CN116726156A - 一种口服酵母介导的产气荚膜梭菌α毒素重组DNA疫苗 - Google Patents
一种口服酵母介导的产气荚膜梭菌α毒素重组DNA疫苗 Download PDFInfo
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Abstract
本发明公开了一种口服酵母介导的产气荚膜梭菌α毒素重组DNA疫苗,通过重组酿酒酵母靶向递送携带plc基因定点突变片段或截短后的C‑末端结构域片段的表达载体,随着该表达载体在树突状细胞内激活相应片段的转录和翻译,并由宿主机制呈递抗原,宿主机体的黏膜免疫反应激活,从而通过相应的中和抗体对产气荚膜梭菌引起的感染进行预防。
Description
技术领域
本发明属于基因工程技术领域,涉及预防产气荚膜梭菌感染的免疫制剂,具体涉及一种靶向肠道树突状细胞(DCs)递送产气荚膜梭菌α毒素抗原基因的口服重组酵母DNA疫苗。
背景技术
产气荚膜梭菌(Clostridium perfringens)广泛存在于自然界,是一种革兰氏阳性棒状厌氧菌,菌体呈现短杆状,没有鞭毛、无法运动、能形成孢子,在厌氧环境中生长状态良好,37℃~45℃为其最适的生长温度。产气荚膜梭菌的菌体表面能产生明显的荚膜,是一种条件性病原体,其分泌的肠毒素通过肠黏膜吸收,与人类的食源性疾病和动物疾病有关,可导致人体肌肉坏死(气性坏疽)、坏死性肠炎和食物中毒,以及在动物体内引起各种急性的胃肠道中毒,尤其在仔猪和羔羊中导致高发病率和高死亡率,给养殖业造成重大的经济损失。
产气荚膜梭菌分为A型、B型、C型、D型、E型、F型和G型,这7种类型的产气荚膜梭菌菌株都分泌α毒素(CPA)。α毒素是最基本、最重要的毒力因子,在产气荚膜梭菌致病过程中有着重要的作用。α毒素是一种含锌磷脂酶C,具有酶和毒素的双重性质,已被证实是引起气性坏疽和坏死性肠炎的主要毒素,能够破坏细胞膜,并引起溶血和组织坏死。另外α毒素也被称为鞘磷脂酶,能够水解神经鞘磷脂,从而导致膜结构紊乱。
以酵母作为载体的口服制剂,例如中国专利201310647359.3、202011262351.1,不仅能有效地将功能性核酸分子(shRNA、表达载体)输送到黏膜免疫系统,以激活保护性黏膜乃至全身免疫反应,而且输送载体本身也安全无害。但对于产气荚膜梭菌感染而言,通过口服酵母以期有效的介导黏膜免疫反应和全身体液免疫反应、对机体提供安全保护,则并不容易实现,主要原因包括:用于表达抗原分子的DNA需要借助酵母递送至抗原递呈细胞(APCs),例如DC细胞,并在DC细胞中表达和抗原加工,该过程中载体的递送效率、载体表达后形成的抗原分子的呈递效率成为影响抗原免疫效果的重要因素。
目前已经公开的有关产气荚膜梭菌的基因工程疫苗主要包括以具有若干个氨基酸突变的毒素蛋白、毒素蛋白亚基或不同毒素的融合蛋白为有效成分的减毒或灭活免疫制剂,例如201811619231.5(涉及第176位组氨酸突变为天冬酰胺的α毒素)、202111141441.X(涉及包含α毒素C-末端结构域的融合蛋白)。而开发产气荚膜梭菌相应的DNA或mRNA疫苗仍属于技术难题。
发明内容
本发明的目的在于提供一种口服酵母介导的产气荚膜梭菌α毒素重组DNA疫苗,增强经酵母递送的产气荚膜梭菌α毒素抗原基因在激活黏膜免疫反应、产生中和抗体中的效率。
为达到上述目的,本发明采用了以下技术方案:
一种口服重组酵母免疫制剂,该免疫制剂包括酵母菌株以及由该酵母菌株携带的产气荚膜梭菌α毒素重组表达载体,所述重组表达载体包括用于编码突变的产气荚膜梭菌α毒素的突变型plc基因序列或用于编码截短的产气荚膜梭菌α毒素的截短型plc基因序列,所述突变型plc基因序列和截短型plc基因序列包括产气荚膜梭菌α毒素的C-末端结构域的编码序列。
优选的,所述突变的产气荚膜梭菌α毒素为第126位组氨酸突变为甘氨酸(即H126G)的产气荚膜梭菌α毒素。
优选的,所述截短的产气荚膜梭菌α毒素为产气荚膜梭菌α毒素的C-末端结构域。
优选的,所述重组表达载体还包括CMV启动子和polyA尾,重组表达载体具有CMV启动子-突变型plc基因序列-polyA尾的基因表达盒元件排列,或者重组表达载体具有CMV启动子-截短型plc基因序列-polyA尾的基因表达盒元件排列。
优选的,所述重组表达载体是将突变型plc基因序列或截短型plc基因序列克隆到载体骨架而得到的。
优选的,所述载体骨架来源于酵母穿梭型表达载体JMB84-CMV,JMB84-CMV载体的核苷酸序列如SEQ.ID.NO.5所示。
优选的,所述酵母菌株为酿酒酵母。
优选的,所述重组表达载体通过醋酸锂转化法(LiAc)导入到酵母菌株中。
优选的,所述免疫制剂为DNA疫苗、用于增强体液免疫(sIgA、IgG)的佐剂或用于促进树突状细胞成熟的佐剂。
上述产气荚膜梭菌α毒素重组表达载体或口服重组酵母免疫制剂(如DNA疫苗)在制备用于防治产气荚膜梭菌感染的药物中的应用。
优选的,所述药物用于预防动物(如猪、羊)急性胃肠道中毒。
本发明的有益效果体现在:
本发明提供的口服免疫制剂中,重组酵母可将其携带的产气荚膜梭菌α毒素重组表达载体于胃肠道环境进行靶向递送,从而通过激活免疫系统使机体高效产生相应的中和抗体,保护黏膜表面乃至整个机体免受产气荚膜梭菌感染引发的毒力作用。
进一步的,本发明采用的产气荚膜梭菌α毒素重组表达载体含有定点突变或截短的产气荚膜梭菌α毒素(第126位组氨酸突变为甘氨酸的产气荚膜梭菌α毒素,或截短的产气荚膜梭菌α毒素)的编码序列,可以在口服相应重组酵母后产生更高滴度的sIgA,以及更快地激活黏膜免疫和系统免疫。
附图说明
图1为片段CPAH126G的扩增产物琼脂糖凝胶电泳(A)和测序(B)图:左侧泳道为Marker,右侧两泳道为目的片段的2个重复。
图2为JMB84-CMV-H126G载体BamHI/SphI双酶切鉴定的电泳图:泳道M为Marker,泳道1、2为阳性质粒的两个重复。
图3为JMB84-CMV-H126G载体的图谱。
图4为片段CPA247-370的扩增产物琼脂糖凝胶电泳图:泳道1、2为目的片段的2个重复;泳道M为Marker。
图5为JMB84-CMV-CPA247-370载体BamHI/KpnI双酶切鉴定的电泳图:泳道M为Marker;泳道1、2为阳性质粒的两个重复。
图6为JMB84-CMV-CPA247-370载体的图谱。
图7为小鼠免疫及采样流程。
图8为ELISA检测血液中的α毒素特异性抗体IgG的结果:ns P>0.05;*P<0.05;**P<0.01;***P<0.001。
图9为ELISA检测肠黏膜分泌的α毒素特异性抗体sIgA的结果:ns P>0.05;*P<0.05;**P<0.01;***P<0.001。
图10A为经定点突变(即H126G)后的α毒素氨基酸序列:框内标记突变后的氨基酸。
图10B为无突变的α毒素氨基酸序列。
具体实施方式
下面结合附图和实施例对本发明做进一步的详细说明,所述实施例是对本发明的解释,而不是对本发明保护范围的限制。
一、重组酿酒酵母菌株的制备
1.JMB84-CMV-H126G载体的构建
1.1根据GenBank中的产气荚膜梭菌α毒素基因(plc;登录号:AY823400.1),合成plc序列(约为1110bp,编码图10B中的氨基酸序列),克隆至pUC57载体(金唯智生物科技有限公司(苏州分公司)),得到重组载体pUC57-plc。
1.2通过PCR定点突变的方法,将合成的plc序列中编码α毒素第126位的组氨酸的碱基突变为编码甘氨酸的碱基,获得突变后的plc基因片段CPAH126G,相应的突变α毒素的氨基酸序列如图10A所示。
以上步骤1.2的具体流程如下:
(1)首先以合成的plc序列为模板设计引物,用上游引物H126G-a和下游引物H126G-c进行PCR反应获得片段I的产物(在上游引物H126G-a中加入有BamHI限制性酶切位点,参见表1-1);用上游引物H126G-b和下游引物H126G-d进行PCR反应获得片段II的产物(在下游引物H126G-d中加入有SalI限制性酶切位点,参见表1-1)。其中,PCR反应的扩增体系见表1-2、表1-3。
表1-1.引物名称及序列
注:斜体加粗部分为酶切位点,小写字母代表保护碱基,框内标记突变后的碱基
表1-2.片段I的PCR扩增体系
表1-3片段II的PCR扩增体系
(2)将扩增后得到的PCR产物分别进行琼脂糖凝胶电泳,然后切胶回收,以达到纯化产物的目的。以纯化后的片段I和片段II为模板,引物H126G-a为上游引物,引物H126G-d为下游引物,通过PCR反应将两个片段(片段I、片段II)拟合,最终获得片段CPAH126G的产物(电泳结果如图1A所示)。其中,PCR反应的扩增体系见表1-4。
表1-4.片段CPAH126G的PCR扩增体系
(3)将PCR产物进行琼脂糖凝胶电泳检测后,切胶回收,测序(图1B),测定浓度(50-120ng/μL)之后保存于4℃,用于下一步表达载体的构建。
以上步骤1.2中扩增采用的PCR程序为:94℃预变性5min;94℃变性30s,65℃退火30s,72℃延伸2min,执行30个循环;然后72℃终延伸10min,10℃保持。
1.3将用于引入定点突变H126G的plc序列克隆到JMB84-CMV载体中,具体步骤如下:
(1)将片段CPAH126G和保存的JMB84-CMV载体(SEQ.ID.NO.5)分别用限制性核酸内切酶BamHI和SalI进行双酶切(37℃水浴锅中酶切2h),1.2%琼脂糖凝胶进行纯化,紫外灯下切胶并用胶回收试剂盒对目的条带进行回收。其中,JMB84-CMV载体酶切体系见表1-5,片段CPAH126G酶切体系见表1-6。
表1-5.载体酶切反应体系
表1-6.片段酶切反应体系
(2)将胶回收获得的经过酶切的CPAH126G和载体骨架用Nanodrop 2000超微量检测仪测定浓度,并按3:1的摩尔比例用T4连接酶进行连接(25℃反应45min)。其中,连接体系见表1-7。
表1-7.连接反应体系
(3)连接产物通过化学转化法转化大肠杆菌DH5α,涂板于氨苄青霉素(Amp)抗性的固体LB培养皿,倒置于37℃恒温培养箱过夜培养。
(4)挑取单克隆,摇菌,提取质粒。提取得到的质粒使用限制性内切酶BamHI和SphI进行酶切鉴定(阳性质粒鉴定结果如图2所示)。将阳性质粒送到北京奥科生物公司进行测序分析,保存测序正确的质粒(质粒图谱如图3所示),即JMB84-CMV-H126G载体,该载体中包括由CMV启动子(使重组表达载体能够在哺乳动物细胞中表达出相应的蛋白,但在酵母菌中不表达)、含有定点突变H126G的α毒素的编码序列,以及bGHpoly(A)构成的重组产气荚膜梭菌α毒素表达盒。
2.JMB84-CMV-CPA247-370载体的构建
以1.1合成的plc序列为模板设计引物,利用上游引物CPA247-370-F和下游引物CPA247-370-R(引物序列见表2-1),通过PCR反应扩增(扩增体系见表2-2)plc序列中用于编码α毒素的C-末端结构域(即CPA蛋白氨基酸序列的第247至370位)的基因序列,扩增的目的片段记为CPA247-370。所述C-末端结构域的氨基酸序列如图10B的第247-370位所示。
表2-1.引物名称及序列
注:斜体加粗部分为酶切位点,小写字母代表保护碱基
表2-2.PCR扩增体系
注:PCR程序设置同上
PCR产物进行琼脂糖凝胶电泳(结果如图4所示),切胶回收,测序,测定浓度(50-120ng/μL),按以下步骤构建载体:
(1)将片段CPA247-370和JMB84-CMV载体分别用限制性核酸内切酶BamHI和SalI进行酶切(37℃水浴锅中酶切2h),1.2%琼脂糖凝胶进行纯化,紫外灯下切胶并用胶回收试剂盒对目的条带进行回收。其中,JMB84-CMV载体酶切体系同表1-5,片段CPA247-370酶切体系见表2-3。
表2-3.片段酶切切反应体系
(2)用T4连接酶按照经过酶切的CPA247-370和载体骨架摩尔比为3:1的比例,连接片段和载体骨架(在25℃反应45min)。其中,连接体系见表2-4。
表2-4.连接反应体系
(3)连接产物通过化学转化法转化大肠杆菌DH5α,并涂板于氨苄青霉素(Amp)抗性的固体LB培养皿,倒置于37℃恒温培养箱过夜培养。
(4)挑取单克隆,摇菌,提取质粒,提取得到的质粒使用限制性内切酶BamHI和KpnI进行酶切鉴定(阳性质粒鉴定结果如图5所示)。将阳性质粒送到北京奥科生物公司进行测序分析,保存测序正确的质粒(质粒图谱如图6所示),即JMB84-CMV-CPA247-370,该载体中包括由CMV启动子、截短的α毒素(即上述C-末端结构域)的编码序列,以及bGH poly(A)构成的重组产气荚膜梭菌α毒素表达盒。
3.构建重组酵母
分别将JMB84-CMV载体、JMB84-CMV-H126G载体、JMB84-CMV-CPA247-370载体转化酿酒酵母,鉴定筛选之后,得到携带相应质粒的重组酿酒酵母菌株。具体步骤如下:
(1)将实验室保存的尿嘧啶缺陷(ura)型酿酒酵母JMY1(MATa,ade2-1;ura3-1;his3-11;trp1-1;leu2-3112;can1-100),用其他尿嘧啶缺陷型酿酒酵母菌株也可以获得同样的转化效果)划线接种在酵母全营养(YEP)固体培养基,置于30℃恒温培养箱中培养3d。
(2)挑取JMYI单克隆于2mL酵母YEP液体培养基中,30℃、200rpm摇床震荡培养过夜。
(3)第二天按1:50的比例接种到5mL酵母YEP液体培养基中,继续震荡培养至OD为0.5左右时,取2mL于离心管中3000rpmn离心,收集菌体,后用ddH2O清洗两遍,再用900μL的ddH2O重悬菌体,用醋酸锂转化法将三种不同质粒分别转化酿酒酵母JMYI,具体转化步骤如下:
准备1mL鲑鱼精DNA(ssDNA)在沸水中处理5min,然后置于冰上待用;重悬的酵母菌体中加入100μL醋酸锂(1.0mol/L),颠倒混匀,30℃水浴锅处理15min,离心收集菌体,弃去上清;准备50μL的质粒溶液(质粒约2ng,其余用ddH2O补齐),用质粒溶液重悬收集的菌体,再加入36μL醋酸锂、25μL处理好的ssDNA和240μL PEG6000(50%),涡旋混匀,42℃热激45min,12000rpm离心2min,弃去上清,加入1mL酵母YEP液体培养基重悬,而后于30℃恒温摇床中180rpm复苏1h;之后5000rpm离心5min,弃去上清,用100μL酵母YEP液体培养基重悬,而后涂板在预热的尿嘧啶缺陷型的酵母SD培养基(SD-ura),置于30℃恒温培养箱,培养3-4d,观察酵母生长情况。
长出单菌落后,挑取酵母单克隆,接种于5mL液体SD-ura培养基中,30℃、220rpm震荡培养过夜,而后用酵母质粒提取试剂盒提取酵母质粒。由于提取得到的酵母质粒的浓度较低,无法直接检测鉴定,因此将提取的酵母质粒吸取5μL,通过化学转化法导入大肠杆菌JM109。挑取单克隆,摇菌,提取质粒,提取出来的质粒进行酶切鉴定。鉴定正确的三种重组酿酒酵母菌株(分别携带JMB84-CMV载体、JMB84-CMV-H126G载体、JMB84-CMV-CPA247-370载体)保存于-80℃备用。
二、小鼠免疫实验
1.小鼠饲喂重组酵母
根据实验安排选择84只体重和大小相近的6周龄雌性小鼠,随机分为4组:PBS组、携带空载体JMB84-CMV的重组酿酒酵母组(命名为JMB84组)、携带JMB84-CMV-H126G载体的重组酿酒酵母组(命名为H126G组)、携带JMB84-CMV-CPA247-370载体的重组酿酒酵母组(命名为C247-370组),每组21只,具体分组及免疫方案见表3-1。
表3-1.小鼠口服免疫实验方案
将-80℃保存的重组酿酒酵母菌株复苏,分别于250mL液体SD-ura培养基中30℃、220rpm震荡培养3d,收集菌体,用PBS重悬清洗两遍,而后将菌体重悬于PBS中,并在显微镜下计数,调整至浓度为5×1010cfu/mL。
每次口服免疫时,除了PBS组,其他各组每只小鼠每天分别口服对应的重组酿酒酵母重悬液100μL(即口服5×109cfu重组酿酒酵母菌体),PBS组口服100μL PBS。口服免疫三次,一次连续口服3d,参见图7。
在口服重组酿酒酵母的第0d、7d、14d、21d、28d、35d、42d,每组随机选择3只小鼠采集血清和肠黏膜液。具体步骤如下:
对每次从每组中随机选择的三只小鼠进行心脏采血,采集的血液样品在4℃静置2h,4℃离心机4000rpm离心10min,收集血清,保存于-20℃。
解剖小鼠腹部,取下小鼠小肠部分,将小肠内容物清理干净,用剪刀剪成10cm左右的小段,然后用2mL PBS反复冲洗,收集冲洗液于2mL EP管中,后在4℃离心机12000rpm离心5min,收集上清液(命名为肠黏膜液),保存于-20℃。
2.特异性抗体的检测
通过96孔酶标板,用间接ELISA法测定血液中的特异性IgG水平和肠黏膜的分泌型IgA(sIgA)水平,详细操作步骤如下。
2.1血液特异性抗体IgG的检测
(1)用PBS缓冲液洗涤酶标板3次,将重组CPA蛋白(序列如图10B所示,模拟a毒素)用包被缓冲液(北京索莱宝科技有限公司)按梯度稀释至浓度分别为2μg/mL、4μg/mL、6μg/mL、8μg/mL、10μg/mL,96孔板的竖列按照梯度分别每孔加入100μL,同时设置只加包被缓冲液的空白孔(每个梯度做两个重复),4℃包被18h;
(2)倒出液体,用洗涤液(北京索莱宝科技有限公司)清洗三次,每次每孔约300μL洗涤液并静置30s,清洗后拍干;
(3)每孔加入封闭液(5%脱脂奶粉)200μL,37℃封闭2h,随后倒出液体,继续清洗3次后拍干;
(4)以0d收集的小鼠血清为阴性血清,以42d收集的H126G组和CPA247-370组的小鼠血清为阳性血清,血清按照1:50、1:100、1:200、1:500的比例稀释,按照每个抗原(重组CPA蛋白)的梯度,96孔板横排按梯度每孔加入100μL血清,在37℃条件下反应2h,洗涤液清洗5遍,拍干;
(5)每孔加入100μL按照1:2000比例稀释的HRP结合的山羊抗鼠IgG(二抗;康为世纪生物科技有限公司),37℃反应45min,倒出液体,洗涤液清洗5遍,拍干;
(6)避光条件下,每孔加入100μL的TMB(四甲基联苯胺)显色液,37℃避光反应15min;
(7)接着每孔加入50μL终止液,并在5min内使用酶标仪在450nm和630nm(参考波长)波长条件下测量OD值。选择阴性血清OD值小于0.1,阳性血清与阴性血清比值最大的对应稀释比例为最佳稀释倍数(血清1:50,抗原包被浓度6μg/mL)。
根据得到的最佳稀释倍数,分别检测各组小鼠在0d、7d、14d、21d、28d、35d和42d的血液中的特异性IgG抗体,每个时间点做两个复孔。
采用双因素方差分析的邓尼特多重比较检验,进行每次采样的各组差异性比较。
2.2肠黏膜特异性抗体sIgA的检测
(1)选取抗原(重组CPA蛋白)包被浓度为6μg/mL,96孔酶标板每孔加入100μL,4℃包被18h后,用洗涤液清洗;
(2)封闭液37℃封闭,继续用洗涤液清洗;
(3)收集的小鼠肠黏膜液不经稀释加入封闭后的酶标板,每孔100μL,37℃反应2h,洗涤液清洗5遍,拍干;
(4)加入按1:5000稀释的HRP结合的山羊抗小鼠IgA(二抗;康为世纪生物科技有限公司),37℃反应30min,洗涤液清洗5遍,拍干;
(5)加入TMB显色液,37℃条件下15min,再加入终止液,在5min内使用酶标仪测得450nm波长下的OD值。
按以上方法测得各组小鼠在0d、7d、14d、21d、28d、35d和42d的肠黏膜分泌的特异性IgA(sIgA)。
3.实验结果分析
参见图8,经ELISA检测小鼠血液中的特异性抗体,结果显示:口服重组酿酒酵母之后,两实验组(H126G组、C247-370组)的小鼠血液中的特异性抗体IgG水平呈不断上升的趋势,在经过第二次免疫后,在28d收集的血清中特异性IgG的水平达到峰值,与其他两对照组有显著的差异(P<0.001);空白对照组(PBS组)和实验对照组(JMB84组)小鼠的血清特异性IgG较免疫前(0d)无显著的差异。两实验组之间对比发现,H126G组的小鼠血清特异性IgG水平与C247-370组之间并无显著的差异(P>0.05)。上述结果说明,定点突变型的α毒素基因形式与截短型的α毒素(上述C-末端结构域)基因形式在用作构建DNA疫苗时,都能诱导产生较高水平的抗体;二者诱导产生的抗体水平并无显著差异,即二者作为抗原基因的免疫原性无明显差异。
参见图9,经ELISA检测小鼠肠黏膜特异性抗体,结果显示:与空白对照组和实验对照组相比,小鼠口服重组酿酒酵母后,其肠黏膜特异性sIgA的水平明显上升,在第一次免疫后的第一周(即7d),收集的两实验组的肠黏膜液中特异性sIgA的水平与空白对照组(PBS组)并无显著差异(P>0.05);而在免疫后的第二周(14d)两实验组检测出明显的肠黏膜特异性sIgA水平升高,且与空白对照组和实验对照组有明显的差异;在第二次免疫后,实验组的肠黏膜特异性sIgA水平在第28d达到最高值。
图8和图9的结果还说明,实验组重组酿酒酵母诱导了更高滴度的sIgA分泌,且系统免疫与黏膜免疫同步达到峰值(都在28天),表明实验组重组酿酒酵母诱导肠黏膜免疫激活的同时,也成功诱导激活了系统免疫。
以上实验中,小鼠经饲喂含plc基因两种不同形式的重组酿酒酵母(携带JMB84-CMV-H126G载体的重组酿酒酵母、携带JMB84-CMV-CPA247-370载体的重组酿酒酵母)后,其血清和肠黏膜液中均检测到了特异性抗体,提示携带抗原基因的重组酿酒酵母能够有效激活肠道的特异性黏膜免疫反应,使黏膜免疫系统分泌相应的特异性抗体,诱导特异性黏膜免疫应答,并且通过刺激相关的T细胞和B细胞诱导产生全身的体液免疫应答。
三、重组酿酒酵母菌株作为DNA疫苗的应用
上述重组酿酒酵母菌株通过携带构建的质粒(含有用于引入定点突变H126G的plc序列或含有与截短后的C-末端结构域片段对应的plc序列),经宿主口服后穿过其胃肠道环境,由DC细胞识别和吞噬该重组酿酒酵母,使其携带的质粒(靶向DC细胞递送相应的含有产气荚膜梭菌α毒素抗原基因的DNA)在宿主中转录和翻译(将表达载体引流至细胞核进行转录,转录后的mRNA在细胞质进行翻译,产生相关的蛋白),以激活黏膜免疫与系统免疫,并在宿主机体诱导产生特异性免疫应答,清除宿主体内的产气荚膜梭菌α毒素(在免疫后sIgA抗体和IgG抗体水平明显升高,并且两种抗体水平同步达到峰值,使全身免疫组织联系成为一个有机整体,达到一种平衡状态),从而为防治产气荚膜梭菌引起的肠道疾病提供新的途径。
总之,本发明使用酿酒酵母作为DNA疫苗的递送载体,在应用中无需体外生产相关减毒、灭活或者蛋白疫苗(过程繁琐、成本高且有一定的安全风险),通过口服DNA疫苗可以有效的诱导黏膜免疫应答和全身免疫应答,从而保护养殖的畜禽(如仔猪、羔羊)免受产气荚膜梭菌感染。
Claims (10)
1.一种口服重组酵母免疫制剂,其特征在于:该免疫制剂包括酵母菌株以及由该酵母菌株携带的产气荚膜梭菌α毒素重组表达载体,所述重组表达载体包括用于编码突变的产气荚膜梭菌α毒素的突变型plc基因序列或用于编码截短的产气荚膜梭菌α毒素的截短型plc基因序列,所述突变型plc基因序列和截短型plc基因序列包括产气荚膜梭菌α毒素的C-末端结构域的编码序列。
2.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述突变的产气荚膜梭菌α毒素为第126位组氨酸突变为甘氨酸的产气荚膜梭菌α毒素。
3.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述截短的产气荚膜梭菌α毒素为产气荚膜梭菌α毒素的C-末端结构域。
4.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述重组表达载体还包括CMV启动子和polyA尾,重组表达载体具有CMV启动子-突变型plc基因序列-polyA尾的基因表达盒元件排列,或者重组表达载体具有CMV启动子-截短型plc基因序列-polyA尾的基因表达盒元件排列。
5.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述重组表达载体是将突变型plc基因序列或截短型plc基因序列克隆到载体骨架而得到的。
6.根据权利要求5所述一种口服重组酵母免疫制剂,其特征在于:所述载体骨架来源于JMB84-CMV载体,该载体的核苷酸序列如SEQ.ID.NO.5所示。
7.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述酵母菌株为酿酒酵母。
8.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述重组表达载体通过醋酸锂转化法导入到酵母菌株中。
9.根据权利要求1所述一种口服重组酵母免疫制剂,其特征在于:所述免疫制剂为DNA疫苗、用于增强体液免疫的佐剂或用于促进树突状细胞成熟的佐剂。
10.一种产气荚膜梭菌α毒素重组表达载体或如权利要求1所述的口服重组酵母免疫制剂在制备用于防治产气荚膜梭菌感染的药物中的应用。
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