CN107266578A - 鱼华支睾吸虫病口服疫苗及其制备与应用 - Google Patents
鱼华支睾吸虫病口服疫苗及其制备与应用 Download PDFInfo
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- CN107266578A CN107266578A CN201610217516.0A CN201610217516A CN107266578A CN 107266578 A CN107266578 A CN 107266578A CN 201610217516 A CN201610217516 A CN 201610217516A CN 107266578 A CN107266578 A CN 107266578A
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Abstract
本发明属于生物技术领域,具体涉及一种鱼华支睾吸虫病口服疫苗及其制备与应用。本发明首先提供了一种融合蛋白,含有枯草芽孢杆菌芽孢衣壳蛋白CotC和华支睾吸虫半胱氨酸蛋白酶CsCP。所述融合蛋白,以枯草杆菌芽孢为载体,制备成鱼华支睾吸虫病口服疫苗,能够在鱼体肠道内成功定植,免疫效果良好。能够切断华支睾吸虫病传播途径,达到有效预防和控制该病流行和传播的目的。选取枯草芽孢作为蛋白抗原提呈系统能很好地避免直接口服纯蛋白经过鱼体胃酸环境易降解的缺陷,保证了疫苗能发挥有效的保护作用。
Description
技术领域
本发明属于生物技术领域,具体涉及一种鱼华支睾吸虫病口服疫苗及其制备与应用。
背景技术
华支睾吸虫病(clonorchiasis)又称肝吸虫病,是我国流行范围最广、受威胁人口最多、危害程度最大的食源性人兽共患寄生虫病,该病主要由于食入含华支睾吸虫(Clonorchissinensis,C.sinensis)感染期囊蚴的淡水鱼或虾所致。华支睾吸虫感染可引起胆道炎症、胆管阻塞,甚至胆管癌、肝硬化及肝癌等疾病。据WHO报道,华支睾吸虫感染者患胆管癌风险是正常人的58倍,患肝硬化风险是正常人的30倍,患肝癌风险是正常人15倍。2009年3月,华支睾吸虫被WHO确定为胆管癌的直接病因学因素。当前全球约有1500万名华支睾吸虫感染者,主要分布于东亚,包括中国、韩国、越南和俄罗斯等地。其中中国感染者近1300万名,数量居全球首位,占总数的85%以上,且仍有增加趋势,其中广东省感染情况最为严重,约有接近600万华支睾吸虫感染者,流行区感染率高达60%。
由于流行病区居民喜食生鱼的习惯长期存在且很难改变,而华支睾吸虫的保虫宿主种类及分布广泛等多种因素影响,近年来华支睾吸虫病发病率居高不下,已严重影响到人群健康和淡水鱼养殖业的经济效益。目前我国针对肝吸虫病的防治策略很多,但未取得满意的控制疾病传播的效果。由于该病主要是由于人食入含华支睾吸虫感染期囊蚴的淡水鱼或虾所致,故预防华支睾吸虫病应抓住经口感染这一环节,以预防为主、将关口前移,防止食入活囊蚴是防治本病的关键。近几年淡水鱼华支睾吸虫囊蚴的感染率居高不下,2010年广东省江门市新会区淡水鱼华支睾吸虫囊蚴的感染率达到86.57%,2015年黑龙江省肇源县淡水鱼华支睾吸虫囊蚴感染率为36.47%,其中麦穗鱼、柳根鱼和船丁鱼的感染率分别为92.45%(98/106)、78.95%(15/19)和52.00%(13/25)。因此防治淡水鱼感染华支睾吸虫对阻断华支睾吸虫病的传播具有重要意义。
疫苗是预防感染性疾病最有效的方式之一。目前除本实验室外尚无关于华支睾吸虫病疫苗的专利和期刊报道。目前市面上使用的抗感染疫苗可分为传统的减毒活疫苗、灭活疫苗,和新型的亚单位疫苗(如蛋白疫苗、多肽疫苗)、核酸疫苗等。减毒活疫苗效果虽好,但减毒阈值不易控制;DNA疫苗效果好,生产成本低,但有遗传毒性和基因变异等问题;亚单位疫苗具有安全性高,纯度高、稳定性好,产量高等优点,故亚单位疫苗(蛋白/多肽疫苗)是重要研究对象。
鱼类疫苗的免疫方式主要有注射、浸泡和口服三种。目前大多数采用浸泡免疫和肌肉注射免疫两种方法接种,普遍以注射为主,口服免疫很少。注射免疫具有剂量准确及诱导免疫应答强等优点,但这种免疫方式容易造成鱼体受伤,不适合应用于大面积养殖及多种疾病的防治,且对规格较小的幼鱼不适用,同时腹腔注射可能导致生长阻滞、内脏粘连等问题;浸泡免疫省时方便,适合大规模操作及难以注射的鱼苗,但通常吸收效果不好,如何使疫苗进入鱼体是浸泡法面临的最大难题。相比之下,口服免疫途径对鱼体无损伤,安全易行,操作简单,不受鱼体大小的限制,能避免注射引起的应激及致死反应,且使用量较浸泡免疫少,是最实用的免疫方法,但存在抗原的吸收效率低、免疫效果不稳定等问题。近年来,随着国内外学者对鱼的胃肠黏膜免疫机理的研究,普遍认为口服免疫可以引起鱼体产生很强的黏膜免疫应答,且操作方便,可能与其引起鱼体肠道一些新的关键分子反应相关,如IgM、IgT、IgZ及pIgR等,这使得鱼的口服疫苗的研制成为研究热点。考虑到大规模推广的可操作性和安全性,口服免疫是一条较好的途径。硬骨鱼类已有相对成熟的免疫系统,包括免疫器官、免疫细胞和免疫分子。除了胸腺、脾等重要的淋巴细胞组织,鱼类皮肤、鳃和消化道的上皮组织中也存在淋巴细胞、巨噬细胞和各类粒细胞,共同组成了良好的抵抗外来病菌侵害的屏障系统。外源性的疫苗分子通过不同途径进入鱼体后诱导鱼体产生抗体IgM,当鱼体再次接触该病原体时IgM能够中和病原体,进而可以预防鱼类病害。鱼的肠道黏膜分为上皮层和固有层,淋巴细胞如巨噬细胞、粒细胞及浆细胞等存在于整个鱼体消化道,具有黏膜免疫独立完成免疫应答的细胞基础。
口服疫苗由于要通过胃酸屏障和消化道严苛的环境,而大部分蛋白抗原免疫原性都很差,通过胃肠道途径时会变性和被蛋白酶所降解,不能刺激强力有效的免疫应答,而且蛋白疫苗免疫效果较差,需与蛋白载体偶联或加入具有免疫增强作用的佐剂才能发挥较满意效果,因此要选择有效的载体作为抗原递呈系统,它不仅仅能将抗原递呈在疫苗载体表面,而且能够避免在胃液中降解。
选择枯草芽孢杆菌(Bacillus subtilis,B.subtilis)芽孢作为口服疫苗的载体递呈抗原。枯草芽孢杆菌在营养缺乏时,引起一系列芽孢生成基因启动,形成代谢相对静止特殊状态——芽孢。枯草芽孢杆菌芽孢作为口服疫苗载体,具有很多常规疫苗载体不可比拟的优点:(1)枯草芽孢杆菌芽孢的生物安全性高,其属革兰阳性菌,不产生毒素,而且还是常用的益生菌之一,已用作为人的功能性食品、动物饲料添加剂等;(2)芽孢的稳定性极好:芽孢对干燥、高温、高压、氧化、及酸、碱、毒物等不良环境的抵抗力强,能够长期在逆环境中生存,能够避免蛋白抗原在胃液中被降解;(3)枯草芽孢杆菌芽孢有独特的免疫学特性:口服的芽孢在动物肠道能被胃肠道相关淋巴组织(GALT)识别,增强抗原的免疫原性,发挥免疫佐剂作用,增强机体的非特异性和特异性免疫应答;(4)枯草芽孢杆菌有完善的分泌系统,能将外源重组蛋白直接分泌至培养液中,大大减少了表达产物的分离纯化步骤,已应用于许多重组蛋白的表达,其生产成本低,对营养要求不高,生长快速,适于现代工业发酵生产。枯草芽孢杆菌芽孢现已在畜牧业和渔产业得到广泛应用。故枯草芽孢以其良好的益生菌生物学特性,成为口服疫苗最佳的候选载体之一。据文献报道枯草芽孢杆菌芽孢的外衣壳蛋白种类较多,包括CotB、CotC、CotG、CotZ和CotA等。
目前国内外尚无关于预防鱼华支睾吸虫病疫苗的专利和期刊文献。
发明内容
为了克服现有技术中所存在的问题,本发明的目的在于提供一种鱼华支睾吸虫病口服疫苗及其制备与应用。
为了实现上述目的以及其他相关目的,本发明采用如下技术方案:
本发明的第一方面,提供一种融合蛋白,含有枯草芽孢杆菌芽孢衣壳蛋白CotC和华支睾吸虫半胱氨酸蛋白酶CsCP。
优选地,所述枯草芽孢杆菌芽孢衣壳蛋白CotC的氨基酸序列对应于如SEQ ID NO.1所示核苷酸序列所编码的氨基酸序列。
进一步优选地,所述枯草芽孢杆菌芽孢衣壳蛋白CotC的编码核苷酸序列如SEQ ID NO.1所示,具体为:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTTGATTTGTTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAATATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTAC。
优选地,所述华支睾吸虫半胱氨酸蛋白酶CsCP的氨基酸序列对应于如SEQ ID NO.2所示核苷酸序列所编码的氨基酸序列。
进一步优选地,所述华支睾吸虫半胱氨酸蛋白酶CsCP的编码核苷酸序列如SEQ IDNO.2所示,具体为:
AGCAACATTCCTGAATCAGAAAATGCCCGGCAATTGTACGAAGAGTTCAAGCTGAAGTACAAGAAGTCCTATTCAAATGACGACGACGAATATAGATTCAGAGTTTTCAAGGACAACCTATTGCGCATAAAGCAATTTCAAAACATGGAGCGGGGAACGGCAAAGTACGGTGTGACTCAGTTTTCCGACCTCACAGCTCAGGAGTTCAAGGTTAGGTACCTAAGATCAAAATTTGGTGGTGTCCCTGTGGACAGAGAACCGGTTCCATTCATACGAATGGATGTGGATGACGACAACTTCGACTGGCGAAATCATGGTGCTGTGGGACCCGTATTGGACAAAGGAGATTGCGGATCGTGCTGGGCATTTTCCGCGGTTGGAAACATCGAGGGTCAATGGTTCCGTAAAACTGACAATCTTTTGCAGTTAAGCGAACAGCAACTCCTTGATTGCGATGAGGTGGATGAAGGTTGCAACGGCGGCACTCCTCAACAGGCTTTCAAGCAAATTTTGGGAATGGGCGGGTTGCAGCTGGACTCCGACTACCCATATGAGGGGAGAGAAGGGCAGTGCAGAATGGTGCCATCAAAGGTCAAGGTCTATATTAACGGCTCGAAAATTCTGCCGGAAGATGAGCAAATCCAGGCTCAAATGCTCAAGGAAACCGGGCCTTTTAGCTCCGCTCTAAATGCACTTTCCCTGCAATTTTACACGGAAGGGATTTTGCATCCCCTGCCAGCGCTTTGCGATGCCCAATCGTTAAACCATGCTGTTTTAACTGTCGGTTACGGGAAAGAAGGCAGGCTACCATACTGGACGGTTAAAAACAGTTGGAGCACTATGTTTGGCGAAAATGGTTACTTCCGTATTTACCGTGGAGACGGCCCCTGTGGAATCAATACCCTAGTTTCCACCTCGATCATCTTGTGA。
优选地,所述融合蛋白的氨基酸序列中含有如SEQ ID NO.3所示核苷酸序列所编码的氨基酸序列。
进一步优选地,所述融合蛋白的编码核苷酸序列中含有如SEQ ID NO.3所示的核苷酸序列。如SEQ ID NO.3所示的核苷酸序列具体为:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTTGATTTGTTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAATATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACAAGCTTAGCAACATTCCTGAATCAGAAAATGCCCGGCAATTGTACGAAGAGTTCAAGCTGAAGTACAAGAAGTCCTATTCAAATGACGACGACGAATATAGATTCAGAGTTTTCAAGGACAACCTATTGCGCATAAAGCAATTTCAAAACATGGAGCGGGGAACGGCAAAGTACGGTGTGACTCAGTTTTCCGACCTCACAGCTCAGGAGTTCAAGGTTAGGTACCTAAGATCAAAATTTGGTGGTGTCCCTGTGGACAGAGAACCGGTTCCATTCATACGAATGGATGTGGATGACGACAACTTCGACTGGCGAAATCATGGTGCTGTGGGACCCGTATTGGACAAAGGAGATTGCGGATCGTGCTGGGCATTTTCCGCGGTTGGAAACATCGAGGGTCAATGGTTCCGTAAAACTGACAATCTTTTGCAGTTAAGCGAACAGCAACTCCTTGATTGCGATGAGGTGGATGAAGGTTGCAACGGCGGCACTCCTCAACAGGCTTTCAAGCAAATTTTGGGAATGGGCGGGTTGCAGCTGGACTCCGACTACCCATATGAGGGGAGAGAAGGGCAGTGCAGAATGGTGCCATCAAAGGTCAAGGTCTATATTAACGGCTCGAAAATTCTGCCGGAAGATGAGCAAATCCAGGCTCAAATGCTCAAGGAAACCGGGCCTTTTAGCTCCGCTCTAAATGCACTTTCCCTGCAATTTTACACGGAAGGGATTTTGCATCCCCTGCCAGCGCTTTGCGATGCCCAATCGTTAAACCATGCTGTTTTAACTGTCGGTTACGGGAAAGAAGGCAGGCTACCATACTGGACGGTTAAAAACAGTTGGAGCACTATGTTTGGCGAAAATGGTTACTTCCGTATTTACCGTGGAGACGGCCCCTGTGGAATCAATACCCTAGTTTCCACCTCGATCATCTTGTGA。
本发明的第二方面,提供了一种制备前述融合蛋白的方法,包括如下步骤:
(1)获得目的基因;
(2)构建重组质粒:将步骤(1)所得目的基因以及载体质粒采用相同的限制性内切酶切割,然后进行连接,获得重组质粒;
(3)将步骤(2)所得重组质粒导入感受态细胞,转化;
(4)筛选阳性克隆,并将正确的重组质粒转化宿主感受态细胞;
(5)诱导表达融合蛋白。
优选地,步骤(1)中,所述目的基因核苷酸序列含有枯草芽孢杆菌芽孢衣壳蛋白CotC的编码核苷酸序列和华支睾吸虫半胱氨酸蛋白酶CsCP的编码核苷酸序列。
优选地,步骤(1)中,所述枯草芽孢杆菌芽孢衣壳蛋白CotC的编码核苷酸序列如SEQ ID NO.1所示。所述华支睾吸虫半胱氨酸蛋白酶CsCP的编码核苷酸序列如SEQ ID NO.2所示。
进一步优选地,步骤(1)中,所述目的基因的核苷酸序列如SEQ ID NO.3所示,具体为:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTTGATTTGTTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAATATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACAAGCTTAGCAACATTCCTGAATCAGAAAATGCCCGGCAATTGTACGAAGAGTTCAAGCTGAAGTACAAGAAGTCCTATTCAAATGACGACGACGAATATAGATTCAGAGTTTTCAAGGACAACCTATTGCGCATAAAGCAATTTCAAAACATGGAGCGGGGAACGGCAAAGTACGGTGTGACTCAGTTTTCCGACCTCACAGCTCAGGAGTTCAAGGTTAGGTACCTAAGATCAAAATTTGGTGGTGTCCCTGTGGACAGAGAACCGGTTCCATTCATACGAATGGATGTGGATGACGACAACTTCGACTGGCGAAATCATGGTGCTGTGGGACCCGTATTGGACAAAGGAGATTGCGGATCGTGCTGGGCATTTTCCGCGGTTGGAAACATCGAGGGTCAATGGTTCCGTAAAACTGACAATCTTTTGCAGTTAAGCGAACAGCAACTCCTTGATTGCGATGAGGTGGATGAAGGTTGCAACGGCGGCACTCCTCAACAGGCTTTCAAGCAAATTTTGGGAATGGGCGGGTTGCAGCTGGACTCCGACTACCCATATGAGGGGAGAGAAGGGCAGTGCAGAATGGTGCCATCAAAGGTCAAGGTCTATATTAACGGCTCGAAAATTCTGCCGGAAGATGAGCAAATCCAGGCTCAAATGCTCAAGGAAACCGGGCCTTTTAGCTCCGCTCTAAATGCACTTTCCCTGCAATTTTACACGGAAGGGATTTTGCATCCCCTGCCAGCGCTTTGCGATGCCCAATCGTTAAACCATGCTGTTTTAACTGTCGGTTACGGGAAAGAAGGCAGGCTACCATACTGGACGGTTAAAAACAGTTGGAGCACTATGTTTGGCGAAAATGGTTACTTCCGTATTTACCGTGGAGACGGCCCCTGTGGAATCAATACCCTAGTTTCCACCTCGATCATCTTGTGA
优选地,步骤(2)中,所述载体质粒为pEB03质粒。
所述PEB03质粒为现有技术中已有质粒。例如可参考文献“production ofn-acetyl-d-neuraminic acid by use is of an efficient spore surface display system”。
优选地,步骤(2)中,所述限制性内切酶为SalI、SacI。
优选地,步骤(3)中,所述感受态细胞为枯草芽孢杆菌WB600感受态细胞。
优选地,步骤(4)中,所述宿主感受态细胞为枯草芽孢杆菌WB600感受态细胞。
优选地,步骤(5)中,所述诱导采用DSM芽孢培养基诱导。
本发明的第三方面,提供了前述融合蛋白在制备鱼华支睾吸虫病口服疫苗中的用途。
本发明的第四方面,提供了一种鱼华支睾吸虫病口服疫苗,含有前述融合蛋白。
优选地,所述鱼华支睾吸虫病口服疫苗还含有疫苗载体,所述疫苗载体选用枯草芽孢杆菌芽孢。
进一步优选地,所述鱼华支睾吸虫病口服疫苗还含有鱼肝油或豆油。
进一步优选地,所述鱼华支睾吸虫病口服疫苗还含有饲料。
与现有技术相比,本发明具有如下有益效果:
本发明通过广泛而深入的研究获得的融合蛋白,以枯草杆菌芽孢为载体,制备成鱼华支睾吸虫病口服疫苗,能够在鱼体肠道内成功定植,免疫效果良好。能够切断华支睾吸虫病传播途径,达到有效预防和控制该病流行和传播的目的。选取枯草芽孢作为蛋白抗原提呈系统能很好地避免直接口服纯蛋白经过鱼体胃酸环境易降解的缺陷,保证了疫苗能发挥有效的保护作用。
附图说明
图1:WB600-pEB03-CotC-CsCP菌液PCR扩增CsCP鉴定阳性克隆结果图,1-6:WB600-pEB03-CotC-CsCP菌液。
图2:WB600-pEB03-CotC-CsCP双酶切鉴定结果图,1-3:pEB03-CotC-CsCP质粒;1’-3’:pEB03-CotC-CsCP经HindⅢ+SacⅠ双酶切,箭头所指为CsCP基因;CsCP:930bp,pEB03:5kb。
图3:SDS-PAGE检测诱导芽孢不同时间点、超声沉淀(C)及上清(S)中融合蛋白CotC-CsCP表达情况,M:蛋白Marker;箭头所指为融合蛋白CotC-CsCP(43.8KDa)。CotC:8.8KDa,CsCP:35KDa。
图4:Western Blotting检测诱导不同时间点、超声沉淀(C)及上清(S)中融合蛋白CotC-CsCP表达情况。
图5:免疫荧光检测重组芽孢经过诱导后CsCP的表达情况,将诱导表达后的WB600-pEB03-CotC-CsCP及WB600-pEB03-CotC的重组芽孢均用大鼠抗rCsCP的抗血清孵育,再用然后加入Cy3标记的荧光二抗IgG,DAPI染核,分别在白光及荧光下观察蛋白表达情况(放大倍数1×400)。
图6:ELISA检测口服免疫草鱼不同时间点抗CsCP特异性IgM的水平,ELISA方法检测106,107and 108cfu/g CotC-CsCP和107cfu/g CotC枯草芽孢杆菌芽孢口服免疫草鱼后草鱼血清(A)、胆汁(B)、肠黏液(C)和体表黏液(D)中的特异性IgM的抗体水平。*是指有统计学差异:*p<0.05,**p<0.01。
图7:草鱼肠道内芽孢存在情况的鉴定,草鱼口服重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP),于第10周取鱼的前肠和后肠分别检测芽孢及重组质粒的存在情况;加入无菌生理盐水将肠道组织研磨成匀质,68℃水浴10min后,将其均匀涂布在含有壮观霉素抗性的LB固体培养板培养约12h;A-a.草鱼前肠匀浆涂布菌落生长情况;A-b.草鱼后肠匀浆涂布菌落生长情况;在培养板上随机挑取一定数量的单克隆,进行菌落PCR验证融合基因CotC-CsCP的存在情况;B.1Marker;1-5,检测草鱼前肠匀浆涂布菌落CotC-CsCP情况;6-22,检测草鱼后肠匀浆涂布菌落CotC-CsCP情况;(投喂普通饲料组);24,阳性对照(WB600-pEB03-CotC-CsCP菌株)。
图8:草鱼口服免疫8W前肠及后肠的肠道上皮淋巴细胞及肠绒毛生长情况
A:前肠,B:后肠;说明:重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)能使鱼体肠道上皮淋巴细胞增多及肠绒毛增长,有效提高肠道免疫力,促进对食物吸收能力。
图9:草鱼口服免疫8W血清中谷草转氨酶(AST/GOT)及谷丙转氨酶(ACT/GPT)的水平,提示:重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)对草鱼的肝胰脏无毒害作用。
具体实施方式
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或者按照各制造商所建议的条件。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明,本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明,具体可参见Sambrook等
MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel等,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODSIN ENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999;和METHODS IN MOLECULAR BIOLOGY,Vol.119,ChromatinProtocols(P.B.Becker,ed.)Humana Press,Totowa,1999等。
实施例1
1.主要溶液配制
枯草芽孢杆菌电转化A培养基:LB培养基+0.5M山梨醇,pH=7.2。
枯草芽孢杆菌电转化B培养基:0.5M山梨醇,0.5M甘露醇,10%甘油。
枯草芽孢杆菌电转化C培养基:0.5M山梨醇,0.5M甘露醇。
LB液体培养基:NaCl 10g,酵母提取物5g,胰蛋白胨10g,加入超纯水至1,000ml,调整pH至7.0,121℃高压灭菌20min。
LB固体培养基:NaCl 10g,酵母提取物5g,胰蛋白胨10g,琼脂粉15g,加入超纯水至1,000ml,调整pH至7.0,121℃高压灭菌20min;待温度降至约50~55℃(需要时加入相应的抗生素溶液),进行倒板。
DSM芽孢培养基(1,000ml):8g Difco营养肉汤,1g KCl,0.25g MgSO4.7H2O,0.002gMnCl2.4H2O;调整pH=7.0,高压灭菌,使用时加入过滤除菌的0.1M CaCl2至终浓度5×10-4M;同时加入过滤除菌的0.1M FeSO4至终浓度1×10-6M,芽孢培养时不需加抗生素。
2.枯草芽孢杆菌WB600基因组DNA的提取
1)-80℃冰箱取出枯草芽孢杆菌WB600菌株,划LB平板,37℃培养过夜;
2)挑取单克隆菌落接种于5ml LB培养基中,37℃摇床培养过夜;
3)按1:100比例取1ml菌液转入100ml LB培养基中,37℃摇床培养过夜;
4)4℃离心收集细菌(重约0.5g),使用含0.5%溶菌酶的EDTA液吹悬菌体,37℃静置30分钟;
5)加入10%SDS至终浓度0.5%,吹匀,65℃水浴静置30分钟;
6)再加入与步骤5)中所用10%SDS等体积的酚:氯仿:异戊醇混合液(酚:氯仿:异戊醇体积比为25:24:1)抽提两次,每次冰浴15分钟,12000g离心5分钟,弃上清;
7)用0.6倍体积异丙醇沉淀基因组DNA,室温30min。12000g离心10分钟,弃上清;
8)用70%乙醇洗涤沉淀,晾干,DNA溶于500μl TE缓冲液中,加RNase至终浓度100μg/ml,65℃水浴静置30分钟;重复此步骤两次。
9)将DNA溶于100μl TE缓冲液中,取5μl样品进行琼脂糖凝胶电泳鉴定,其余样品-20℃保存备用。
3.枯草芽孢杆菌/大肠杆菌穿梭表达载体的构建
3.1重组质粒pBluescript II SK(+)-CotC的构建
根据芽孢衣壳CotC编码序列和载体pBluescript II SK(+)多克隆位点,设计特异性基因扩增引物,将CotC目的片段从枯草芽孢杆菌基因组DNA中扩出后与载体pBluescript IISK(+)重组,获得重组质粒pBluescript II SK(+)-CotC,引入多克隆位点。基因工程技术在大肠杆菌复制菌株DH5α中操作,重组质粒测序鉴定无误后常规保存。
所述CotC目的片段的核苷酸序列如SEQ ID NO.1所示,具体为:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTTGATTTGTTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAATATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTAC。
引物如下:
上游引物P1:5′-CATGTCGACTGTAGGATAAATCGTT-3′(SEQ ID NO.4),下划线为Sal I酶切位点,CAT为保护性碱基;
下游引物P2:5′-CGGAAGCTTGTAGTGTTTTTTATGC-3′(SEQ ID NO.5),下划线为Hind III酶切位点,CGG为保护性碱基。
3.2重组质粒pBluescript II SK(+)-CotC-CsCP的构建
CotC与pBluescript II SK(+)重组后,根据CsCP编码序列和载体pBluescript II SK(+)以及载体pEB03多克隆位点,设计特异性基因扩增引物,将CsCP编码序列连接至pBluescript II SK(+)-CotC,获得重组质粒pBluescript II SK(+)-CotC-CsCP。基因工程技术在大肠杆菌复制菌株DH5α中操作,重组质粒测序鉴定无误后常规保存。
所述CsCP编码序列的核苷酸序列如SEQ ID NO.2所示,具体为:
AGCAACATTCCTGAATCAGAAAATGCCCGGCAATTGTACGAAGAGTTCAAGCTGAAGTACAAGAAGTCCTATTCAAATGACGACGACGAATATAGATTCAGAGTTTTCAAGGACAACCTATTGCGCATAAAGCAATTTCAAAACATGGAGCGGGGAACGGCAAAGTACGGTGTGACTCAGTTTTCCGACCTCACAGCTCAGGAGTTCAAGGTTAGGTACCTAAGATCAAAATTTGGTGGTGTCCCTGTGGACAGAGAACCGGTTCCATTCATACGAATGGATGTGGATGACGACAACTTCGACTGGCGAAATCATGGTGCTGTGGGACCCGTATTGGACAAAGGAGATTGCGGATCGTGCTGGGCATTTTCCGCGGTTGGAAACATCGAGGGTCAATGGTTCCGTAAAACTGACAATCTTTTGCAGTTAAGCGAACAGCAACTCCTTGATTGCGATGAGGTGGATGAAGGTTGCAACGGCGGCACTCCTCAACAGGCTTTCAAGCAAATTTTGGGAATGGGCGGGTTGCAGCTGGACTCCGACTACCCATATGAGGGGAGAGAAGGGCAGTGCAGAATGGTGCCATCAAAGGTCAAGGTCTATATTAACGGCTCGAAAATTCTGCCGGAAGATGAGCAAATCCAGGCTCAAATGCTCAAGGAAACCGGGCCTTTTAGCTCCGCTCTAAATGCACTTTCCCTGCAATTTTACACGGAAGGGATTTTGCATCCCCTGCCAGCGCTTTGCGATGCCCAATCGTTAAACCATGCTGTTTTAACTGTCGGTTACGGGAAAGAAGGCAGGCTACCATACTGGACGGTTAAAAACAGTTGGAGCACTATGTTTGGCGAAAATGGTTACTTCCGTATTTACCGTGGAGACGGCCCCTGTGGAATCAATACCCTAGTTTCCACCTCGATCATCTTGTGA。
引物如下:
上游引物P1:5′-CGCAAGCTTATGTCGATCCTCAAAATTAC-3′(SEQ ID NO.6),下划线为Hind III酶切位点,CGC为保护性碱基;
下游引物P2:5′-CGTGAGCTCCTAACAGAGTGTACTC-3′(SEQ ID NO.7),下划线为SacⅠ酶切位点,CGT为保护性碱基。
3.3重组质粒pEB03-CotC-CsCP的构建
CsCP编码序列连接至pBluescript II SK(+)-CotC后,采用SalI、SacI酶切位点将CotC-CsCP目的基因片段整体从重组质粒pBluescript II SK(+)-CotC-CsCP上切下,连接至pEB03获得重组质粒pEB03-CotC-CsCP。
基因工程技术在大肠杆菌复制菌株DH5α中操作,重组质粒测序鉴定无误后常规保存。
4.重组质粒pEB03-CotC-CsCP电转化枯草芽孢杆菌WB600,获得WB600-pEB03-CotC-CsCP
1)接菌WB600于3ml LB培养基中,37℃,250rpm,培养过夜;
2)将2.6ml菌液转入40ml电转化A培养基中,37℃,250rpm,摇至OD值0.8-0.9;
3)菌液冰水浴10min,5,000g,4℃离心5min;
4)用50ml预冷的电转化B培养基吹悬菌体,5,000g,4℃离心5min,重复此步骤4次;
5)将菌体吹悬于1ml电转化B培养基,分装10管,每个EP管分装100μl;
6)将100μl WB600感受态细胞中加入5μl(约1μg)重组质粒pEB03-CotC-CsCP,冰上静置2min;
7)将100μl感受态细胞吸入到预冷的电击杯中(1mm),调整电转化仪参数:2.0kV,1mm,25μF,200Ω,4.5-5.0ms,电击一次;
8)电击结束后吸出电击杯中感受态细胞转移至新的EP管中,立即加入1ml电转化C培养基,37℃,150rpm,复苏3h,涂LB平板(壮观霉素抗性),37℃温箱中培养20h;
9)挑取单克隆至LB培养基中(壮观霉素抗性),37℃,250rpm,培养过夜;获得WB600-pEB03-CotC-CsCP;
10)WB600-pEB03-CotC-CsCP菌液PCR及提取质粒双酶切鉴定阳性克隆。
WB600-pEB03-CotC-CsCP菌液PCR扩增CsCP鉴定阳性克隆结果如图1所示。
WB600-pEB03-CotC-CsCP双酶切鉴定结果如图2所示。
此外,本发明的发明人还对WB600-pEB03-CotC-CsCp摇菌提质粒并对其中CotC-CsCP的核酸序列进行测序。结果与CotC-CsCP目的基因片段的核苷酸序列一致。
CotC-CsCP目的基因片段核苷酸序列如SEQ ID NO.3所示,具体为:
TGTAGGATAAATCGTTTGGGCCGATGAAAAATCGGCTCTTTATTTTGATTTGTTTTTGTGTCATCTGTCTTTTTCTATCATTTGGACAGCCCTTTTTTCCTTCTATGATTTTAACTGTCCAAGCCGCAAAATCTACTCGCCGTATAATAAAGCGTAGTAAAAATAAAGGAGGAGTATATATGGGTTATTACAAAAAATACAAAGAAGAGTATTATACGGTCAAAAAAACGTATTATAAGAAGTATTACGAATATGATAAAAAAGATTATGACTGTGATTACGACAAAAAATATGATGACTATGATAAAAAATATTATGATCACGATAAAAAAGACTATGATTATGTTGTAGAGTATAAAAAGCATAAAAAACACTACAAGCTTAGCAACATTCCTGAATCAGAAAATGCCCGGCAATTGTACGAAGAGTTCAAGCTGAAGTACAAGAAGTCCTATTCAAATGACGACGACGAATATAGATTCAGAGTTTTCAAGGACAACCTATTGCGCATAAAGCAATTTCAAAACATGGAGCGGGGAACGGCAAAGTACGGTGTGACTCAGTTTTCCGACCTCACAGCTCAGGAGTTCAAGGTTAGGTACCTAAGATCAAAATTTGGTGGTGTCCCTGTGGACAGAGAACCGGTTCCATTCATACGAATGGATGTGGATGACGACAACTTCGACTGGCGAAATCATGGTGCTGTGGGACCCGTATTGGACAAAGGAGATTGCGGATCGTGCTGGGCATTTTCCGCGGTTGGAAACATCGAGGGTCAATGGTTCCGTAAAACTGACAATCTTTTGCAGTTAAGCGAACAGCAACTCCTTGATTGCGATGAGGTGGATGAAGGTTGCAACGGCGGCACTCCTCAACAGGCTTTCAAGCAAATTTTGGGAATGGGCGGGTTGCAGCTGGACTCCGACTACCCATATGAGGGGAGAGAAGGGCAGTGCAGAATGGTGCCATCAAAGGTCAAGGTCTATATTAACGGCTCGAAAATTCTGCCGGAAGATGAGCAAATCCAGGCTCAAATGCTCAAGGAAACCGGGCCTTTTAGCTCCGCTCTAAATGCACTTTCCCTGCAATTTTACACGGAAGGGATTTTGCATCCCCTGCCAGCGCTTTGCGATGCCCAATCGTTAAACCATGCTGTTTTAACTGTCGGTTACGGGAAAGAAGGCAGGCTACCATACTGGACGGTTAAAAACAGTTGGAGCACTATGTTTGGCGAAAATGGTTACTTCCGTATTTACCGTGGAGACGGCCCCTGTGGAATCAATACCCTAGTTTCCACCTCGATCATCTTGTGA。
5枯草芽孢杆菌融合表达华支睾吸虫半胱氨酸蛋白酶(融合蛋白)的检测
5.1 SDS-PAGE
挑取单克隆菌落接种于5ml LB培养基中(壮观霉素抗性),37℃摇床培养过夜。按1:100体积比转入500ml DSM DSM芽孢培养基(不含抗生素),37℃,250rpm,培养24h,于0h、6h、12h、24h各取1ml DSM芽孢培养基离心处理细菌沉淀进行12%SDS-PAGE。于24h后停止诱导,离心DSM芽孢培养基,用SDS-DTT液吹悬细菌沉淀,37℃水浴中静置2h,12,000g离心10min;1M Tris(pH=8.0)洗涤芽孢沉淀,12,000g离心10min,共洗涤6次;将芽孢悬于破壳缓冲液,冰上超声5min,12,000g离心10min,分离上清蛋白和衣壳蛋白进行12%SDS-PAGE,考马斯亮蓝R250染色2h后脱色,观察融合蛋白CotC-CsCP在枯草芽孢杆菌中的表达及其在芽孢中的定位。结果如图3所示,说明融合蛋白CotC-CsCP在枯草芽孢杆菌中成功表达,并且随着诱导时间的增长表达增多。
5.2 Western blotting
将上述诱导0h、6h、12h、24h留取的全菌沉淀及超声破壁分离的芽孢上清蛋白和衣壳蛋白在进行12%SDS-PAGE后继续进行Western blotting。将蛋白凝胶上目的融合蛋白融合蛋白CotC-CsCP电转移至PVDF膜,然后分别将PVDF膜与大鼠抗CsCP血清及羊抗大鼠IgG孵育,鉴定融合蛋白CotC-CsCP在枯草芽孢杆菌中的表达及其在芽孢衣壳的定位。结果如图4所示。
5.3免疫荧光检测芽孢表面融合蛋白的表达
1)取50μl诱导24h的芽孢液加入1ml固定液中,室温静置30min,冰上静置1h;
2)12,000g离心2min,用PBS反复洗涤沉淀3次;
3)加入80μl GTE溶液,吹悬芽孢沉淀后涂于凹玻片内,37℃温箱烘干;
4)取出干燥玻片,浸于-20℃冰甲醇5min,冰丙酮30s,等待干燥;
5)干燥玻片上滴加正常山羊血清封闭,4℃过夜;
6)PBST洗涤玻片,5min/次,共洗三次,玻片上滴加大鼠抗CsCP血清,室温孵育2h;
7)PBST洗涤玻片,5min/次,共洗三次,往玻片上滴加羊抗大鼠Cy3标记的IgG,室温孵育1h;
8)PBST洗涤玻片,5min/次,共洗三次,往玻片上滴加组织淬灭剂,室温孵育30min;
9)PBST洗涤玻片,5min/次,共洗三次,显微镜下观察。
结果如图5所示。
6.枯草芽孢杆菌大量培养
1)按照1:100接菌于40ml LB培养基中(壮观霉素抗性),37℃摇床培养过夜。于次日转入4L DSM芽孢培养基中(不含抗生素),37℃摇床培养24h;
2)12,000g离心10min,弃上清留取沉淀,加入溶菌酶至终浓度4mg/ml反复吹悬沉淀,室温静置30min;
3)用1M NaCl洗涤芽孢,同时加加入PMSF至终溶度1mM,充分混匀;
4)12,000g离心10min,弃上清留取沉淀,再用1M KCl洗涤芽孢同时加加入PMSF至终溶度1mM,充分混匀;
5)去离子水洗涤芽孢三次,12,000g离心10min,弃上清留取沉淀;
6)40ml去离子水吹悬芽孢沉淀,65℃水浴静置1h以杀灭残留的枯草芽孢杆菌繁殖体;
7)取3μl芽孢用去离子稀释,显微镜下计数,每升DSM芽孢培养基可获得约1×1011个芽孢,将芽孢于-20℃保存备用。通过本部分枯草芽孢杆菌大量培养获得了重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP),所述疫苗中含有融合蛋白CotC-CsCP以及枯草杆菌芽孢。
7.重组枯草芽孢杆菌芽孢疫苗的投喂
本制品属于口服疫苗,适合随时投喂,不限鱼龄。从鱼苗时期喂起能达到更好的预防鱼感染华支睾吸虫病的效果。
对于刚开口的鱼花,可将重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)按一定的比例与开口料或轮虫混合进行投喂。每隔约半个月投喂一次,持续投喂四次以上,最后一次可间隔一个月,能达到良好的预防效果。对于体型稍大的鱼,可将重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)与饲料混合进行投喂,为了防止芽孢扩散,可加入少许的鱼肝油或大豆油进行包被。投喂量约为108cfu/g的开口料或饲料。大规模野外鱼苗投喂实验提示:重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)能成功预防草鱼感染肝吸虫。
本发明的发明人将重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)以不同剂量1×106cfu/g、1×107cfu/g及1×108cfu/g混合饲料投喂给草鱼。并同时设立对照组CotC枯草芽孢杆菌芽孢疫苗1×107cfu/g、以及阴性对照组(阴性对照组为投喂普通饲料)。WB600-pEB03-CotC的构建方法,包括步骤:(1)构建重组质粒PEB03-cotC:根据芽孢衣壳CotC编码序列和载体PEBO3多克隆位点,设计特异性基因扩增引物,将cotC目的片段从枯草杆菌基因组DNA中扩出后与PEBO3重组,获得重组质粒PEBO3-cotC作为对照;(2)参照上述4的方法将步骤(1)所得重组质粒pEB03-CotC电转化枯草杆菌WB600,获得WB600-pEB03-CotC,菌液PCR及提取质粒双酶切鉴定阳性克隆;(3)参照上述5和6的方法表达CotC蛋白,并将其制备成CotC枯草芽孢杆菌芽孢疫苗作为对照组。
具体的,重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)日投喂剂量为鱼体重的2%,投喂时间上午09:30,下午16:30,采用定点定位定时的投喂方式;分别检测免疫2W(2周)、4W(4周)、6W(6周)及8W(8周),及免疫后2W(2周)相应指标的变化情况。
ELISA检测每个时间点血清、体表黏液、胆汁及肠道黏液特异性IgM水平,结果如图6所示。说明重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)能够提高鱼体血清、胆汁、肠黏液及体表黏液中特异性IgM的水平,引起鱼体局部黏膜免疫及系统性免疫反应。
草鱼口服重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP),于第10周取鱼的前肠和后肠分别检测芽孢及重组质粒的存在情况。加入无菌生理盐水将肠道组织研磨成匀质,68℃水浴10min后,将其均匀涂布在含有壮观霉素抗性的LB固体培养板培养约12h。结果如图7所示,A-a.投喂疫苗组草鱼前肠匀浆涂布菌落生长情况;A-b.投喂疫苗组草鱼后肠匀浆涂布菌落生长情况。在培养板上随机挑取一定数量的单克隆,进行菌落PCR验证融合基因CotC-CsCP的存在情况,B.1Marker;1-5,检测投喂疫苗组草鱼前肠匀浆涂布菌落CotC-CsCP情况;6-22,检测投喂疫苗组草鱼后肠匀浆涂布菌落CotC-CsCP情况;23,阴性对照(投喂普通饲料组);24,阳性对照(WB600-pEB03-CotC-CsCP菌株)。因此,肠道涂板法检测提示:枯草杆菌芽孢能顺利到达草鱼后肠。
HE染色检测肠道上皮内淋巴细胞及肠绒毛生长情况,结果如图8所示,说明:重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP)能使鱼体肠道上皮淋巴细胞增多及肠绒毛增长,有效提高肠道免疫力,促进对食物吸收能力。因此,肠道HE染色法提示:重组芽孢对鱼肠道健康有促进作用。
微板法检测重组枯草杆菌芽孢口服免疫草鱼后血清中的ACT/GPT和AST/GOT等指标的水平,如图9所示,提示:芽孢疫苗对鱼体的肝脏无损伤作用。
草鱼口服重组枯草芽孢杆菌芽孢疫苗(WB600-pEB03-CotC-CsCP),于第10周取鱼20只,随机分为两组。送至中国疾病控制预防中心寄生虫病预防控制所(NIPD)检测口服免疫后草鱼体内的华支睾吸虫囊蚴。结果如表1所示。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明方法的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员,在不脱离本发明的精神和范围的情况下,当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化,均为本发明的等效实施例;同时,凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变,均仍属于本发明的技术方案的范围内。
Claims (10)
1.一种融合蛋白,其特征在于,所述融合蛋白含有枯草芽孢杆菌芽孢衣壳蛋白CotC和华支睾吸虫半胱氨酸蛋白酶CsCP。
2.根据权利要求1所述的融合蛋白,其特征在于,所述枯草芽孢杆菌芽孢衣壳蛋白CotC的氨基酸序列对应于如SEQ ID NO.1所示核苷酸序列所编码的氨基酸序列。
3.根据权利要求1所述的融合蛋白,其特征在于,所述华支睾吸虫半胱氨酸蛋白酶CsCP的氨基酸序列对应于如SEQ ID NO.2所示核苷酸序列所编码的氨基酸序列。
4.根据权利要求1所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列中含有如SEQ ID NO.3所示核苷酸序列所编码的氨基酸序列。
5.一种制备如权利要求1~4任一权利要求所述融合蛋白的方法,包括如下步骤:
(1)获得目的基因;
(2)构建重组质粒:将步骤(1)所得目的基因以及载体质粒采用相同的限制性内切酶切割,然后进行连接,获得重组质粒;
(3)将步骤(2)所得重组质粒导入感受态细胞,转化;
(4)筛选阳性克隆,并将正确的重组质粒转化宿主感受态细胞;
(5)诱导表达融合蛋白。
6.根据权利要求5所述的方法,其特征在于,步骤(1)中,所述目的基因核苷酸序列含有枯草芽孢杆菌芽孢衣壳蛋白CotC的编码核苷酸序列和华支睾吸虫半胱氨酸蛋白酶CsCP的编码核苷酸序列。
7.根据权利要求5所述的方法,其特征在于,步骤(2)中,所述限制性内切酶为SalI、SacI。
8.根据权利要求5所述的方法,其特征在于,步骤(3)中所述感受态细胞为枯草芽孢杆菌WB600感受态细胞;步骤(4)中所述宿主感受态细胞为枯草芽孢杆菌WB600感受态细胞。
9.如权利要求1~4任一权利要求所述融合蛋白在制备鱼华支睾吸虫病口服疫苗中的用途。
10.一种鱼华支睾吸虫病口服疫苗,含有如权利要求1~4任一权利要求所述融合蛋白。
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