合疫苗的纯化
(1)用高速离心机12000 rpm,10 min收菌,并将菌体转至50 mL离心管中。
(2)菌体用0.9%NaCl洗涤一次,然后再加入Binding buffer重悬菌体至OD600≈200。
(3)加入蛋白酶抑制剂(终浓度为1 mM)和溶菌酶(终浓度为1 mg/mL)。冰浴30min。
(4)将离心管放在冰上,用超声破碎仪破碎菌体细胞。超3S,停2S,累计超声时间约40 min,直至液体呈棕褐色且液体流动性变好。所述的蛋白酶抑制剂购买于thermo fisherscientific公司。
(5)加入少量的DNA酶和RNA酶(终浓度为5 μg/mL),冰浴15 min。
(6)4℃,8000 rpm离心15 min,将上清转至另一50 mL离心管备用(可置于-80℃保存2-3天)。
用AKTA Primeplus系统进行蛋白纯化,步骤如下:
(1)配制IMAC buffer A、IMAC buffer B、去内毒素buffer、AEC bufferA、AECbufferB以及20%的无水乙醇。以上试剂均需过水膜处理并超声30 min。
(2)打开仪器,在load模式下清洗机器管路,待显示屏上曲线都平稳后装上镍离子鳌合层析柱。
(3)换用IMAC buffer A再次进行清洗,直至曲线再次平稳。
(4)将超声破碎后的上清液过0.22 μm滤膜,并进样至上样环中。此时如果曲线有所波动继续在此模式下用IMAC buffer A冲洗至平稳。
(5)将仪器调至inject模式,使上样环中的样品与镍柱结合。直至上样环中的样品完全与镍柱结合。
(6)重新调至load模式,先用去内毒素buffer冲洗40个柱体积再用IMAC buffer A继续冲洗,直至曲线再次平稳。
(7)调至梯度洗脱模式,使IMAC buffer B浓度在6个柱体积内从0%上升至100%进行梯度洗脱目的蛋白。当显示屏上指示UV的曲线出现明显的吸收峰时,收集出峰时所对应的收样管中的样品即为目的蛋白。
(8)样品放入透析袋并在透析液中置于4℃冰箱透析2h以除去咪唑。
(9)用镍离子亲和层析纯化完的蛋白用离子交换色谱柱再次进行纯化。将镍离子鳌合层析柱换成离子交换色谱柱,用AEC buffer A冲洗管路。
(10)将蛋白上样至上样环,待曲线平稳后调至inject模式使蛋白与离子交换色谱柱充分结合。
(11)用AEC buffer A冲洗10个柱体积。
(12)用AEC buffer A和AEC buffer B进行梯度洗脱目的蛋白。当显示屏上指示UV的曲线出现明显的吸收峰时,收集出峰时所对应的收样管中的样品即为我们的目的蛋白(如SEQ ID NO:9所示),加10%的甘油-80℃保存。
实施例5
糖蛋白结合疫苗检测——western blot检测:
(1)将 1mL 菌液离心后弃上清并用 100 μL ddH2O重悬,加入等量的2×SDS上样缓冲液,沸水浴10分钟。
(2)将(1)中制备的样品离心后取上清加入SDS-PAGE胶上样孔中,通过电泳(80V,20,120V,1.5小时)将蛋白或者脂多糖分离。
(3)利用转膜仪,由上到下按照滤纸、胶、PVDF 膜、滤纸的顺序,70V恒压约1小时,从而将蛋白转入PVDF膜上。
(4)将膜浸于WB封闭液中,37℃孵育1小时。
(5)孵育一抗(用 TBST 按照一定比例稀释),按照一定比例稀释),37℃温箱中1小时。
(6)将膜用TBST溶液洗3次,每7分钟。
(7)孵育HRP标记的二抗(用TBST按照一定比例稀释),37℃温箱中1小时。
(8)再次将膜用TBST 溶液洗3次,每7分钟。
(9)将WB发光试剂中A液和B液按照1:1体积混匀,孵于膜表面(蛋白),体积混匀,孵于膜表面(蛋白),体积混匀,孵于膜表面(蛋白),体积混匀,孵于膜表面(蛋白),利用WB成像仪检测。
实施例6
LPS提取和银染
(1)将细菌培养于 37 ℃三角瓶中 220 rpm 培养12小时后 8000 rpm 离心8分钟收菌,弃上清。沉淀用预冷的PBS洗3次, 最后一次离心之后弃上清再空离3分钟。
(2)按照每克湿重的菌体加入3 mL 蒸馏水的比例重悬菌体,置于冰上蒸馏水的比例重悬菌体,置于冰上3分钟 后再放入68 ℃水浴锅中 30分钟,如此往复3次。
(3)加入等体积的90%苯酚溶液,在 68℃水浴下剧烈震荡30分钟
(4)150000 rpm,离心20分钟,小心吸取上层水相部分。加入等量蒸馏水,再次在68℃水浴下剧烈震荡30分钟后离心,取水相部分。
(5)加入-20℃预冷的无水乙醇使无水乙醇的体积达到75% (v/v),-20 ℃静置20min,之后15000 rpm离心10 min,弃上清。室温挥发干。
(6)按比例在ddH2O中加入cutsmart buffer,每50 μl体系中加入加入10 mg/mlRNase和DNase I各4 μl,37℃消化2小时,之后加入10 mg/ml蛋白酶K 4μl,56℃消化2小时
(7)重复(5)
(8)加入适量去离子水重溶沉淀,即为LPS水溶液
(9)待测样品与等体积的2×银染上样缓冲液混匀,沸水中煮5分钟后上样,通过SDS-PAGE进行分离。80 V恒压20分钟后,120 V恒压约1.5小时,直到溴酚蓝跑出胶的最下沿。
(10)将胶小心取下,浸没于固定液中,在摇床中缓慢震荡15分钟,之后换固定液再震荡15分钟。
(11)固定后将胶置于增敏液中,缓慢晃动30分钟。
(12)用ddH2O洗3次,每次15分钟。
(13)洗完后将胶放入硝酸银溶液中,反应20分钟。
(14)再用ddH2O洗两次,每次1分钟。
(15)胶置于显影液中,视颜色终止反应。当出现条带后,倒掉显影液,加入终止液,终止10分钟。
(16)再用ddH2O清洗3次,每次5分钟。之后扫描仪进行扫描。
实施例7
肠道外致病大肠杆菌糖蛋白结合疫苗的应用:动物免疫实验
5周的CD-1雌鼠(维通利华实验动物有限公司)进行免疫实验及抗菌实验,具体过程如下:
(1)将小鼠随机分为3组,分别为pbs组,阳性对照组和实验组。
(2)分别在第0,14,28天进行免疫。每只小鼠每次注射量总体积为100 μL,包括2.5μg的糖蛋白结合疫苗(以多糖量计算)以及等体积的弗氏佐剂,皮下注射。其中第一次免疫用弗氏完全佐剂,后两次均为弗氏不完全佐剂。负对照组均注射等体积的PBS。阳性对照组为108经65℃、30 min灭活的死菌。
(3)末次免疫结束7天后,对每组中的每只小鼠尾静脉取血,3000 rpm,4°C离心得到血清置于-80°C备用。
(4)进行抗菌实验。对三组小鼠每只小鼠注射最小致死量CFU溶于1×PBS溶液的O5/O7血清型的致脑膜炎大肠杆菌,尾静脉注射。24小时后尾静脉取血于抗凝管中稀释涂布LB固体平板,37°C,待平板长出单菌落后计数。连续观察4天小鼠的生存状况。
(5)结果显示经过糖蛋白结合疫苗注射过的小鼠成活率较高,达到了70%(见图7)。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制,尽管参照上述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的范围和精神。
SEQUENCE LISTING
<110> 南开大学
<120> 一组肠道外致病大肠杆菌糖蛋白结合疫苗的基因工程大肠杆菌的构建及应用
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gctacaagca gagaactatg acgc 24
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<211> 25
<212> DNA
<213> 人工序列
<400> 10
aagcatctgc gggatttaca gttcg 25
Claims (1)
1.肠道外致病大肠杆菌糖蛋白结合疫苗的制备方法,其特征在于:采用重组菌进行糖蛋白疫苗细胞发酵合成,具体步骤如下:
步骤一:删除 E.coli K-12 MG1655菌株中的 zwf 、 pfkB 、 lacZ 、 manA 、 pykA 、waaL 、ECA和O16基因簇;基于CRISPR-Cas9的方法删除 zwf 、 pfkB 、 lacZ 、 manA 和pykA 基因得菌株OS12,删除OS12中O抗原连接酶WaaL、肠杆科共有抗原ECA以及原始已失活的O16抗原基因簇,构建得到底盘细胞OSLA;
步骤二:含有肠道外致病大肠杆菌ExPEC-O5/O7抗原合成基因簇分别导入上述底盘细胞OSLA,命名为OSLA-O5和OSLA-O7;
步骤三:将质粒pCas分别导入转化到宿主菌OSLA-O5、OSLA-O7中,获得携带该质粒的宿主菌;
以基因组为模板,设计sgRNA、霍乱毒素亚基B基因 ctxB 和寡糖转移酶基因片段 pglL和相应的P15启动子和BBa_B1004终止子以及同源臂,通过Gibson组装的方法体外构建pTarget质粒,并转化感受态细胞DH5α;提取测序正确的pTarget质粒;
向携带质粒pCas的宿主菌中转化pTarget质粒,电击复苏后涂布于LB+壮观霉素(50ug/ml)+L-(+)-arabinose(10mM)的平板,获得目的基因插入正确的重组菌;
步骤四:将所得细胞工厂在20 ml含有葡萄糖 20-100 g/L,甘油20-100 g/L的NBSYT培养基中37℃,220 rpm过夜活化;其中,NBSYT培养基(1L)包括Tryptone(胰蛋白胨):1-10g,Yeast Extract(酵母提取物):1-5g,(NH4)2HPO4(磷酸二氢钾):1-3 .5g,K2HPO4:1-3 .5g,KH2PO4•3H2O:1-6 .5g;活化菌转入1 L 含葡萄糖 20-100g/L,甘油20-100 g/L的NBSYT培养基,37℃,220rpm培养至 OD 600 ≈0.6,并转至30℃,180 rpm培养,约72h后,离心收集菌体,超高压破碎仪破碎细胞,借助于AKTA Primeplus蛋白纯化工作站大量纯化糖蛋白,能够用作疫苗原液。
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