CN116496406A - 激活tlr-5的幽门螺旋杆菌融合抗原及其应用 - Google Patents
激活tlr-5的幽门螺旋杆菌融合抗原及其应用 Download PDFInfo
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Abstract
本发明涉及一种激活TLR‑5的幽门螺旋杆菌融合抗原及其应用，利用该融合抗原可产生能够阻止幽门螺旋杆菌自由运动的鞭毛抗体，还可通过激活TLR‑5信号通路，增强了B细胞产生抗体的能力，提高抗体效价。
Description
技术领域
本发明涉及一种激活TLR-5的幽门螺旋杆菌融合抗原及其应用。
背景技术
幽门螺旋杆菌（Helicobacter pylori，Hp）是一种伴随人类进化的革兰阴性细菌。目前，治疗幽门螺旋杆菌感染是联合应用抗生素。但是，即使用抗生素清除感染者胃内的幽门螺旋杆菌后，还会再次感染。反复感染，反复用药，将导致肠道菌群失调，还容易引起细菌耐药，使治疗效果和清除率显著降低。如果所有患者都服用抗生素治疗，大量患者排泄出的抗生素也容易导致环境其它耐药菌株的出现。为了解决上述问题，前期我们用抗体牛乳治疗幽门螺旋杆菌感染，通过临床试验，取得了较好的清除效果。
TLRs（Toll like receptors，TLRs）是迄今认识的第一类，并且是被认为最重要的天然免疫识别受体，能够识别各种与免疫相关的微生物配体，被称为识别病原体“感受器”。TLRs在天然免疫防御中起重要作用，同时也能调节适应性免疫，是连接天然免疫和适应性免疫的桥梁。目前发现的TLRs成员有TLR1-11，表达于除骨髓细胞以外的多种类型的细胞。TLRs可通过激活NF-κB途径上调DC和B细胞的MHC-I/II、CD80和CD86分子的表达，提高了DC和B细胞的抗原提呈能力，从而增强了激活CD4+Th的能力，激活的CD4+Th反过来又可以增强B细胞分泌产生抗体的能力。此外，TLRs的信号通路的激活可上调滤泡B细胞的BCR信号通路和滤泡辅助T细胞（Tfh）CD40信号通路的激活，增强了生发中心的B细胞进一步分化为长寿浆细胞或记忆B细胞的能力。由于浆细胞也表达大量TLRs，浆细胞TLRs信号通路的激活也可增强其产生抗体的能力。因此，为了提升疫苗的免疫效果，先进的疫苗设计需要充分考虑TLRs的作用。
幽门螺旋杆菌拥有4~8条单极鞭毛，运动活泼，可帮助细菌迅速穿过黏稠的黏液层到达黏膜表面并在粘液层运动，同时帮助细菌逃避胃酸的杀菌作用。抗幽门螺旋杆菌鞭毛蛋白抗体可以阻止细菌自由运动，细菌因此不能逃避胃酸的杀菌作用。幽门螺旋杆菌的鞭毛蛋白可被TLR-5所识别，由于人B细胞不表达TLR-5，由此降低了人体对幽门螺旋杆菌的免疫清除作用，是幽门螺旋杆菌能在人体胃内持续存在的原因之一。
发明内容
本发明的目的是提供一种可阻止幽门螺旋杆菌自由运动的鞭毛抗体，通过激活TLR-5信号通路，增强了B细胞产生抗体的能力，提高抗体效价的激活TLR-5的幽门螺旋杆菌融合抗原及其应用。
本发明采用如下技术方案：
一种激活TLR-5的幽门螺旋杆菌融合抗原，其包括如SEQ ID No.1所示氨基酸序列。
一种编码上述激活TLR-5的幽门螺旋杆菌融合抗原的核酸分子，其包括如SEQ IDNo.7所示的核苷酸序列。
一种包含上述核酸分子的载体。
一种包含上述核酸分子的基因工程菌。
一种利用激活TLR-5的幽门螺旋杆菌融合抗原制备的多克隆抗体。
一种利用激活TLR-5的幽门螺旋杆菌融合抗原制备的疫苗。
一种激活TLR-5的幽门螺旋杆菌融合抗原在制备刺激奶牛的免疫应答反应的奶牛疫苗中的应用。
一种利用激活TLR-5的幽门螺旋杆菌融合抗原制备的免疫牛乳。
本发明的有益效果在于：本发明制备出能提高牛奶抗体效价的多价抗原，利用本发明的融合抗原免疫奶牛后，不但获得了鞭毛抗体，阻止了幽门螺旋杆菌的自由运动，还能通过激活TLR-5信号通路，增强了B细胞产生抗体的能力，抗体的效价提高了1.2~1.6倍，为大规模生产抗体用于临床提供了技术保障，降低了生产成本和医疗成本。
附图说明
图1为蛋白总离子一级、二级流色谱图。
图2为融合蛋白His标签图。
图3为融合蛋白质谱图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
一、融合多肽的表达
1、通过蛋白组学分析临床分离株外膜蛋白的表达，筛选出高表达外膜蛋白，如图1所示。
2、融合多肽表达载体的构建
2.1 通过NetMHCpan -4.1软件、ABCpred软件和DNAstar软件，并结合牛的BoLA-IIa基因，从高表达外膜蛋白种筛选出4个评分最高的氨基酸序列（如SEQ ID No.3~6所示）与鞭毛蛋白序列（如SEQ ID No.2所示）进行人工基因融合，该人工融合基因片段两端分别带有EcoRI（GAATTC）-XbaI（TCTAGA）酶切位点，其核苷酸序列如SEQ ID No.7所示，对应的氨基酸序列如SEQ ID No.1所示。
2.2 酶切空骨架载体pPiczaA表达载体，对载体酶切产物进行琼脂糖凝胶电泳，回收目的条带。
酶切体系：
10×buffer 2μL
EcoRI 1μL
XbaI 1μL
Plasmid 2~3μL
Add ddH2O to 20μL。
2.3 合成基因与表达载体的连接
得到骨架载体回收产物后，根据同源重组的原理，利用无缝拼接试剂盒将合成基因克隆到pPiczaA载体中，克隆位点：EcoRI（GAATTC）-XbaI（TCTAGA）。
2.4 转化涂板及菌斑鉴定
无缝拼接后产物5~10μL转化至100μL DH5a感受态，42℃金属浴，热激1min，冰上迅速预冷2min，在超净工作台中，加入600μL无抗培养基，37℃摇床振荡培养1h，取适量菌液涂布在含有Zeocin+抗性的平板上，在恒温培养箱中倒置培养12~16 h。菌落PCR后鉴定阳性克隆。
2.5 阳性克隆摇菌及质粒提取
挑选3~4个单菌落摇菌，加入含有Zeocin+的培养基摇菌过夜（8mL LB液体培养基），然后参照质粒抽提试剂盒进行质粒抽提。
3、制备融合多肽
3.1 转化
（1）将回收的质粒用 SacI 进行线性化。
（2）将线性化后的质粒电转至酵母感受态细胞X33中，涂YPDS（含100μg/mL Zeocin+）平板，30℃倒置培养约2~3天。
3.2 菌落PCR鉴定
待单克隆菌落形成，利用PCR技术检测单克隆。
3.3 小量表达测试
（1）接种：挑选10个PCR验证为阳性克隆菌落接种至BMGY培养基中，28.5℃培养至OD600=2~6。
（2）诱导：更换 BMMY培养基进行诱导（1%甲醇），并将重悬后的菌液放至28.5℃培养70h后检测。
（3）样品制备：取100μL培养物12000rpm离心5min后，取80μL上清于1.5mL离心管，加入20μL 5×Loading Buffer，沸水浴10min。
3.4 捕捉His标签
（1）SDS-PAGE，将胶样品一分为二（一半用于曝光捕捉His标签，一半用于质谱鉴定）；
（2）一半转膜；
（3）将转膜结束后的PVDF膜放在封闭液中，在摇床上封闭1个小时；
（4）用PBST洗去封闭液（3次，每次5-10min），孵育一抗（1∶3000），4℃摇晃孵育过夜；
（5）第二天将孵育好的PVDF膜用PBST洗3遍，每次5-10min；
（6）孵育二抗（1∶1000），避光孵育1个小时（温和震荡），PBST洗3次，避光；
（7）曝光，看是否含有His标签（结果如图2所示）。
3.5 质谱鉴定
（1）将另一半胶烤染，放在摇床上染色半小时；
（2）脱色，每隔1h换1次脱色液，然后4℃孵育过夜；
（3）第二天用手术刀片切下胶上目标条带，切成1mm的小块，置于1.5mL EP管中；
（4）每管加入300μL色液室温脱色，清洗至透明，去除上清；每1~2h建议更换一次脱色液以加快脱色进程；根据不同情况，脱色一般需要4~8h；
（5）干胶：加入300μL 100%ACN ThermoMixer震荡5min至胶粒变白，吸去ACN，冻干3min；
（6）每管加入300μL 10mM DTT/50mM NH4HCO3，振荡混匀至胶块泡胀透明，56℃，1h，随后弃上清；
（7）干胶：加入300μL 100%ACN ThermoMixer震荡5min至胶粒变白，吸去ACN，冻干3min；
（8）每管加入300μL 60mM IAA/50mM NH4HCO3，避光振荡混匀至胶块泡胀透明，暗处反应30min；
（9）干胶：加入300μL 100%ACN ThermoMixer震荡5min至胶粒变白，吸去ACN，冻干3min；
（10）每管加入50mM碳酸氢铵溶液50~80μL，再加入1~2μg胰酶（或者将两者混合后再加入样品中，淹没胶条），用玻璃棒将凝胶挤碎，37℃温育6h以上；
（11）每管加入200μL含0.1% FA乙腈震荡5分钟，吸取上清液至干净的EP管中；
（12）凝胶中再加入 30μL 0.1%FA震荡 5分钟，再加入 200μL含0.1% FA 乙腈震荡5分钟，吸取上清，并将两次上清液合并，冻干3小时以上；
（13）除盐
甲醇活化：200μL甲醇，离心室温1200g，5min，弃流出液；
Buffer B：200μL，2次，4000g，2min
Buffer A：200μL，3次，6000g，2min
将样品稀释至200μL，加入到除盐小柱中，2000g，5min；
Buffer A：200μL，3次，6000g，2min
Buffer B：180μL，2次离心，2000g，2min；4000g，2min。
将两次洗脱液放置新的EP管中，冻干。
3.6 上质谱，结果如图3所示。
二、多克隆抗体牛乳的制备
1、融合多肽疫苗的制备
（1）接种：把阳性克隆X33工程菌接种至BMGY培养基中，28.5℃培养至OD600≥6。
（2）诱导：更换 BMMY培养基进行诱导（1%甲醇），并将重悬后的菌液放至28.5℃培养70h后检测。
（3）取培养物，12000rpm离心5min后，取上清。
2、蛋白纯化仪器纯化融合多肽
（1）先将上清用0.22微米的滤器过滤，然后将蛋白浓度稀释到1mg/mL；
（2）连接纯化柱，先用超纯水洗泵，3~5柱体积；
（3）用结合缓冲液洗3~5个柱体积；
（4）上样10个柱体积，然后用结合缓冲液再洗5~10个柱体积；
（5）用洗脱液洗，一旦见到起峰，立即暂停洗脱2min，让其充分反应，然后开始收集；
（6）收集完成后，将柱子拆卸下来用结合缓冲液冲洗5~10个柱体积，然后用20%的乙醇冲洗；
（7）收集好的融合多肽疫苗放入-80℃冰箱保存，备用。
3、融合多肽免疫奶牛
选择体质健康的3~4周岁的孕牛，分别于分娩前63d、42d、7d及分娩后21d免疫，总计4次；首次用6mg TLR-5融合多肽+2mg钥孔血蓝蛋白+氢氧化铝佐剂配制成4mL，于奶牛颈部两侧肌肉注射；后三次用3mg TLR-5融合多肽+1mg钥孔血蓝蛋白+氢氧化铝佐剂配制成4mL免疫。根据抗体效价检测结果再进一步做加强免疫。对照组用无TLR-5融合多肽进行免疫（制备过程同前）；首次用6mg无TLR-5融合多肽+2mg钥孔血蓝蛋白+氢氧化铝佐剂配制成4mL，于奶牛颈部两侧肌肉注射；后三次用3mg无TLR-5融合多肽+1mg钥孔血蓝蛋白+氢氧化铝佐剂配制成4mL免疫。阴性对照为PBS+钥孔血蓝蛋白+氢氧化铝佐剂配制。
4、ELISA检测牛乳抗体效价
（1）乳清的制备
把牛奶样品放入50mL圆底离心管中，44000g，4℃离心30分钟, 去除脂肪层；然后取脱脂牛奶放入新的离心管中，44000g离心30分钟，4℃；收集清亮乳清，在-20℃等分储存备用。
（2）间接ELISA检测抗体效价
先用2.5%戊二醛溶液150μL/孔预处理96孔板1小时，37℃，超纯水洗涤4次，每次甩净拍干；根据SEQ ID No.3~6所示氨基酸序列，人工合成多肽（每条10mg）；然后用合成多肽和表达的融合多肽100μL（0.05mg/mL）分别包被酶标板，37℃至干燥，用PBST（含0.5ml/LTweem 20的PBS缓冲液）洗涤液洗板3次，每次3min，每次甩净拍干；用3%BSA的PBST封闭1小时，洗涤同上；加不同稀释度的乳清，100μL/孔，每样3个复孔，37℃温育1小时；洗涤3次后加入兔抗牛IgG-HRP（1:8000），100μL/孔，37℃温育1小时；洗涤3次后加四甲基联苯胺底物工作液100µl，37℃反应15分钟后每孔加50μL H2SO4（2mol/L）终止反应，立即在酶标仪上测450nm 波长的吸光度值，以OD450 0.5的最大稀释倍数为抗体效价（是阴性孔OD值的2.1倍以上）。TLR-5融合多肽组抗体总效价（1:1600）是对照组（1:1200）的1.3倍（结果见表1）。
表1 TLR-5组与无TLR-5组的抗体效价比较
。
5、收集和消毒牛乳
收集抗体效价达到和稳定在1:1200以上的牛乳；在62.8~65.6℃，30min或者71.7℃，15s消毒牛乳。
6、低温喷雾干燥成奶粉
离心脱脂后用低温冷冻喷雾机干燥成奶粉。
Claims (8)
1.一种激活TLR-5的幽门螺旋杆菌融合抗原，其特征在于，其包括如SEQ ID No.1所示氨基酸序列。
2.一种编码如权利要求1所述的激活TLR-5的幽门螺旋杆菌融合抗原的核酸分子，其特征在于，其包括如SEQ ID No.7所示的核苷酸序列。
3.一种包含如权利要求2所述的核酸分子的载体。
4.一种包含如权利要求2所述的核酸分子的基因工程菌。
5.一种利用如权利要求1所述的激活TLR-5的幽门螺旋杆菌融合抗原制备的多克隆抗体。
6.一种利用如权利要求1所述的激活TLR-5的幽门螺旋杆菌融合抗原制备的疫苗。
7.一种如权利要求1所述的激活TLR-5的幽门螺旋杆菌融合抗原在制备刺激奶牛的免疫应答反应的奶牛疫苗中的应用。
8.一种利用如权利要求1所述的激活TLR-5的幽门螺旋杆菌融合抗原制备的免疫牛乳。
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