CN112575030A - 一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法 - Google Patents
一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法 Download PDFInfo
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Abstract
本发明公开了一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法。本发明能够利用浮萍表达系统简便、高效表达草鱼呼肠孤病毒口服亚单位疫苗,本方法包括外源基因的扩增、农杆菌植物双元表达载体的构建、浮萍愈伤组织遗传转化体系的建立,并使用PCR、荧光显微观察、GUS染色和Western blotting等实验技术验证外源基因的表达。本发明能利用浮萍高效表达草鱼呼肠孤病毒亚单位疫苗,制备的疫苗可直接口服免疫草鱼,可减少因草鱼呼肠孤病毒感染而造成的经济损失,也便于操作,节省成本,更具有推广和实际应用价值。
Description
技术领域
本发明属于草鱼呼肠孤病毒口服亚单位疫苗的制备技术领域,具体涉及一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法。
背景技术
草鱼是我国淡水渔业中重要的养殖品种之一,我国每年的草鱼总产量约占国内淡水鱼类养殖总产量的25%左右,占世界草鱼养殖总产量的90%以上。但是,草鱼抵抗病原能力较差,在生长的各个阶段均易遭受疾病侵袭,其中草鱼出血病是严重制约草鱼养殖业可持续发展的主要障碍。草鱼呼肠孤病毒(Grass carp reovirus,GCRV)是引起草鱼出血病的病原,该疾病流行地区广泛,发病率高,死亡率高达90%以上,是草鱼健康养殖中的瓶颈问题。
科学使用疫苗是预防该病最有效的手段,其中,口服疫苗由于操作简单,且能够减少对鱼类的机械损伤和应激,受到国内外研究者的广泛关注。但是,口服疫苗易受胃肠道内消化酶的降解,因此,可有效避免口服疫苗被降解和消化的疫苗递送系统是口服疫苗发挥作用的基础和保障。植物真核表达系统在蛋白表达过程中的修饰和加工更加完善,且在一定程度上可减少消化道对疫苗的破坏,所以,植物作为口服疫苗的生产和递送系统成为研究的热点。其中,浮萍作为单子叶水生漂浮植物,因繁殖速度快,蛋白表达量高等优势备受青睐。利用浮萍表达系统制备草鱼呼肠孤病毒亚单位疫苗,抗原免疫原性更高,同时浮萍的细胞壁属于天然的“生物胶囊”,作为递送系统可减少胃肠道对疫苗的降解。此外,利用浮萍制备的草鱼呼肠孤病毒亚单位疫苗,可直接饲喂草鱼,操作方便,价格低廉,更适于应用和推广。近些年来,浮萍在作为植物生物反应器和递送系统等研究方面取得了令人瞩目的进展,但是,国内外尚无利用浮萍表达系统制备草鱼出血病口服亚单位疫苗的相关报道。
发明内容
本发明解决的技术问题是提供了一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法。
本发明为解决上述技术问题采用如下技术方案,一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法,其特征在于具体过程为:
步骤S1:将Ⅱ型草鱼呼肠孤病毒培养于L8824细胞系中,培养后收集细胞培养液,离心取上清提取病毒RNA,通过反转录获得cDNA模板,根据GenBank中提交的S11序列设计引物,使用PCR扩增技术获得S11基因,随后将扩增的S11基因片段构建到pCAMBIA1303载体中,再进行双酶切和测序验证;
步骤S2:将验证正确的重组质粒pCAMBIA1303-S11与农杆菌EHA105感受态细胞混匀,冰浴后放入液氮中速冻1 min,然后在37℃水浴中放置5 min,接着冰浴2 min,然后加入1 mL YEP液体培养基,28℃、160 rpm振荡培养4 h,然后均匀涂布到含卡那霉素的LB固体培养基上,挑选单克隆进行PCR检测,阳性菌保存于15wt%甘油并放置在-80℃保藏备用;
步骤S3:将转有重组质粒的土壤农杆菌EHA105在50 mL YEP液体培养基中复苏摇菌至OD为0.5-0.8,离心后使用B5培养基重悬,加入乙酰丁香酮和吐温,混匀活化农杆菌,然后将脱菌的深绿色结实紧密的稀脉萍胚性愈伤块移入农杆菌悬浮液中,培养30 min后使用滤纸吸干愈伤组织表面的菌液,然后将愈伤组织转移到B5培养基中暗培养两天,待淡色的菌圈出现时,超声波清洗愈伤组织后使用筛选培养基筛选13-16 d,当愈伤组织达到5-6 mm移入再生培养基培养28-32 d获得再生植株。
进一步限定,步骤S1中所述pCAMBIA1303载体含有GUS和mGFP5双标签,用于方便外源基因表达的验证。
进一步限定,步骤S1中所述PCR扩增技术获得S11基因的过程中扩增S11基因节段序列的引物为S11-F:CGGGGTACCCGCCACCATGGAACCAGCAAAACCATTG,S11-R:TGAGATCCAGGGACAGTCTGAGAAAGATGAGCTATAAGGATCCGCG,其中正向引物包括kpn1酶切位点GGGTACCC和kozak序列GCCACCATGG,反向引物包含BamH1酶切位点GGATCC和内质网滞留信号肽序列TCTGAGAAAGATGAGCTA。
本发明与现有技术相比具有以下优点和有益效果:本发明可以高效的以浮萍作为表达系统大量表达口服草鱼呼肠孤病毒亚单位疫苗,利用该系统制备的疫苗免疫原性更好,同时该疫苗受浮萍细胞壁的保护,也更容易递送至后肠引起机体免疫反应。浮萍是草鱼的天然饵料,将浮萍作为口服疫苗的递送载体,无需后期的加工,可直接口服免疫草鱼,便于操作,成本较低,更具有应用和推广价值。
附图说明
图1是实施例步骤S1中扩增的S11基因序列PCR电泳图,其中泳道M为核酸分子Marker,1为实验组S11基因扩增结果,2为对照组实验结果;
图2是实施例步骤S1中双元表达载体pCAMBIA1303-S11双酶切验证图,其中泳道1为pCAMBIA1303-S11双酶切电泳图,泳道2为pCAMBIA1303-S11载体电泳图;
图3是实施例步骤S3中转化了农杆菌的浮萍愈伤组织;
图4是实施例步骤S4中在RNA水平使用PCR技术验证土壤农杆菌转染浮萍效果,其中泳道M为核酸分子Marker,泳道1和2为对照组,泳道3和4为扩增得到的S11基因节段;
图5是实施例步骤S4中在DNA水平使用PCR技术验证土壤农杆菌转染浮萍效果,其中泳道M为核酸分子Marker,泳道1为扩增得到的S11基因节段,泳道2为对照组;
图6是实施例步骤S4中使用荧光观察技术验证浮萍转染效果,其中A图为对照组浮萍叶基部,B图为实验组浮萍叶基部;C图为对照组浮萍叶边缘,D图为实验组浮萍叶边缘;
图7是实施案例S4中使用GUS染色技术验证浮萍转染效果,其中A和B图分别为对照组叶基部和叶边缘,C和D为实验组叶基部和叶边缘;
图8是实施案例S4中使用Western blotting实验技术验证浮萍转染效果,其中泳道1是实验组,泳道2是对照组。
具体实施方式
以下通过实施例对本发明的上述内容做进一步详细说明,但不应该将此理解为本发明上述主题的范围仅限于以下的实施例,凡基于本发明上述内容实现的技术均属于本发明的范围。
实施例
步骤S1:Ⅱ型草鱼呼肠孤病毒GCRV S11基因节段的扩增
将保存的II型草鱼呼肠孤病毒GCRV-HN14培养于L8824细胞系(草鱼肝脏细胞)中,培养一周后收集细胞培养液,5000 g离心5 min,取上清提取病毒RNA(具体方法参照TRNzol说明书(北京天根公司)),通过反转录扩增cDNA获得模板,根据GenBank中提交的S11基因节段序列(MK675085.1)设计引物S11-F:CGGGGTACCCGCCACCATGGAACCAGCAAAACCATTG,S11-R:TGAGATCCAGGGACAGTCTGAGAAAGATGAGCTATAAGGATCCGCG,其中正向引物包括kpn1酶切位点GGGTACCC和kozak序列GCCACCATGG,反向引物包含BamH1酶切位点GGATCC和内质网滞留信号肽序列TCTGAGAAAGATGAGCTA。使用PCR扩增获得S11基因并使用DNA回收试剂盒进行回收(具体方法参照DNA片段凝胶回收试剂盒(宝日医生物技术(北京)有限公司))。将回收的S11基因与pMDTM19-T 载体在16℃连接8 h,随后转化DH5α感受态细胞,挑单克隆进行测序,测序成功后的质粒pMD19-T-S11用于构建植物双元表达载体。用内切酶KpnI和BamHI分别双酶切pMD19-T-S11和pCAMBIA1303空载体,然后用T4连接酶将S11基因节段连接到pCAMBIA1303载体上(载体中含有GUS标签序列和mGFP5标签序列),随后将重组质粒pCAMBIA1303-S11进行双酶切和测序验证。
步骤S2:农杆菌植物双元表达载体的构建
吸取2 µL验证正确的重组质粒pCAMBIA1303-S11与50 µL农杆菌EHA105感受态细胞轻轻混匀,冰浴30 min后放入液氮中速冻1 min, 然后在37℃水浴中放置5 min,接着迅速冰浴2 min,然后加入1 mL YEP液体培养基,28℃、160 rpm振荡培养4 h,然后均匀涂布到含卡那霉素的LB固体培养基上,挑选单克隆进行PCR检测,阳性菌保存于15wt%甘油并放置在-80℃保藏备用。
步骤S3:浮萍愈伤组织遗传转化体系的建立
将转有重组质粒的土壤农杆菌EHA105在50 mL YEP液体培养基中复苏摇菌至OD为0.5-0.8,4000 g离心5 min,使用20 mL B5培养基重悬,加入100 μM乙酰丁香酮(AS)和0.2wt% 的吐温(Tween-20),充分混匀后,活化农杆菌30 min。然后将脱菌的深绿色紧致结实的稀脉浮胚性愈伤块移入20 mL农杆菌悬浮液的容器中(真空抽滤,3次,每次1 min,0.05-0.06 Mpa)。培养30 min后使用干净滤纸吸干愈伤组织表面的菌液,然后将愈伤组织转移到含2.0 mg/L的2,4-二氯苯氧乙酸(2,4-D)的B5培养基中暗培养两天,待淡淡菌圈出现,超声波清洗愈伤组织1-3次后使用含有300 mg/L头孢霉素和潮霉素的筛选培养基中筛选15 d左右,当愈伤组织达到5-6 mm移入再生培养基培养30 d左右可获得再生植株。将再生植株移入含1 mg/L卡那霉素的B5培养基上进行单株扩繁。
步骤S4:外源基因的表达结果
扩繁植株使用PCR、荧光显微观察、GUS染色和Western blotting等实验技术验证外源基因的表达结果,详细步骤如下:
1)PCR:提取再生植株DNA(具体方法参照DNA提取试剂盒(北京天根公司)),根据S11节段序列设计特异性引物(F:ATGGAACCAGCAAAACC;R:GGTACTGTCCCTGGATCTCAGG)。PCR反应体系如下:2 μL 10×缓冲液、1.62 μL dNTPs、0.62 μL正向引物、0.62 μL反向引物、0.5μL Taq酶、1 μL模板,补水至20 μL;PCR反应程序:94℃,5 min;94℃,1 min ;60℃,1 min;72℃,1 min;72℃,7 min。提取再生植株RNA(具体方法参照TRNzol说明书(北京天根公司)),反转录成cDNA(具体方法参照反转录试剂(北京宝日医生物技术有限公司)),按照上述方法进行PCR反应。
2)GUS染色:将再生植株浸于预冷90wt%丙酮中固定10 min,使用GUS染色底液(0.5M PBS,pH 7.2;10 mM EDTA;0.1wt% Triton X-100;100 mM 铁氰化钾;100 mM亚铁氰化钾)漂洗3次,每次5min;将植株放置于1.5 mL离心管中,加入染色液(1 mL 0.5 M PBS,pH 7.2;0.2 mL 10 mM EDTA;0.1 mL 0.1wt% Triton X-100;0.2 mL 100 mM 铁氰化钾;0.2 mL100 mM亚铁氰化钾;10.4 mg X-gluc;定容至10 mL)浸过材料,抽气10 min,并染色过夜,然后用体积分数为50%、60%、70%、80%、90%乙醇依次脱水30 min后,在无水乙醇中放置1 h后显微观察。
3)荧光显微观察步骤:将植株放在纯水中洗净后,使用干净滤纸将植株表面的水系吸干后,将植株放在载玻片上,放置在荧光显微镜下观察并拍照。
4)Western blotting具体步骤:将冲洗干净的浮萍放置在液氮中研磨,然后抽提蛋白(具体方法参照植物组织蛋白提取试剂盒(北京索莱宝科技有限公司)),将蛋白浓度调至200 μg/mL,吸取10 μL与等体积的上样缓冲液混匀,煮沸10 min。使用移液枪将样品加至样品槽中,然后进行电泳。电泳结束后将蛋白样品转入PVDF膜中,使用5wt%的牛血清白蛋白(BSA)封闭4 h,使用含有0.2wt% Tween-20的PBS溶液(PBST)清洗三次后,使用抗VP35蛋白的多克隆抗体孵育2 h,使用PBST清洗三次,将PVDF放入碱性磷酸酶标记的羊抗鼠(体积比1:4000,西格玛奥德里奇(上海)贸易有限公司)中,孵育2 h,孵育结束后使用PBST清洗3次,将PVDF膜放置在含有四唑硝基蓝(NBT)和5-溴- 4-氯-3-吲哚-磷酸(BCIP)的发色液(100mM Tris;100 mM 氯化钠;5 mM 氯化镁,pH 9.5)中,染色5 min后使用超纯水终止反应,清洗PVDF,随后观察并拍照。
以上实施例描述了本发明的基本原理、主要特征及优点,本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明原理的范围下,本发明还会有各种变化和改进,这些变化和改进均落入本发明保护的范围内。
SEQUENCE LISTING
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Claims (3)
1.一种利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法,其特征在于具体过程为:
步骤S1:将Ⅱ型草鱼呼肠孤病毒培养于L8824细胞系中,培养后收集细胞培养液,离心取上清提取病毒RNA,通过反转录获得cDNA模板,根据GenBank中提交的S11序列设计引物,使用PCR扩增技术获得S11基因,随后将扩增的S11基因片段构建到pCAMBIA1303载体中,再进行双酶切和测序验证;
步骤S2:将验证正确的重组质粒pCAMBIA1303-S11与农杆菌EHA105感受态细胞混匀,冰浴后放入液氮中速冻1 min,然后在37℃水浴中放置5 min,接着冰浴2 min,然后加入1 mLYEP液体培养基,28℃、160 rpm振荡培养4 h,然后均匀涂布到含卡那霉素的LB固体培养基上,挑选单克隆进行PCR检测,阳性菌保存于15wt%甘油并放置在-80℃保藏备用;
步骤S3:将转有重组质粒的土壤农杆菌EHA105在50 mL YEP液体培养基中复苏摇菌至OD为0.5-0.8,离心后使用B5培养基重悬,加入乙酰丁香酮和吐温,混匀活化农杆菌,然后将脱菌的深绿色结实紧密的稀脉萍胚性愈伤块移入农杆菌悬浮液中,培养30 min后使用滤纸吸干愈伤组织表面的菌液,然后将愈伤组织转移到B5培养基中暗培养两天,待淡色的菌圈出现时,超声波清洗愈伤组织后使用筛选培养基筛选13-16 d,当愈伤组织达到5-6 mm移入再生培养基培养28-32 d获得再生植株。
2.根据权利要求1所述的利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法,其特征在于:步骤S1中所述pCAMBIA1303载体含有GUS和mGFP5双标签,用于方便外源基因表达的验证。
3.根据权利要求1所述的利用浮萍表达系统制备草鱼呼肠孤病毒口服亚单位疫苗的方法,其特征在于:步骤S1中所述PCR扩增技术获得S11基因的过程中扩增S11基因节段序列的引物为S11-F:CGGGGTACCCGCCACCATGGAACCAGCAAAACCATTG,S11-R:TGAGATCCAGGGACAGTCTGAGAAAGATGAGCTATAAGGATCCGCG,其中正向引物包括kpn1酶切位点GGGTACCC和kozak序列GCCACCATGG,反向引物包含BamH1酶切位点GGATCC和内质网滞留信号肽序列TCTGAGAAAGATGAGCTA。
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CN115161340B (zh) * | 2022-08-17 | 2024-04-02 | 中国科学院成都生物研究所 | 浮萍遗传转化方法及应用与利用浮萍表达细胞因子的方法 |
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