CN114409804A - 一种大肠杆菌肠毒素多表位融合蛋白及其制备方法和应用 - Google Patents
一种大肠杆菌肠毒素多表位融合蛋白及其制备方法和应用 Download PDFInfo
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- CN114409804A CN114409804A CN202210019708.6A CN202210019708A CN114409804A CN 114409804 A CN114409804 A CN 114409804A CN 202210019708 A CN202210019708 A CN 202210019708A CN 114409804 A CN114409804 A CN 114409804A
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Abstract
本发明涉及基因工程技术领域，公开了一种大肠杆菌肠毒素多表位融合蛋白及其制备方法和应用，本发明以产肠毒素大肠杆菌不耐热肠毒素A(LTa)作为骨架蛋白，将产肠毒素大肠杆菌耐热肠毒素A(STa)和B(STb)的中和表位替换LTa免疫原性较弱的表位，通过融合表达制备重组蛋白；所得重组蛋白能够高效表达，且可以将STa和STb的表位展示出来，有效解决了因STa和STb分子量较小导致的免疫原性较弱、表达量差等问题。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及了一种大肠杆菌肠毒素多表位融合蛋白及其制备方法和应用。
背景技术
产肠毒素大肠杆菌(ETEC)是引起仔猪腹泻的主要病原菌。据报道，全世界每年因ETEC造成仔猪死亡约占一半以上，给生猪养殖业造成巨大的损失。ETEC主要的致病因素是其产生的肠毒素，包括耐热型肠毒素A(STa)、耐热型肠毒素B(STb)、不耐热型肠毒素A(LTa)和不耐热型肠毒素B(LTb)。其中，耐热型肠毒素具有较强的致病性，可使仔猪肠道黏膜萎缩，脱落，诱发炎症，导致仔猪腹泻，进而死亡。除此之外，STa和STb还能够污染水源、食物等，有研究表明ETEC是发展中国家幼儿死亡的重要原因之一，也是人类公共卫生的一个重要危险。然而，由于STa和STb分子量较小，免疫原性较弱，表达量差，毒性强等问题，目前市场上，还未开发出针对STa和STb的有效的疫苗或中和活性的抗体。研究如何能够展示STa和STb抗原表位，已经成为一个研究热点。
多表位融合抗原是一种基于抗原结构和表位的疫苗技术，为多价疫苗的开发提供了新的平台。通过模拟抗原的天然表位并呈递多个异源中和表位，多表位融合抗原允许单个免疫原(蛋白质)携带来自多种毒力决定簇的多种抗原性元素(表位或肽)，从而具有广泛的免疫原性，可以广泛开发免疫原性多价疫苗。
发明内容
有鉴于此，本发明利用LTa作为骨架蛋白，将STa和STb中和表位展示在LTa上得到重组蛋白，能有效解决STa和STb分子量较小，免疫原性较弱，表达量差，毒性强等问题，并有望改善仔猪腹泻频发、死亡率高、疫苗保护效率差等现状。
本发明的技术方案具体如下：
本发明第一方面提供了一种大肠杆菌肠毒素多表位融合蛋白，具体为如下任一：
(i).氨基酸序列为SEQ ID NO:1的蛋白质；
(ii).将SEQ ID NO:1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
(iii).在(i)或(ii)所述蛋白质的N端和/或C端连接标签后得到的蛋白质。
本发明第二方面提供了制备上述大肠杆菌肠毒素多表位融合蛋白的方法，包括以下步骤：
S1.克隆LTa基因，筛选LTa免疫原性较弱的表位，克隆STa的中和表位和STb的中和表位，将所述STa和STb的中和表位替换LTa免疫原性较弱的表位；
S2.将替换后的基因克隆在载体上，构建重组载体，转入宿主菌株中进行诱导表达；
S3.提取纯化目的蛋白。
优选地，所述LTa免疫原性较弱的表位的氨基酸序列如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4和SEQ ID NO:5所示；所述STa中和表位的氨基酸序列如SEQ ID NO:6所示，克隆所述STa中和表位所用的引物序列如SEQ ID NO:10和SEQ ID NO:11所示；所述STb中和表位的氨基酸序列SEQ ID NO:7所示，克隆所述STb中和表位所用的引物序列如SEQ ID NO:12和SEQ ID NO:13所示。
优选地，所述载体为PET质粒，包括但不限于：PET28a、PET30a、PET32a。
优选地，所述宿主菌株为大肠杆菌，包括但不限于：BL21、DH5α、JM109。
优选地，所述诱导条件为：0.1～1.0mmol/LIPTG，16℃～37℃。
优选地，所述步骤S3具体为：离心收集菌体细胞，破碎菌体收集包涵体，在变性缓冲液中裂解包涵体，再使用复性缓冲溶液透析。
更加优选地，所述变性缓冲液的组成为：15～25mM PB缓冲液(pH 7.2～7.6)、0.4～0.6M NaCl和7～9M尿素；所述复性缓冲液的组成为：15～25mM PB缓冲液(pH 8.0～9.0)、4～6％甘油、0.04～0.06mM氧化型谷胱甘肽、0.4～0.6mM还原型谷胱甘肽和尿素，所述尿素为6M、4M、2M、1M或0M。
更加优选地，所述透析过程为：将纯化的变性目的蛋白装入透析袋，连续用含有6、4、2、1和0mol/L尿素的复性缓冲液在4℃分步缓慢透析，其中最后两步不加甘油，每步透析12～24h。
本发明第三方面请求保护编码上述大肠杆菌肠毒素多表位融合蛋白的核酸分子。
本发明第四方面请求保护含有上述核酸分子的重组载体，包括但不限于：重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
本发明第五方面请求保护包含有上述大肠杆菌肠毒素多表位融合蛋白的疫苗。
本发明第六方面请求保护上述大肠杆菌肠毒素多表位融合蛋白在制备产肠毒素大肠杆菌LTa、STa和STb毒素的抗体中的应用。
本发明的有益效果为：本发明提供的多表位融合蛋白以不耐热型肠毒素A(LTa)作为骨架蛋白，且其兼具佐剂功能，很好地将STa和STb的中和表位呈现出来；融合蛋白特异性好、能够明显提升传统方法的效率，对解决ETEC临床常见多血清问题具有重要的意义。
附图说明
图1为实施例1制备的融合蛋白的Western blotting检测图，其中，A图是验证STa表位的表达结果，B图为验证STb表位的表达结果，泳道M是蛋白Marker；泳道1是LTa-STa-STb重组蛋白；泳道2是LTa重组蛋白。
具体实施方式
为了更好的理解本发明，下面结合具体实施例进一步阐明本发明的内容，但以下实施例用于说明本发明，但不用来限制本发明的范围。
若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW，Molecular Cloning：a Laboratory Manual，2001)，或按照制造厂商说明书建议的条件。
实施例1
1)肠毒素LTa菌毛基因克隆及筛选其免疫原性较弱表位
将临床分离到的产肠毒素大肠杆菌菌株作为菌液模板，利用PCR克隆其菌毛LTa基因。
PCR反应体系如下：
反应程序如下：
PCR引物序列如下：
F-LTa：5’-CGAATTCGCTGATTGGACGGAAGGTC-3’(SEQ ID NO:8)；
R-LTa：5’-CCTCGAGACTATAAATAACGGTGATAG-3’(SEQ ID NO:9)。
2)STa和STb的中和表位克隆
反应体系如下：
反应程序如下：
PCR引物序列如下：
F-STa：5’-CTGTATTATCTCCCCTCTTTTAGC-3’(SEQ ID NO:10)；
R-STa：5’-ATAACATGGAGCACAGGCAGG-3’(SEQ ID NO:11)；
F-STb：5’-ATGAAAAAGAATATCGCATTTC-3’(SEQ ID NO:12)；
R-STb：5’-GCATCCTTTTGCTGCAACC-3’(SEQ ID NO:13)。
3)构件重组质粒pET28a-LTa-STa-STb
通过载体和目的基因进行双酶切，体系如下：
酶切条件：37℃，水浴酶切3h。
分别将双酶切的产物进行纯化回收，利用T4连接酶进行连接，体系如下：
连接条件：混匀后16℃连接过夜，转入大肠杆菌感BL21(DE3)受态细胞中。
4)LTa-STa-STb融合蛋白的表达与纯化
提取阳性单克隆质粒，化转至大肠杆菌BL21(DE3)感受态细胞中，复苏后涂布于固体培养基过夜培养。次日，挑取单个克隆于SB-羧苄培养基中培养，加入IPTG(0.4mmol/L、25℃)诱导过夜表达；次日，离心收集菌体细胞，用超声破碎仪裂解细胞，收集沉淀，利用变性缓冲溶液、复性缓冲溶液得到高纯度的多表位融合蛋白；在本实施例中，变性缓冲溶液的组成为：20mM PB缓冲液(pH 7.4)、0.5M NaCl和8M尿素，复性缓冲溶液的组成为20mM pH8.0PB缓冲液、5％甘油、0.05mM氧化型谷胱甘肽、0.5mM还原型谷胱甘肽和尿素(6、4、2、1、0M)。经氨基酸测序分析，所得融合蛋白的氨基酸序列如SEQ ID NO:1所示。
5)利用Western Blotting验证LTa展示STa与STb中和表位的结果
将融合蛋白经SDS-PAGE分离后，使用双蒸水轻轻地漂洗凝胶，然后将胶块转移至电泳缓冲液中。将PVDF膜置于甲醇(100％)中浸泡数分钟，至下而上，分别放置海绵、滤纸、PVDF膜、胶块、滤纸、海绵；将组装好的胶块架子置于已经放置在电泳仪的内槽中，将电泳仪置于盛有冰水混合物的容器中准备进行电转，转移参数：100V，60min左右。待转膜完成后，将凝胶块转移至盛有凝胶染色的托盘中；向盛有PVDF膜容器中，加入封闭缓冲液(5％脱脂乳)使其能够没过膜，且不含有气泡，摇床上室温孵育1h。将电转后仪器、海绵、滤纸等清洗后，放置空闲地方，晾干；弃去封闭液，使用TBST将PVDF膜摇床上洗涤3次，每次大约10min，加入使用5％脱脂乳稀释的鼠抗大肠杆菌耐热肠毒素(1:2500)，排除气泡后平放于摇床，室温孵育1～2h；使用离心管将一抗收集起来以备下次使用，用TBST漂洗PVDF膜3次，每次大约10min，将多余或非特异性结合的抗体洗掉；再加入山羊抗鼠的二抗(1:10,000)室温孵育1h，然后再采用上述洗涤方式进行洗涤后，弃去TBST漂洗液，同时将PVDF膜转移较小的容器中，加入TBST溶液，室温洗涤；使用ECL发光液进行显色。
鼠抗产肠毒素大肠杆菌不耐热毒素STa和STb惠赠于扬州大学兽医学院，段强德老师。
酶标记的山羊抗小鼠的二抗体，购自Sigam-Aldrich公司，商品编号：AP308P。
ECL发光液，为HRP底物发光液，购自Sigam-Aldrich公司，商品编号：WBULS0100。
检测结果如图1所示，从实验结果可知所选STa和STb表位能够嵌合在LTa骨架蛋白上。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
SEQUENCE LISTING
<110> 湖北神地生物科技有限公司
<120> 一种肠杆菌肠毒素多表位融合蛋白及其制备方法和应用
<130> 2022.1.4
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<170> PatentIn version 3.5
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<212> PRT
<213> LTa-STa-STb融合蛋白的氨基酸序列
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Met Lys Asn Ile Thr Phe Ile Phe Phe Ile Leu Leu Ala Ser Pro Leu
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Tyr Ala Asn Gly Asp Lys Leu Tyr Cys Cys Glu Leu Cys Cys Asn Pro
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Ala Cys Ala Gly Cys Tyr Gly Leu Met Pro Arg Gly His Asn Glu Tyr
35 40 45
Phe Asp Arg Gly Thr Gln Met Asn Ile Cys Cys Glu Leu Cys Cys Asn
50 55 60
Pro Ala Cys Ala Gly Cys Tyr Arg Tyr Asp Asp Gly Tyr Val Lys Lys
65 70 75 80
Asp Leu Cys Glu His Tyr Ala His Leu Ala Gly Gln Ser Ile Leu Ser
85 90 95
Gly Tyr Ser Thr Tyr Tyr Ile Tyr Val Ile Ala Thr Ala Pro Asn Met
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Phe Asn Val Asn Asp Val Leu Gly Val Tyr Lys Lys Asp Leu Cys Glu
115 120 125
His Tyr Val Ser Ala Leu Gly Gly Ile Pro Tyr Ser Gln Ile Tyr Gly
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Trp Tyr Arg Val Asn Phe Gly Val Ile Asp Glu Arg Leu His Arg Asn
145 150 155 160
Arg Glu Tyr Arg Asp Arg Tyr Tyr Arg Asn Leu Asn Ile Ala Pro Ala
165 170 175
Glu Asp Gly Tyr Arg Leu Ala Gly Phe Pro Pro Asp His Gln Ala Trp
180 185 190
Arg Glu Glu Pro Trp Ile His His Ala Pro Gln Gly Cys Gly Asn Ser
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Ser Arg Thr Ile Thr Gly Asp Thr Cys Asn Glu Glu Thr Gln Asn Leu
210 215 220
Ser Thr Ile Tyr Leu Arg Lys Tyr Gln Ser Lys Val Lys Arg Gln Ile
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Phe Ser Asp Tyr Gln Ser Glu Val Asp Ile Tyr Asn Arg Ile Arg Asn
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Glu Leu
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<213> 人工序列
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Arg Ala Asp Ser Arg Pro Pro Asp Glu Ile Lys Arg Ser Gly
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Ser Thr Ser Leu Ser Leu Arg Ser
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Ser Pro His Pro Tyr Glu Gln Glu
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Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr
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Lys Lys Asp Leu Cys Glu His Tyr
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Claims (10)
1.一种大肠杆菌肠毒素多表位融合蛋白，其特征在于，为如下任一：
(i).氨基酸序列为SEQ ID NO:1的蛋白质；
(ii).将SEQ ID NO:1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的蛋白质；
(iii).在(i)或(ii)所述蛋白质的N端和/或C端连接标签后得到的蛋白质。
2.编码如权利要求1所述大肠杆菌肠毒素多表位融合蛋白的核酸。
3.包含权利要求2所述核酸的重组载体。
4.一种包含权利要求1所述的大肠杆菌肠毒素多表位融合蛋白的疫苗。
5.权利要求1所述的大肠杆菌肠毒素多表位融合蛋白在制备产肠毒素大肠杆菌LTa、STa和STb毒素的抗体中的应用。
6.一种制备权利要求1所述大肠杆菌肠毒素多表位融合蛋白的方法，其特征在于，包括如下步骤：
S1.克隆LTa基因，筛选LTa免疫原性较弱的表位，克隆STa的中和表位和STb的中和表位，将所述STa和STb的中和表位替换LTa免疫原性较弱的表位；
S2.将替换后的基因克隆在载体上，构建重组载体，转入宿主菌株中进行诱导表达；
S3.提取纯化目的蛋白。
7.根据权利要求6所述制备大肠杆菌肠毒素多表位融合蛋白的方法，其特征在于，所述LTa免疫原性较弱的表位的氨基酸序列如SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4和SEQID NO:5所示；所述STa的中和表位的氨基酸序列如SEQ ID NO:6所示，克隆所述STa的中和表位所用的引物序列如SEQ ID NO:10和SEQ ID NO:11所示；所述STb的中和表位的氨基酸序列SEQ ID NO:7所示，克隆所述STb的中和表位所用的引物序列如SEQ ID NO:12和SEQ IDNO:13所示。
8.根据权利要求6所述制备大肠杆菌肠毒素多表位融合蛋白的方法，其特征在于，所述宿主菌株为大肠杆菌，所述诱导的条件为：0.1～1.0mmol/LIPTG、16℃～37℃。
9.根据权利要求6所述制备大肠杆菌肠毒素多表位融合蛋白的方法，其特征在于，步骤S3具体为：离心收集菌体细胞，破碎菌体收集包涵体，在变性缓冲液中裂解包涵体，再使用复性缓冲溶液透析。
10.根据权利要求9所述制备大肠杆菌肠毒素多表位融合蛋白的方法，其特征在于，所述变性缓冲液的组成为：15～25mM PB缓冲液、0.4～0.6M NaCl和7～9M尿素；所述复性缓冲液的组成为：15～25mM PB缓冲液、4～6％甘油、0.04～0.06mM氧化型谷胱甘肽、0.4～0.6mM还原型谷胱甘肽和尿素，所述尿素为6M、4M、2M、1M或0M。
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|---|
| DANA RAUSCH等: "Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)", 《VETERINARY MICROBIOLOGY》 * |
| TI LU等: "Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 赵浩飞等: "猪源产肠毒素大肠杆菌疫苗的研究进展", 《畜牧与兽医》 * |
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Denomination of invention: A multi epitope fusion protein of Escherichia coli enterotoxin and its preparation method and application Granted publication date: 20221021 Pledgee: Hubei Bank Co.,Ltd. Jingshan Branch Pledgor: HUBEI SHENDI AGRICULTURE BRANCH TRADE CO.,LTD. Registration number: Y2024980009282 |