CN114806994A - 表达sat2型口蹄疫病毒结构蛋白vp1的枯草芽孢杆菌及应用 - Google Patents
表达sat2型口蹄疫病毒结构蛋白vp1的枯草芽孢杆菌及应用 Download PDFInfo
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- CN114806994A CN114806994A CN202210629321.2A CN202210629321A CN114806994A CN 114806994 A CN114806994 A CN 114806994A CN 202210629321 A CN202210629321 A CN 202210629321A CN 114806994 A CN114806994 A CN 114806994A
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- bacillus subtilis
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Abstract
本发明属于生物医药领域,涉及一种表达SAT2型口蹄疫病毒结构蛋白VP1的枯草芽孢杆菌及应用。本发明将表达SAT2型口蹄疫病毒结构蛋白VP1的重组质粒转化至枯草芽孢杆菌WB800N,获得了一种表达SAT2型口蹄疫病毒结构蛋白VP1的重组枯草芽孢杆菌;所述重组枯草芽孢杆菌在高效表达SAT2型口蹄疫病毒结构蛋白VP1的同时,具有良好的免疫反应性,能够诱导机体产生体液免疫应答和细胞免疫应答。还可以作为黏膜疫苗,经口服免疫后,产生较高水平的抗原特异性IgG抗体及黏膜sIgA抗体,升高外周血CD4+和CD8+T淋巴细胞的比例,刺激脾脏淋巴细胞增殖,并促进细胞因子IFN‑γ、IL‑2和IL‑4的表达。
Description
技术领域
本发明涉及生物制药领域,具体涉及一种表达SAT2型口蹄疫病毒结构蛋白VP1的枯草芽孢杆菌及应用。
技术背景
口蹄疫(Foot-and-mouth disease,FMD)是由口蹄疫病毒(Foot-and-mouthdisease virus,FMDV)引起的一种急性、热性、高度接触性传染病,主要感染猪、牛、羊等偶蹄类动物,给畜牧养殖业造成巨大经济损失。FMDV共有7个血清型,即O型、A型、亚洲1型(Asia1)、南非1型(Southern African Territories 1,SAT1)、南非2型(SAT2)、南非3型(SAT3)和C型,我国主要流行O型和A型。南非型口蹄疫地域性较为明显,主要集中在撒哈拉沙漠以南,但近几年的报告表明,SAT2型口蹄疫已跨越非洲,在中东地区蔓延和爆发,传入东南亚和我国的风险较高,对我国养殖业造成潜在威胁。因此,急需针对SAT2型FMDV开展疫苗研发,为我国口蹄疫防控进行技术储备。
目前防控口蹄疫最经济有效的措施是免疫疫苗,口蹄疫灭活疫苗是我国口蹄疫防控的主导产品,为我国口蹄疫防控做出了巨大贡献,但自身也有一些不足,例如传统制造可能病毒灭活不完全,存在着散毒风险和安全隐患,且灭活疫苗通常无法诱导机体产生有效的黏膜免疫应答。因此,研发更加安全、高效、绿色的口蹄疫疫苗具有重要意义。
黏膜是保护机体免受病原体入侵的第一道防线,FMDV主要通过呼吸道及消化道的黏膜入侵机体,因此,诱导机体产生黏膜免疫应答对口蹄疫的预防和治疗尤为重要。而且黏膜疫苗与传统疫苗相比,有以下优点:黏膜免疫的接种方式(点眼、饮水、灌胃、舌下、滴鼻、喷雾)多样化且较为简单,无需专业技术人员就可完成免疫接种,能降低免疫过程中对动物的造成的疼痛及应激;大多数黏膜免疫方式均适用于大规模疫苗接种、且给药时无需针头和注射器,不会产生血源性疾病传播的风险。因此,口蹄疫黏膜疫苗是一种极具潜力的新型疫苗。
枯草芽孢杆菌作为一种益生菌,是较好的黏膜疫苗递送载体的候选者。虽然目前使用枯草芽孢杆菌递送抗原蛋白已有大量研究,但是并没有确切有效的口服黏膜疫苗被用于口蹄疫防控。
发明内容
针对上述技术问题,首先,在本发明研究过程中发现,以SAT2型口蹄疫病毒结构蛋白VP0制备重组枯草芽孢杆菌WB800N/pHT43-VP0时,虽然能够高效表达SAT2型口蹄疫病毒结构蛋白VP0,具有良好的免疫原性,但是所诱导机体产生的体液免疫应答和细胞免疫应答水平较低,且不能诱导机体产生黏膜免疫应答,无法作为黏膜疫苗使用;而且以pMA5载体构建表达SAT2型口蹄疫病毒结构蛋白VP1的重组质粒,虽然可成功电转化至宿主细菌B.S168,但是目的蛋白VP1不能表达;以pWB980载体构建表达SAT2型口蹄疫病毒结构蛋白VP1的重组质粒时,VP1基因无法成功连接至载体;以pHY300PLK载体构建表达SAT2型口蹄疫病毒结构蛋白VP1的重组质粒,虽然可成功电转化至宿主细菌WB600,但是目的蛋白VP1的表达量极低。而本发明意外发现,只有构建的表达SAT2型口蹄疫病毒结构蛋白VP1的重组枯草芽孢杆菌WB800N/pHT43-VP1,能够诱导机体产生体液免疫应答和细胞免疫应答,且能够诱导机体产生较高水平的黏膜免疫应答,可用于SAT2型口蹄疫黏膜疫苗的制备。具体包括以下内容:
第一方面,本发明提供了一种表达SAT2型口蹄疫病毒结构蛋白VP1的重组枯草芽孢杆菌WB800N/pHT43-VP1,所述重组枯草芽孢杆菌WB800N/pHT43-VP1是将编码SAT2型口蹄疫病毒结构蛋白VP1的基因或表达SAT2型口蹄疫病毒结构蛋白VP1的重组载体/质粒导入枯草芽孢杆菌WB800N获得。
优选地,所述重组枯草芽孢杆菌WB800N/pHT43-VP1的制备方法为:全基因合成SAT2型口蹄疫病毒结构蛋白VP1基因;将SAT2型口蹄疫病毒结构蛋白VP1基因连接至pHT43质粒中,构建重组质粒pHT43-VP1;将重组质粒pHT43-VP1转化枯草芽孢杆菌WB800N感受态细胞获得重组枯草芽孢杆菌WB800N/pHT43-VP1。
优选地,所述扩增SAT2型口蹄疫病毒结构蛋白VP1基因的引物为:
pHT43-VP1-F:atcagccgtaggatccATGACTACCTCGGCGGGAGAAGGCGCAGA,其中,小写部分为同源臂基因序列;
pHT43-VP1-R:tcattaggcgggctgcTCAATGGTGGTGATGGTGATGCAGGGTCTGTC,其中小写部分为同源臂基因序列;斜体部分为6×His标签基因序列。
优选地,所述SAT2型口蹄疫病毒结构蛋白VP1的基因序列如SEQ ID NO.1所示。
优选地,所述转化为电转化。
优选地,所述电转化为:
(1)向枯草芽孢杆菌WB800N感受态细胞中加重组质粒pHT43-VP1,冰浴后加入电转杯,电转仪设置为2.5kv、25μF、2000Ω、持续时间4.5ms,电击1次;
(2)取出电转杯,加入复苏培养基RM,37℃、200rpm复苏3h,取200μL菌液涂布于Cm抗性(10μg/mL)的2×YT固体平板培养基上,37℃过夜培养。
优选地,所述枯草芽孢杆菌WB800N感受态细胞的制备方法为:
(1)挑取枯草芽孢杆菌WB800N单菌落接种于LB培养基中,37℃、200rpm培养18h;
(2)取步骤(1)所述过夜培养物转接至GM液中,使OD600=0.2左右,于37℃、200rpm培养至OD600=1.0左右;
(3)取步骤(2)中的全部菌液冰浴,4℃、5000rpm离心8min,弃掉上层废液,收集下层沉淀中的菌体;
(4)4℃预冷电转缓冲液ETM,每次加入40mL电转缓冲液ETM洗涤步骤(3)所述菌体,4℃、5000rpm离心8min,弃掉上层废液,保留下层沉淀,反复3次;
(5)将步骤(4)洗涤后的菌体重悬于500μL步骤(4)所述的ETM中,-80℃冻存备用。
第二方面,本发明提供了上述第一方面所述的重组枯草芽孢杆菌WB800N/pHT43-VP1在制备SAT2型口蹄疫病毒疫苗中的应用。
第三方面,本发明提供了上述第一方面所述的重组枯草芽孢杆菌WB800N/pHT43-VP1在制备预防或治疗SAT2型口蹄疫病毒感染药物中的应用。
优选地,所述重组枯草芽孢杆菌WB800N/pHT43-VP1加入药学上可接受的载体制成口服液、注射剂、喷雾剂、滴剂、贴剂、散剂、片剂、颗粒剂、胶囊剂、乳剂、混悬剂或软膏剂中的任一种。
本发明的有益效果是:本发明将表达SAT2型口蹄疫病毒结构蛋白VP1的重组质粒转化至枯草芽孢杆菌WB800N,获得了一种能够表达SAT2型口蹄疫病毒结构蛋白VP1的重组枯草芽孢杆菌;所述重组枯草芽孢杆菌在高效表达SAT2型口蹄疫病毒结构蛋白VP1的同时,具有良好的免疫反应性,能够诱导机体产生体液免疫应答和细胞免疫应答。还可以作为黏膜疫苗,经口服免疫后,产生较高水平的抗原特异性IgG抗体及黏膜sIgA抗体,升高外周血CD4+和CD8+T淋巴细胞的比例,刺激脾脏淋巴细胞增殖,并促进细胞因子IFN-γ、IL-2和IL-4的表达。
附图说明
图1本发明的SAT2型FMDV结构蛋白VP1重组枯草芽孢杆菌表达载体pHT43-VP1的构建示意图;
图2SAT2型FMDV结构蛋白VP1基因的扩增结果;
图3pHT43质粒进行双酶切结果;
图4重组质粒pHT43-VP1的PCR鉴定结果图;
图5重组质粒pHT43-VP1的MegAlign软件比对分析结果;
图6重组枯草芽孢杆菌WB800N/pHT43-VP1的PCR鉴定结果图;
图7以His标签单克隆抗体为一抗进行Western blot检测的结果图;
图8以SAT2型FMDV VP1兔多抗血清TDR为一抗进行Western blot检测图;
图9血清中IgG的ELISA检测结果;
图10肺洗液中黏膜样品中sIgA的ELISA检测结果;
图11肠洗液中黏膜样品中sIgA的ELISA检测结果;
图12外周血CD4+和CD8+T淋巴细胞亚群检测结果;
图13脾淋巴细胞增殖试验结果;
图14细胞因子水平检测结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。下述实施例中的实验方法若无特别说明,均为常规方法。下述实施例中所用的试剂材料等,如无特殊说明,均为市售购买。
本发明中涉及使用的现有核苷酸序列均能在已发表的本领域学术刊物及GenBank中检索而得,本领域普通技术人员均能在参考本领域教科书或实验手册的基础上完成本发明涉及的分子生物学和细胞生物学试验。
实施例1重组枯草芽孢杆菌WB800N/pHT43-VP1的制备
1.SAT2型FMDV结构蛋白VP1重组枯草芽孢杆菌表达质粒的构建
重组枯草芽孢杆菌表达载体pHT43-VP1的构建示意图如图1所示;具体如下:
(1)引物设计与合成:根据GenBank公布的FMDV-SAT2-VII-VP1(序列号:JX014256)基因为参考序列,经南京金斯瑞生物公司合成重组质粒GS1803U-pUC57,并将重组质粒转化至Top10中保存为模板GS1803U-pUC57-Top10,设计引物,引物由西安擎科生物科技有限公司合成:
pHT43-VP1-F:atcagccgtaggatccATGACTACCTCGGCGGGAGAAGGCGCAGA,其中,小写部分为同源臂基因序列;
pHT43-VP1-R:tcattaggcgggctgcTCAATGGTGGTGATGGTGATGCAGGGTCTGTC,其中小写部分为同源臂基因序列;斜体部分为6×His标签基因序列。
PCR扩增VP1基因:以GS1803U-pUC57重组质粒为模板,PCR扩增体系为50μL:即Ex-Taq Mix 25μL,上下游引物各2μL,模板2μL,ddH2O 19μL。PCR扩增程序:95℃预变性3min;95℃变性30s、58℃退火45s、72℃延伸1min,循环30次;72℃延伸10min。PCR产物经1%琼脂糖凝胶电泳检测扩增结果,用DNA凝胶回收试剂盒回收目的片段,样品于-20℃保存。扩增结果如图2所示,其中,M为DNA分子质量标准;1为阴性对照;2为VP1基因PCR扩增产物;结果表明,以重组质粒GS1803U-pUC57为模板,以上述引物进行PCR扩增,在VP1-His-666bp处出现特异性条带,与SAT2型FMDV结构基因VP1的预期大小相符合。
(2)重组质粒pHT43-VP1的构建与鉴定:根据pHT43质粒的酶切位点,利用XbaⅠ和SmaⅠ限制性核酸内切酶对pHT43质粒进行双酶切,酶切产物进行1%琼脂糖凝胶电泳,用胶回收试剂盒回收线性化载体,并且进行重组反应及重组产物转化,取重组产物转化于E.coli DH5α感受态细胞,涂布于含氨苄青霉素抗性的LB平板上,37℃培养12-16h,挑取单菌落培养8h,12000rpm离心收菌体,提取质粒,将提取的重组质粒作为PCR反应的模板,利用上述(1)中所述引物进行PCR扩增鉴定,PCR反应体系及反应条件如上述(1)所述,核酸电泳观察结果扩增产物;将阳性质粒送往生物公司测序,MegAlign软件比对分析结果。
以Xba Ⅰ和Sma Ⅰ限制性核酸内切酶对pHT43质粒进行双酶切,结果如图3所示,其中M为DNA分子质量标准;1为pHT43质粒双酶切产物;结果表明pHT43质粒进行双酶切得到大小约为8057bp的线性化载体,与预期结果相符合。
扩增结果如图4所示,其中M为DNA分子质量标准;1为阴性对照;2为VP1基因的PCR扩增产物;3-5为重组质粒pHT43-VP1的PCR产物。结果表明,扩增出预期大小的目的片段且阴性对照孔无条带,则认为所提取的质粒为阳性重组质粒。
MegAlign软件比对分析结果如图5所示,重组质粒送公司测序后用MegAlign软件分析,与目的基因的同源性均达到100%,表明成功构建出重组质粒pHT43-VP1,即为SAT2型FMDV结构蛋白VP1重组枯草芽孢杆菌表达载体。
2.重组枯草芽孢杆菌WB800N/pHT43-VP1的制备
2.1制备枯草芽孢杆菌WB800N感受态细胞
(1)新鲜平板挑单菌落枯草芽孢杆菌WB800N接种于5mL LB培养基中,37℃、200rpm培养18h;
(2)取2.6mL过夜培养物转接至50mL GM液中,使OD600=0.2左右,37℃、200rpm培养至OD600=1.0左右;
(3)将全部菌液冰浴10min,4℃、5000rpm离心8min,弃掉上层废液,收集下层沉淀中的菌体;
(4)在4℃冰箱预冷电转缓冲液ETM,每次加入40mL洗涤菌体,4℃、5000rpm离心8min,弃掉上层废液,保留下层沉淀,反复漂洗3次;
(5)将洗涤后的菌体重悬于500μL步骤(4)的ETM中,用无菌的1.5mL离心管分装,60μL/管,-80℃冻存备用。
2.2重组质粒转化枯草芽孢杆菌WB800N感受态细胞
(1)向60μL WB800N感受态细胞中加5μL重组质粒,冰浴5min后加入2mm电转杯,电转仪设置为2.5kv、25μF、2000Ω、持续时间4.5ms,电击1次;
(2)取出电转杯,加入1mL复苏培养基RM,37℃、200rpm复苏3h,取200μL菌液涂布于Cm抗性(10μg/mL)的2×YT固体平板培养基上,37℃过夜培养,筛选阳性转化子。
2.3表达SAT2型FMDV结构蛋白VP1的重组枯草芽孢杆菌的鉴定
(1)挑取转化后的平板上的枯草芽孢杆菌单菌落,接种于5mL Cm抗性(10μg/mL)的2×YT液体培养基中,37℃、220rpm过夜培养。
(2)保存甘油菌于-40℃,采用试剂盒提取重组表达质粒,-20℃保存备用。
(3)以提取的重组表达质粒为模板,以步骤上述1中(1)所述的引物进行PCR鉴定,将阳性转化子命名为WB800N/pHT43-VP1。
扩增结果如图6所示,其中M为DNA分子质量标准DL2000;1-5为重组菌WB800N/pHT43-VP1的PCR扩增产物;6为VP1基因PCR扩增产物;7为阴性对照。结果表明,本申请所构建的重组枯草芽孢菌WB800N/pHT43-VP1扩增出VP1-His条带,长约666bp,与预期目的片段大小一致,且阴性对照孔无条带,表明正确构建了能够表达SAT2型FMDV结构蛋白VP1的重组枯草芽孢菌WB800N/pHT43-VP1。
2.4 SAT2型FMDV结构蛋白VP1在重组枯草芽孢杆菌表达质粒的表达与鉴定
(1)SAT2型FMDV结构蛋白VP1的诱导表达:无菌挑取鉴定为阳性的单菌落,接种于5mL Cm抗性的2×YT液体培养基中,37℃、220rpm过夜培养。按2%的比例接种新鲜菌液至100mL Cm抗性的2×YT液体培养基,培养至OD600为0.6左右时,加入100μL浓度为1mol/L的IPTG,37℃继续诱导14h,6000rpm离心10min收集菌体,弃掉上层废液,下层沉淀用PBS溶液重悬。
(2)SAT2型FMDV结构蛋白VP1的Western blot鉴定:①制样:取诱导与未诱导的WB800N/pHT43-VP1重悬液各80μL,加入20μL 5×蛋白loading buffer,充分混匀,100℃金属浴10min,室温静置冷却后,室温离心2min;②电泳:将SDS-PAGE凝胶(12%分离胶)固定在蛋白电泳仪器上,加入足量的1×SDS-PAGE电泳缓冲液,每个泳道的上样量为10μL。80V电压条件下电泳20min,调节电压为120V电泳至结束;③转印:转印液提前置于4℃冰箱预冷,去离子水清洗凝胶表面。裁剪稍大于凝胶面积的PVDF膜,在100%甲醇中浸泡1-2min。叠放好膜与SDS-PAGE凝胶之后放入电泳槽,加入足量的转印液,400mA电流条件下转膜1.5h;④封闭:转印完成后,取出PVDF膜,5%脱脂奶粉室温封闭2h;⑤孵育一抗:加入8mL稀释的His标签鼠单克隆抗体(1:2000稀释)或SAT2型FMDV VP1兔多抗血清TDR(1:500稀释),4℃过夜孵育;⑥孵育二抗:用1×PBST清洗7-10次,加入以8mL稀释的HRP标记的山羊抗鼠IgG抗体(1:5000稀释)或HRP标记的山羊抗兔IgG抗体(1:5000稀释),室温孵育1h;⑦曝光:用1×PBST清洗5次,在PVDF膜上滴加适量ECL显色液,扫描曝光后保存图片。
以His标签单克隆抗体为一抗进行Western blot检测的结果如图7所示,其中M为蛋白质分子标准;1为VP1蛋白;2为WB800N/pHT43诱导后全菌体;3为重组菌WB800N/pHT43-VP1诱导后上清;4为重组菌WB800N/pHT43-VP1诱导后沉淀;5为重组菌WB800N/pHT43-VP1诱导后全菌体;结果表明,超声后上清液、超声后沉淀中的蛋白在25kDa附近均有明显的条带,与目的蛋白VP1-His分子量大小一致。
以SAT2型FMDV VP1兔多抗血清TDR为一抗进行Western blot检测的结果如图8所示,其中M为蛋白质分子标准;1为VP1蛋白;2为重组菌WB800N/pHT43-VP1诱导后上清;3为重组菌WB800N/pHT43-VP1诱导后沉淀;4为重组菌WB800N/pHT43-VP1诱导后全菌体;5为WB800N/pHT43诱导后全菌体;结果表明,超声后上清液、超声后沉淀中的蛋白在25kDa附近均有明显的条带,与目的蛋白VP1-His分子量大小一致。
以上试验结果表明,本发明所述的重组枯草芽孢杆菌能够表达SAT2型FMDV的结构蛋白VP1,且具有良好的免疫反应性。
实施例2重组枯草芽孢杆菌WB800N/pHT43-VP1黏膜疫苗的制备及该疫苗的免疫原性评价
1.重组枯草芽孢杆菌WB800N/pHT43-VP1黏膜疫苗的制备
(1)复苏种子菌:分别将重组枯草芽孢杆菌WB800N/pHT43-VP1,空载菌WB800N/pHT43分别接种于5mL Cm抗性(10μg/mL)的2×YT液体培养基中,37℃、220rpm过夜培养。
(2)种子菌的扩大培养:按2%的比例接种新鲜菌液至50mL Cm抗性的2×YT液体培养基,37℃、220rpm过夜培养。
(3)目的蛋白的诱导表达:按2%的比例接种新鲜菌液至2L Cm抗性的2×YT液体培养基,培养至OD600为0.6左右时,加入2mL浓度为1mol/L的IPTG,37℃继续诱导14h。
(4)制备黏膜疫苗:6000rpm离心10min收集菌体,弃掉上层废液,下层沉淀用PBS溶液重悬。用无菌的PBS洗涤收集的菌体3次,最后将菌体重新悬浮在PBS中,采用10×递减稀释法进行细菌计数(每组设置三个重复,取平均值),调整菌液浓度至5×1010CFU/mL,4℃保存备用。
2.黏膜疫苗的免疫原性评价
(1)小鼠分组、免疫及样品采集:将75只6-8周龄雌性BALB/c小鼠随机分成3组,每组25只,分别WB800N/pHT43-VP1免疫组、WB800N/pHT43阴性对照组和PBS空白对照组,分别免疫重组枯草芽孢杆菌WB800N/pHT43-VP1、空载菌WB800N/pHT43和1×PBS缓冲液,口服免疫每次连续免疫三天进行,即第1d-3d、11d-13d、21d-23d,各组均用灌胃针灌服,免疫剂量为0.2mL(1×1010CFU)。分别从3组中每组抽出随机选取不同时间段(10d、20d、30d、37d、44d)的3只小鼠进行眼眶静脉采血并收集血清,同时收集肠洗液和肺洗液,收集后-20℃冻存备用。
(2)免疫小鼠血清中IgG的ELISA检测
用间接ELISA法测定免疫小鼠血清中IgG抗体水平,具体步骤如下:
①抗原包被:以SAT2型FMDV VP1蛋白作为包被抗原,用包被缓冲液稀释抗原,加入ELISA微量反应板中,200ng/孔,100μL/孔,4℃静置12h。
②封闭:将包被抗原的ELISA反应板取出,弃液,洗板5次,拍干。加入1%BSA,220μL/孔,37℃封闭2h,1×PBST洗涤5次并拍干,-40℃保存备用。
③加待检血清:将采集的小鼠血清样品用血清稀释液进行稀释后加入ELISA反应板中,WB800N/pHT43对照组、PBS组的血清作为阴性对照,100μL/孔,每个样品重复2孔。37℃静置孵育1h,弃液,洗板5次并拍干。
④加二抗:将HRP标记的山羊抗鼠IgG用1×PBST稀释后(1:50000)加入各孔中,100μL/孔,37℃静置孵育1h,弃液,洗板5次并拍干。
⑤显色:加入TMB底物显色液,50μL/孔,37℃避光静置孵育15min。
⑥终止:加入终止液,50μL/孔。5min内在酶标仪上测定OD450值,保存结果。
(3)免疫小鼠黏膜免疫sIgA水平的检测
肠洗液的收集:取样前将小鼠禁食禁水6h,每组选取不同时间段(0d、10d、20d、30d、37d、44d)的3只小鼠,颈椎脱臼处死,固定于泡沫板上,用75%酒精棉球体表消毒,打开腹腔后剪取其小肠(长约为6cm),放入200μL预冷的PBS(1.5mM EDTA,0.2mM PMSF)中,用手术剪碎,再用涡旋仪振荡混匀,4℃、12000rpm离心5min,轻柔吸取上清到1.5mL的离心管中,整个过程均在冰上操作,-70℃保存待用。
肺洗液的收集:将收集完肠洗液的小鼠,继续打开胸腔,剪取肺脏,用预冷的PBS冲洗,去除多余的血液,于200μL预冷的PBS(1.5mM EDTA,0.2mM PMSF)中反复冲洗,收集肺洗液,4℃、12000rpm离心5min,轻柔吸取上清于1.5mL离心管中,-70℃保存待用。
采用间接ELISA法对小鼠的肺洗液和肠洗液中黏膜sIgA进行检测,具体步骤如下:
①抗原包被:以SAT2型FMDV VP1蛋白作为包被抗原,用包被缓冲液稀释抗原,加入ELISA微量反应板中,200ng/孔,100μL/孔,4℃静置12h。
②封闭:将包被抗原的ELISA反应板取出,弃液,洗板5次,拍干。加入1%BSA,220μL/孔,37℃封闭2h,1×PBST洗涤5次并拍干,-40℃保存备用。
③加待检样品:小鼠的肠洗液以0.45μm滤膜过滤,用血清稀释液稀释肠洗液和肺洗液后加入反应板,同时将WB800N/pHT43对照组、PBS组的样品作为阴性对照,100μL/孔,每个样品重复2孔。37℃静置孵育1h,弃液,洗板5次并拍干。
④加二抗:将HRP的山羊抗小鼠IgA抗体以1:10000的比例稀释在1×PBST中,100μL/孔,37℃静置孵育1h,弃液,洗板5次并拍干。
⑤显色:加入TMB底物显色液,50μL/孔,37℃避光静置孵育15min。
⑥终止:加入终止液,50μL/孔。5min内在酶标仪上测定OD450值,保存结果。
(4)外周血CD4+、CD8+T淋巴细胞亚群的流式细胞仪检测
通过毛细管采集免疫33d小鼠的眼球静脉丛抗凝血,分别吸取100μL采集的抗凝血到已标记好的洁净的1.5mL离心管,各管加入12μL抗体(2μL FITC-CD4、5μL APC-CD3、5μLPE-CD8),并设置空白组调流式细胞仪的电压。加入相应抗体后,轻弹离心管管壁,上下颠倒混匀,于冰上避光孵育30min。加入1.3mL红细胞裂解液,室温裂解10min。3500rpm离心10min,弃上清,轻弹离心管管壁,PBS洗涤1次,3500rpm离心5min,弃上清,加入150μL PBS重悬细胞,上机检测。
(5)脾淋巴细胞增殖反应测定
采用CCK-8法检测免疫33d的小鼠脾淋巴细胞的增殖情况,步骤如下:
①免疫33d的小鼠摘眼球处死后在75%乙醇中浸泡5min以内,进行体表消毒。
②在生物安全柜内使用无菌镊子和剪刀小心打开小鼠腹腔,无菌分离出脾脏。将小鼠脾脏放入含5mL组织稀释液的细菌培养皿中,用2mL无菌注射器吸取适量组织稀释液,吹打脾脏至呈淡粉色。再将脾脏放入细胞筛网(400目)中,研磨至只剩白色结缔组织。
③将5mL的脾脏细胞悬浮液轻柔地加入到7mL淋巴细胞分离液上方,注意配平。
④20℃,2000g(accel 0、brake 0)水平离心30min,整个过程约50min。
⑤吸取白膜淋巴细胞层至无菌的15mL离心管,加入含1%1640完全培养基的无菌PBS补足至14mL。4℃,2000g(accel 9、brake 9)水平离心20min。
⑥弃上清,加入1mL红细胞裂解液,室温裂解1-2min,PBS终止裂解,补足至14mL,4℃、2000g(accel 9、brake 9)水平离心20min。。
⑦用500μL 1640培养基重悬淋巴细胞,计数。
⑧每孔接种2.5×105个活细胞,150μL/孔,空白对照组:1640培养基(不含细胞)。
⑨阴性对照组:1640完全培养基,实验组:抗原刺激物(5μg/mL),阳性对照组:ConA(5μg/mL),各组均设置6个重复孔,50μL/孔。
⑩37℃、5%CO2条件下培养72h,加入CCK-8溶液,10μL/孔,再继续培养1-4h,读取OD450值。刺激指数(SI)=(实验组OD值-空白对照组OD值)/(阴性对照组OD值-空白对照组OD值)。
(6)细胞因子水平检测
采用深圳欣博盛公司商品化的IL-2、IL-4与IFN-γ试剂盒按照说明书对免疫后第37d的小鼠血清中细胞因子INF-γ、IL-2和IL-4进行检测,评价重组枯草芽孢杆菌在小鼠体内的免疫效果。
3.实验结果
(1)血清中IgG的ELISA检测结果
血清中的IgG抗体是动物机体二次免疫应答产生的重要抗体,故采用间接ELISA检测免疫小鼠血清中的IgG抗体水平。结果如图9所示,与PBS空白对照组相比,WB800N/pHT43阴性对照组小鼠在口服免疫后IgG抗体水平有小幅度上升,重组枯草芽孢杆菌WB800N/pHT43-VP1免疫组小鼠在加强免疫后IgG抗体水平持续升高,在三免后一周(即第30d)抗体水平达到峰值,且与阴性对照组相比有显著性差异(P<0.001),说明重组枯草芽孢杆菌WB800N/pHT43-VP1可诱导产生较高水平的抗原特异性IgG抗体。
(2)黏膜样品中sIgA的ELISA检测结果
sIgA是黏膜中含量最丰富的免疫球蛋白亚型,参与黏膜局部免疫,主要分泌于肠道黏膜表面,特别是小肠。采集不同时间段小鼠的肺洗液和肠洗液,通过间接ELISA检测免疫小鼠黏膜免疫的sIgA抗体水平,肺洗液中黏膜免疫的sIgA抗体水平结果见图10所示,肠洗液中黏膜免疫的sIgA抗体水平结果见图11所示。小鼠在灌胃免疫空载菌WB800N/pHT43后,肺洗液和肠洗液中sIgA抗体水平略升高,但与PBS空白对照组相比无显著性差异,说明枯草芽孢杆菌活载菌对小鼠的黏膜免疫应答有一定的增强作用。在加强免疫后,重组枯草芽孢杆菌WB800N/pHT43-VP1免疫组小鼠的肺洗液和肠洗液中的sIgA抗体水平随着免疫次数和时间的增加而增高,并在三免后一周(即第30d)抗体水平达到峰值。相较于肺洗液来说,肠洗液中的sIgA抗体水平含量更高,进一步说明了sIgA主要分泌于肠道黏膜表面。表明灌胃免疫重组枯草芽孢杆菌WB800N/pHT43-VP1后能有效诱导小鼠产生局部黏膜免疫应答。
(3)外周血CD4+和CD8+T淋巴细胞亚群检测结果
分离各组小鼠的外周血淋巴细胞,利用流式细胞仪检测CD4+和CD8+T淋巴细胞的百分比。每组计数10000个淋巴细胞,各组取平均值,检测结果见图12。重组枯草芽孢杆菌WB800N/pHT43-VP1免疫组的CD3+、CD4+和CD8+T淋巴细胞的百分比均显著高于WB800N/pHT43阴性对照组;且重组枯草芽孢杆菌WB800N/pHT43-VP1免疫组的CD4+/CD8+比值显著高于WB800N/pHT43阴性对照组。结果表明,通过维持正常范围的CD4+和CD8+T淋巴细胞,重组枯草芽孢杆菌WB800N/pHT43-VP1可刺激T淋巴细胞分型变化,提高小鼠的体液免疫和细胞免疫水平。
(4)脾淋巴细胞增殖试验结果
淋巴细胞在体外培养时经抗原刺激后会增殖,增殖细胞数量可以反应动物机体的细胞免疫功能,也是评价动物机体细胞免疫应答水平的一项重要指标。故在免疫后33d,采集小鼠脾脏后分离脾淋巴细胞,采用CCK-8法测定免疫后小鼠的淋巴细胞增殖能力,结果如图13所示。在ConA刺激下,各组小鼠的脾脏淋巴细胞刺激指数(SI)无显著差异,但在相应抗原的刺激下,重组枯草芽孢杆菌WB800N/pHT43-VP1免疫组小鼠的脾脏淋巴细胞刺激指数显著高于WB800N/pHT43阴性对照组及PBS空白对照组,表明重组枯草芽孢杆菌WB800N/pHT43-VP1可有效刺激脾淋巴细胞增殖。
(5)细胞因子水平检测结果
细胞因子是细胞分泌的一类能执行信号转导的蛋白活性分子,参与机体免疫应答与免疫调节,在抵抗病毒等侵袭的过程中起着重要的调节作用。本试验的细胞因子检测结果见图14。WB800N/pHT43阴性对照组血清中的细胞因子IFN-γ、IL-2和IL-4水平略高于PBS空白对照组,但无显著差异;重组枯草芽孢杆菌WB800N/pHT43-VP1免疫组血清中的IFN-γ、IL-2和IL-4水平极显著高于WB800N/pHT43阴性对照组(P<0.001)。IL-2、IFN-γ由T淋巴细胞(Th1)产生,主要介导细胞免疫反应,而IL-4由T淋巴细胞(Th2)产生,主要调节体液免疫反应。以上结果表明,重组枯草芽孢杆菌WB800N/pHT43-VP1经灌胃免疫小鼠后均能同时诱导机体产生细胞免疫和体液免疫应答。
以上所述仅为本发明的较佳实施例而已,并不用于以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院兰州兽医研究所
<120> 表达SAT2型口蹄疫病毒结构蛋白VP1的枯草芽孢杆菌及应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 648
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
actacctcgg cgggagaagg cgcagatgtt gtcaccacgg acccatcgac acacggtggg 60
aatgttcaag agggtcgacg caaacacacc gacgttgcgt tccttcttga tcgcagtaca 120
cacgttcaca caaacaagac atcttttgtt gtggacctca tggacacaag ggagaaggcg 180
ctcgtaggcg caatcctgcg agcatccacc tactactttt gtgaccttga aattgcatgt 240
gtgggcgacc acacaagggt cttctggcag cccaacgggg caccgcggac tacccagctc 300
ggcgacaacc ctatggtttt tgccaagggc ggcgtaaccc gctttgccat cccgtttacg 360
gctccacaca ggctgctgtc tactgtttac aacggcgagt gtgtctacac caaggccgcc 420
gctgccattc gtggagatcg tgcggcactt gcggcaaagt acgctgacac caaccacact 480
ttgccgccaa ccttcaactt cgggtacgtg accgttgaca aaccagtcga cgtttactac 540
cggatgaaga gggctgagct gtactgccca cgcccactgc tgccagccta caaacacaca 600
gacagagaca gattcgacgc gcccatcggc gtcgaaagac agaccctg 648
Claims (10)
1.一种表达SAT2型口蹄疫病毒结构蛋白VP1的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述重组枯草芽孢杆菌WB800N/pHT43-VP1是将编码SAT2型口蹄疫病毒结构蛋白VP1的基因或表达SAT2型口蹄疫病毒结构蛋白VP1的重组载体/质粒导入枯草芽孢杆菌WB800N获得。
2.如权利要求1所述的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述重组枯草芽孢杆菌WB800N/pHT43-VP1的制备方法为:全基因合成SAT2型口蹄疫病毒结构蛋白VP1基因;将SAT2型口蹄疫病毒结构蛋白VP1基因连接至pHT43质粒中,构建重组质粒pHT43-VP1;将重组质粒pHT43-VP1转化枯草芽孢杆菌WB800N感受态细胞获得重组枯草芽孢杆菌WB800N/pHT43-VP1。
3.如权利要求2所述的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述扩增SAT2型口蹄疫病毒结构蛋白VP1基因的引物为:
pHT43-VP1-F:atcagccgtaggatccATGACTACCTCGGCGGGAGAAGGCGCAGA,其中,小写部分为同源臂基因序列;
pHT43-VP1-R:tcattaggcgggctgcTCAATGGTGGTGATGGTGATGCAGGGTCTGTC,其中小写部分为同源臂基因序列;斜体部分为6×His标签基因序列。
4.如权利要求3所述的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述SAT2型口蹄疫病毒结构蛋白VP1的基因序列如SEQ ID NO.1所示。
5.如权利要求2所述的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述转化为电转化。
6.如权利要求5所述的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述电转化为:
(1)向枯草芽孢杆菌WB800N感受态细胞中加重组质粒pHT43-VP1,冰浴后加入电转杯,电转仪设置为2.5kv、25μF、2000Ω、持续时间4.5ms,电击1次;
(2)取出电转杯,加入复苏培养基RM,37℃、200rpm复苏3h,取200μL菌液涂布于Cm抗性(10μg/mL)的2×YT固体平板培养基上,37℃过夜培养。
7.如权利要求5所述的重组枯草芽孢杆菌WB800N/pHT43-VP1,其特征在于,所述枯草芽孢杆菌WB800N感受态细胞的制备方法为:
(1)挑取枯草芽孢杆菌WB800N单菌落接种于LB培养基中,37℃、200rpm培养18h;
(2)取步骤(1)所述过夜培养物转接至GM液中,使OD600=0.2左右,于37℃、200rpm培养至OD600=1.0左右;
(3)取步骤(2)中的全部菌液冰浴,4℃、5000rpm离心8min,弃掉上层废液,收集下层沉淀中的菌体;
(4)4℃预冷电转缓冲液ETM,每次加入40mL电转缓冲液ETM洗涤步骤(3)所述菌体,4℃、5000rpm离心8min,弃掉上层废液,保留下层沉淀,反复3次;
(5)将步骤(4)洗涤后的菌体重悬于500μL步骤(4)所述的ETM中,-80℃冻存备用。
8.如权利要求1-7任一所述的重组枯草芽孢杆菌WB800N/pHT43-VP1在制备SAT2型口蹄疫病毒疫苗中的应用。
9.如权利要求1-7任一所述的重组枯草芽孢杆菌WB800N/pHT43-VP1在制备预防或治疗SAT2型口蹄疫病毒感染药物中的应用。
10.如权利要求9所述的应用,其特征在于,所述重组枯草芽孢杆菌WB800N/pHT43-VP1加入药学上可接受的载体制成口服液、散剂、片剂、颗粒剂、胶囊剂、乳剂、混悬剂、注射剂、喷雾剂、滴剂、贴剂或软膏剂中的任一种。
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CN116555141B (zh) * | 2023-03-28 | 2023-11-03 | 青岛海华众康科技有限公司 | 一种表达猪塞内卡病毒重组蛋白的枯草芽孢杆菌及其应用 |
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