CN108794628A - 一种抗幽门螺杆菌的卵黄抗体制备方法及应用 - Google Patents
一种抗幽门螺杆菌的卵黄抗体制备方法及应用 Download PDFInfo
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- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
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Abstract
本发明公开了幽门螺杆菌5'‑甲硫腺苷核苷酶在制备抗幽门螺杆菌卵黄抗体中的应用。本发明使用幽门螺杆菌5'‑甲硫腺苷核苷酶作为抗原,制备出特异性对抗幽门螺杆菌5'‑甲硫腺苷核苷酶的卵黄抗体,可以特异性的对抗抑制幽门螺杆菌5'‑甲硫腺苷核苷酶的转化通道,阻止幽门螺杆菌的生长,从而达到有效预防和/或治疗幽门螺杆菌的感染和由幽门螺杆菌的感染所引发的相关疾病。另外,本发明所述抗幽门螺杆菌的卵黄抗体的制备利用了基因工程和蛋白重组的技术,操作简单,成本低,利用工业化大规模生产。
Description
技术领域
本发明属于生物技术领域,具体涉及幽门螺杆菌感染的预防和治疗,更具体地涉及一种抗幽门螺杆菌的卵黄抗体的制备方法及其应用。
背景技术
幽门螺杆菌(Helicobacter pylori)是科学家Barry Marshall和Robin Warren发现的,该菌能够感染胃部组织并且引发溃疡,而这两位研究者也因发现了幽门螺杆菌而获得了2005年的诺贝尔生理学及医学奖。
幽门螺杆菌,是胃溃疡和胃癌的致病因子。据估计,一半的世界人口感染上幽门螺杆菌。很多感染者产生胃炎症状,而且每5名感染者当中,就有1人极大可能患上胃溃疡。在很多情形下,这是一种病情进展快速的致命性疾病。胃炎和胃溃疡虽然可利用抗生素加以治疗,但是通常需要使用两到三种不同类型的抗生素,且并不是对所有的患者都有效。若未得到有效治疗,长期的幽门螺杆菌炎症最终会引发胃癌的产生。而胃癌是一种高度致死性、难以治愈的癌症。在全世界,胃癌每年影响着100万人,每年有超过70万人死于胃癌。目前,胃癌产生的主要原因被认为是幽门螺杆菌(Helicobacter pylori)感染,但缺乏有效治疗胃癌的方法货药物;另一方面,由于抗生素耐药性的不断扩散和增强,幽门螺杆菌的防治变得更加复杂。
近日,一项刊登在国际杂志PNAS上的研究报告中,来自能源部橡树岭国家实验室的研究人员成功对诱发胃癌的细菌的关键酶类进行了解析;研究者指出,阐明酶类的结构以及幽门螺杆菌的代谢和生物学途径可对后期开发治疗幽门螺杆菌感染的新型药物提供新的线索。研究人员对名为5'-甲硫腺苷核苷酶(HpMTAN)的幽门螺杆菌关键酶类进行了研究,发现该酶是幽门螺杆菌合成维生素K2途径(该途径能够帮助细菌进行电子转移)中的一种关键酶,而且维生素K2或许还能够加速HpMTAN酶类同其它大分子之间的相互作用。可见,HpMTAN可开发成预防和/或治疗幽门螺杆菌感染以及幽门螺杆菌感染感染引发的相关疾病的新型靶点。
发明内容
本发明的目的在于提供一种幽门螺杆菌5'-甲硫腺苷核苷酶在制备抗幽门螺杆菌卵黄抗体中的应用。采用幽门螺杆菌5'-甲硫腺苷核苷酶(HpMTAN)为蛋白质抗原,免疫产蛋家禽,并从其产出的蛋中提取卵黄抗体,将其制成疫苗或保健品、食品添加剂进行应用。可有效预防和/或治疗幽门螺杆菌的感染及由幽门螺杆菌感染所引发的相关疾病。
本发明的另一目的在于提供一种抗幽门螺杆菌的卵黄抗体的制备方法。
本发明的再一目的在于提供一种抗幽门螺杆菌的卵黄抗体。
本发明的再一目的在于提供所述抗幽门螺杆菌的卵黄抗体的应用。
本发明的再一目的在于提供一种包含有所述抗幽门螺杆菌的卵黄抗体的检测试剂盒。
本发明的上述目的是通过以下方案予以实现的:
幽门螺杆菌5'-甲硫腺苷核苷酶在制备抗幽门螺杆菌卵黄抗体中的应用。
5'-甲硫腺苷核苷酶对于幽门螺杆菌合成维生素K2途径具有重要的影响,而该途径不仅影响到幽门螺杆菌的电子转移,还会影响到HpMTAN酶类同其它大分子之间的相互作用。当幽门螺杆菌中5'-甲硫腺苷核苷酶无法正常工作,则幽门螺杆菌将受到严重的影响。
一种抗幽门螺杆菌的卵黄抗体的制备方法,所述方法包括如下过程:
S1. 将幽门螺杆菌5'-甲硫腺苷核苷酶基因连接到重组表达载体上诱导蛋白表达,并将得到的蛋白进行分离和纯化,得到幽门螺杆菌5'-甲硫腺苷核苷酶重组蛋白;
S2. 用幽门螺杆菌5'-甲硫腺苷核苷酶重组蛋白对产蛋家禽进行免疫,然后收集免疫后的家禽所产携带抗幽门螺杆菌抗体的蛋;
S3. 从携带抗幽门螺杆菌抗体的蛋的卵黄中提取、纯化抗幽门螺杆菌的抗体,并冷冻、干燥,得到抗幽门螺杆菌的卵黄抗体干粉。
利用幽门螺杆菌5'-甲硫腺苷核苷酶(HpMTAN)免疫产蛋家禽,使产蛋家禽体内产生抗体,并产下在蛋黄内含有抗体的蛋。该产品是产蛋家禽在反复摄取幽门螺杆菌5'-甲硫腺苷核苷酶(HpMTAN)抗原后,当产蛋家禽体内产生抗体时产下的蛋。所产的蛋的蛋黄中就含有抗幽门螺杆菌5'-甲硫腺苷核苷酶(HpMTAN)的抗体。该抗体可以抑制幽门螺杆菌HpMTAN的通道,阻止幽门螺杆菌的生长,减少幽门螺杆菌入侵机体天然的免疫系统和诱发相应的感染,对于胃溃疡和胃癌有预防和治疗作用。
优选地,对家禽进行注射免疫时,每次注射的剂量重组蛋白为400~500ug/只;注射次数为3~6次;每次注射间隔时间为两周。
优选地,步骤S2中对产蛋家禽进行免疫时,幽门螺杆菌5'-甲硫腺苷核苷酶重组蛋白可配以佐剂进行注射免疫。在初次注射5'-甲硫腺苷核苷酶特异性重组蛋白质抗原时,配以等体积的完全弗氏佐剂;从第二次注射开始,每次5'-甲硫腺苷核苷酶特异性重组蛋白质抗原配以等体积的不完全弗氏佐剂进行注射。
优选地,当收集的蛋中抗幽门螺杆菌抗体滴度低于104时,可对产蛋家禽进行强化免疫;所述产蛋家禽为产蛋鸡。
本发明同时还保护上述制备方法得到的抗幽门螺杆菌的卵黄抗体。
所述抗幽门螺杆菌的卵黄抗体在制备预防幽门螺杆菌感染疫苗中的应用。
所述抗幽门螺杆菌的卵黄抗体作为预防幽门螺杆菌感染的添加剂的应用。
优选地,所述添加剂可应用于药物、保健品或食品中。
本发明还保护一种包含所述抗幽门螺杆菌的卵黄抗体的检测幽门螺杆菌的试剂盒。
与现有技术相比,本发明具有以下有益效果:
本发明使用幽门螺杆菌5'-甲硫腺苷核苷酶作为抗原,制备出特异性对抗幽门螺杆菌5'-甲硫腺苷核苷酶的卵黄抗体,可以特异性的对抗抑制幽门螺杆菌5'-甲硫腺苷核苷酶的转化通道,阻止幽门螺杆菌的生长,从而达到有效预防和/或治疗幽门螺杆菌的感染和由幽门螺杆菌的感染所引发的相关疾病。另外,本发明所述抗幽门螺杆菌的卵黄抗体的制备利用了基因工程和蛋白重组的技术,操作简单,成本低,利用工业化大规模生产。
具体实施方式
下面结合具体实施例对本发明做出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1
1、幽门螺杆菌5'-甲硫腺苷核苷酶抗原的制备
(1)基因工程菌HpMTAN接种于含Amp和氯霉素(终浓度为100µg/mL)的LB液体培养基中,37℃振荡培养过夜,次日按体积百分比为1%的接种量转接,37℃培养至OD600为0.6-0.8(发酵培养的温度优选为35~37℃发酵培养),然后加入终浓度为0.1mmol/L的IPTG于16℃(IPTG诱导蛋白表达的条件优选为25℃~30℃的条件下0.1~1.0mM IPTG诱导蛋白表达)诱导表达32 h,冰浴15min,离心收集菌体;用buffer A (20 mM HEPES pH 7.5, 0.5M NaCl,5 mM β-巯基乙醇 and 25 mM咪唑)悬浮离心收集的细菌,超声破碎,条件为:200W,超声3S,间歇12S,30min。15000g离心收集上清,弃去沉淀,得到上清液;
(2)将上清液缓冲A (GE Healthcare)的5 mL HisTrap柱平衡,用5~250mm咪唑的线性梯度洗脱HpMTAN,对含有纯化蛋白的馏分进行了酶切处理,并用4℃ 1mol/L PBS缓冲液(PH=7.2)过夜透析。透析蛋白再次用过HisTrap柱(GE Healthcare),然后将含有HpMTAN的组分过Superdex 200柱得到抗原蛋白。经SDS-PAGE分析,蛋白纯度大于95%~80%。
2、抗幽门螺杆菌特异性IgY(卵黄抗体)的制备
(1)用幽门螺杆菌5'-甲硫腺苷核苷酶(HpMTAN)抗原免疫产蛋母鸡:选取20~40周龄产蛋母鸡24只;将上一步所得抗原溶液与弗氏完全佐剂按照体积比1:1混合乳化,采用皮下多点注射的免疫方式对每只母鸡注射0.8mL;首次免疫两周后进行第一次加强免疫,用等体积的弗氏不完全佐剂与幽门螺杆菌5'-甲硫腺苷核苷酶(HpMTAN)抗原混合乳化,采用皮下多点注射的免疫方式对每只母鸡注射0.8mL,以后每隔两周进行加强免疫一次,一共加强免疫5次,从第一加强免疫开始收集免疫鸡蛋,连续收集三个月的免疫鸡蛋,并存放于4℃;
(2)抗幽门螺杆菌特异性卵黄抗体的提纯:将获得的免疫鸡蛋用酒精消毒,破壳收集蛋黄,测定蛋黄体积,与4℃的冻存蒸馏水1:9混匀,调节pH为5.2,4℃保存过夜;高速冷冻离心后取上清液,用纱布过滤,测定滤液体积;在上述上清液中加入PEG6000粉末使其质量终浓度为3.5%,调pH至7.2,充分混匀,4℃,10000r/min离心20min,过滤后得滤液。滤液中加入PEG6000粉末使其质量终浓度为8.5%,调pH至7.2,充分混匀,4℃,10000r/min离心20min,弃上清得沉淀;沉淀用4℃ 1mol/L PBS缓冲液(PH=7.2)溶解后加入PEG6000粉末使其终浓度为12%,充分混匀,4℃,10000r/min离心20min,弃上清得沉淀;再用PBS溶解,转入透析袋,透析24h以上,收集透析液,在-60℃下真空冷冻干燥12h,制成了抗幽门螺杆菌卵黄抗体的干粉纯品,-20℃保存。
实施例2 抗幽门螺杆菌卵黄抗体的细菌酶标染色
取幽门螺杆菌J99(ATCC700824)作酶标染色,涂片并热固定。将实施例1制得的抗幽门螺杆菌的卵黄抗体用pH7.4、0.01mol/L的PBS分配成3个浓度(1:10、1:100、1:1000)(mg:mL)滴加于玻片上37℃作用30min后充分洗涤。滴加二抗(HRP酶标兔抗鸡IgY)(PromegaCorporation)滴加DAB显色液并显微镜观察,并用未免疫鸡的IgY(免疫前鸡蛋卵黄纯化而来)作为阴性对照。结果表明阴性对照未被染色,3个浓度(1:10、1:100、1:1000)的染色强度分别为+++、+++、+,说明抗幽门螺杆菌的卵黄抗体能和幽门螺杆菌结合,实施例1制备的抗幽门螺杆菌的卵黄抗体可以特异性识别幽门螺杆菌。
实施例3 抗幽门螺杆菌幽门螺杆菌卵黄抗体的效价测定
将实施例1制得的抗幽门螺杆菌的卵黄抗体从1:10倍比稀释至1:51200检测其效价。
ELISA间接酶联免疫实验,将实施例1制得的重组HpMTAN蛋白用包被液稀释至10µg/mL,ELISA板每孔包被100μL,用封膜覆盖96孔酶标板,37℃温育2h,然后甩干孔中液体,用PBST洗涤1次,吸水纸拍干;每孔加入100µL 30mg/mL脱脂奶粉进行封闭,置37℃温育1h,然后甩干孔中液体,用PBST洗涤3次(每次洗涤后留置4 min),用吸水纸拍干;PBST洗板后加入卵黄抗体倍比稀释液(1:160~1:10240),100µL/孔加入酶标反应板中,最后一行加入PBS作为空白对照,37℃温育1h,甩干,用PBST洗涤3次(每次洗涤后留置4min),吸水纸拍干;每孔加入l:3000的兔抗鸡IgY-HRP抗体(Promega Corporation)100 µL,置37℃温育1h。甩干,用PBST洗涤3次(每次洗涤后留置4min),然后用吸水纸拍干;每孔加入新鲜配制的底物液(10mg 3,3’,5,5’-四甲基联苯胺(TMB)溶于5mL无水乙醇,用4.5mL蒸馏水稀释,再加5mL磷酸柠檬酸缓冲液,加样前再加入10µL双氧水)100µL,置37℃温育15min,然后每孔加入50µL 2mol/L H2SO4终止反应。将酶标板置酶标仪490nm长处测OD值。吸光度OD值在对照2倍以上为阳性标准,测定抗体滴度。结果表明,本发明制备的抗幽门螺杆菌的卵黄抗体,抗体滴度可达1:5120以上,表明HpMTAN具有良好抗原性,有望成为一个新的幽门螺杆菌的疫苗分子;同时表明HpMTAN卵黄抗体对幽门螺杆菌具有良好的特异性。
实施例4 抗幽门螺杆菌的卵黄抗体致病微生物检测
根据《中国药典》2015版附录1106“非无菌产品微生物限度检查:控制菌检查法”的检验方法,对实施例1制得的抗幽门螺杆菌的卵黄抗体进行控制菌检查,未检出耐胆盐革兰阴性菌,大肠埃希菌,沙门菌,铜绿假单胞菌,金黄色葡萄球菌,梭菌和白色念珠菌。
结果证明实施例1制得的抗幽门螺杆菌的卵黄抗体安全性高,未携带其他病原菌。
实施例5 抗幽门螺杆菌的卵黄抗体禽流感病毒检测
实施例1制得的抗幽门螺杆菌的卵黄抗体用纯化水配制成1mg/mL溶液,使用禽流感病毒胶体金检测试剂盒进行检测,试验结果显示禽流感病毒阴性。
结果表明实施例1制得的抗幽门螺杆菌的卵黄抗体的安全性高,可制备成为疫苗或食品添加剂、药物添加剂进行使用。
实施例6 一种含抑制的卵黄抗体的酸奶
(1)准备原料:主要原料包括奶粉、木糖醇和抗幽门螺杆菌的卵黄抗体;
(2)配制乳液:称取奶粉125g,在奶粉中加入75℃,1000mL温开水配制成乳液;
(3)均质:将步骤(2)中的乳液在均质机上均质2min,均质机的压力保持在10MPa;
(4)灭菌:将步骤(3)中均质后的乳液在90℃的水浴锅中保温10min进行杀菌处理,然后将其冷却至40℃,得到料液;
(5)接种:在无菌条件下,加入无菌的抑制HpMTAN的卵黄抗体10mg,另将发酵剂,按照1g/1000mL的比例接种到料液中,并将其搅拌均匀,得到接种料液;
(6)发酵:在无菌条件下,将接种料液等量灌装于灭菌的玻璃瓶中并封口,然后将封口好的玻璃瓶放置于42℃恒温培养箱中发酵5h;
(7)后发酵:将步骤(6)中发酵后的玻璃瓶放入1℃-4℃的冰箱中进行后发酵,后发酵至少保持24h,得到含抗幽门螺杆菌的卵黄抗体的酸奶。
实施例7
每100g奶粉中加入10g含抗抗幽门螺杆菌的卵黄抗体的蛋黄粉或1g含抗幽门螺杆菌的卵黄抗体纯化的IgY冻干粉,所得产品可用于婴幼儿和成人预防HP感染,或已感染HP患者的辅助治疗。
实施例8
每100mL牛奶中加入1g含抗幽门螺杆菌的卵黄抗体的纯化IgY冻干粉,所得产品可用于婴幼儿和成人预防HP感染,或已感染HP患者的辅助治疗。
实施例9
抗幽门螺杆菌的卵黄抗体的纯化IgY冻干粉与猴头菇多糖按1:5比例混合,灌装胶囊,可用于保护胃黏膜的保健食品。
实施例10
抗幽门螺杆菌的卵黄抗体的纯化IgY冻干粉添加制剂辅料,制成口服液,颗粒剂片剂或胶囊,可用于治疗因HP感染引起的慢性胃炎、十二指肠溃疡等疾病;也可用于预防HP的感染。
实施例1所制备得到的抗幽门螺杆菌的纯化IgY还可以作为添加剂在其他药物、保健性药品以及各种其他食物中的应用,用以预防幽门螺杆菌感染引发的相关疾病。例如,可将实施例1所制备得到的抗幽门螺杆菌的纯化IgY,可一定比例的溶于漱口水中,预防和治疗由幽门螺杆菌所引起的口臭,等等。另外还可以将实施例1所制备得到的抗幽门螺杆菌的卵黄抗体用于制备幽门螺杆菌的检测试剂盒中,用于快速检测幽门螺杆菌。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,对于本领域的普通技术人员来说,在上述说明及思路的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举。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,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.幽门螺杆菌5'-甲硫腺苷核苷酶在制备抗幽门螺杆菌卵黄抗体中的应用。
2.一种抗幽门螺杆菌的卵黄抗体的制备方法,其特征在于,所述方法包括如下过程:
S1. 采用基因工程重组方法制备5'-甲硫腺苷核苷酶特异性重组蛋白质抗原;
S2. 用5'-甲硫腺苷核苷酶特异性重组蛋白质抗原对产蛋家禽进行免疫,然后收集免疫后的家禽所产的蛋;
S3. 从收集的蛋的卵黄中提取、纯化抗幽门螺杆菌的抗体,并冷冻、干燥,得到抗幽门螺杆菌的卵黄抗体干粉。
3. 根据权利要求2所述抗幽门螺杆菌的卵黄抗体的制备方法,其特征在于,步骤S2中对产蛋家禽进行免疫时,幽门螺杆菌5'-甲硫腺苷核苷酶重组蛋白可配以佐剂进行注射免疫;对家禽进行注射免疫时,每次注射的剂量重组蛋白为400~500ug /只;注射次数为4~6次;每次注射间隔时间为两周。
4.根据权利要求2所述抗幽门螺杆菌的卵黄抗体的制备方法,其特征在于,步骤S1中的重组表达载体系统为pET32b(+)-HpMTAN。
5.根据权利要求2所述抗幽门螺杆菌的卵黄抗体的制备方法,其特征在于,当收集的蛋中抗幽门螺杆菌抗体滴度低于104时,可对产蛋家禽进行强化免疫;所述产蛋家禽为20~40周龄的健康产蛋鸡。
6.一种权利要求2~5任一所述制备方法得到的抗幽门螺杆菌的卵黄抗体。
7.权利要求6所述抗幽门螺杆菌的卵黄抗体在制备预防幽门螺杆菌感染疫苗中的应用。
8.权利要求6所述抗幽门螺杆菌的卵黄抗体作为预防幽门螺杆菌感染的添加剂的应用。
9.根据权利要求8所述应用,其特征在于,所述添加剂可应用于药物、保健品或食品中。
10.一种检测幽门螺杆菌的试剂盒,其特征在于,所述试剂盒包含有权利要求6所述抗幽门螺杆菌的卵黄抗体。
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