CN112402598A - 针对鸭疫里默氏杆菌感染引起的通用型亚单位疫苗 - Google Patents
针对鸭疫里默氏杆菌感染引起的通用型亚单位疫苗 Download PDFInfo
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- CN112402598A CN112402598A CN201910756269.5A CN201910756269A CN112402598A CN 112402598 A CN112402598 A CN 112402598A CN 201910756269 A CN201910756269 A CN 201910756269A CN 112402598 A CN112402598 A CN 112402598A
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Abstract
本发明涉及一种鸭疫里默氏杆菌外膜蛋白BamD的制备方法及其应用。培养大肠杆菌菌株并提取其DNA，设计大肠杆菌外膜蛋白表面抗原区域(21～274aa)基因序列的引物对，以鸭疫里默氏杆菌基因组DNA为模板进行PCR扩增。PCR产物和载体pET‑28a(+)连接并转化E.coli BL21感受态细胞中，采取IPTG诱导的方法，诱导大肠杆菌外膜蛋白rBamD表达。经过纯化最终得到重组的大肠杆菌外膜蛋白rBamD。该重组外膜蛋白具有一定的免疫原性及抗原性，能够有效抵抗鸭疫里默氏杆菌病的感染，以及鸭疫里默氏杆菌所引起的疾病。
Description
技术领域
本发明涉及蛋白质工程和基因工程领域，尤其涉及一种大肠杆菌重组外膜蛋白rBamD的制备方法及其免疫保护功能的应用。
背景技术
1)鸭疫里默氏杆菌(Riemerella anatipestifer,RA)，RA系革兰氏阴性短杆菌，无鞭毛及芽孢，不运动，可产生荚膜，可感染家鸭、鹅、火鸡、雉鸡等，是目前危害水禽养殖业的主要病原菌，广泛分布于世界各地的养鸭场，且感染后较难清除。RA感染途径多为呼吸道感染和皮肤伤口(特别是脚部皮肤)感染，引起一种急性或慢性败血性细菌性传染病，典型病理变化是纤维素性心包炎、肝周炎、气囊炎等。该病的感染率有时可高达90％以上，但由于各血清型之间的毒力存在较大差异，所以死亡率为5％～75％不等，急性型表现为急性败血症死亡，慢性型一般无明显症状，有些可表现为减重、产蛋量下降等。该病病死率最高可达90％，对水禽养殖业，尤其鸭和鹅的养殖造成重大损失。
随着抗生素的长期大量使用，大大加速了致病菌耐抗药性的进化。越来越多的耐药性菌株不断出现。研究表明，鸭疫里默氏杆菌基因组中抗药性基因的比重现在已大量增加，且多重耐药现象极其严重，目前已陷入抗生素大量使用-病原菌耐药性增强-加大使用抗生素-耐药性进一步增强的恶性循环中。同时,抗生素大量使用导致的抗生素残留问题也日益严重。抗生素滥用引起的耐药性问题及抗生素残留问题已经是一个全球性亟需解决的问题，改善动物养殖条件，接种疫苗预防病原菌是减少抗生素使用的有效途径。
RA具有多个血清型，目前已报到的有25个血清型，虽然部分疫苗对本血清型或本致病型菌株具有较好的免疫保护作用，但对其他菌株的交叉免疫保护作用较弱或者没有。鸭疫里默氏杆菌病的爆发往往是多种RA共同作用的结果，这给RA的防治带来很大的挑战。
BamD蛋白由294个氨基酸残基组成，前1～20个氨基酸为信号肽。作为外膜组装复合物的一部分，BamD主要参与外膜蛋白的正确组装。该蛋白21～274aa片段鸭疫里默氏杆菌体内具有高度的保守性，具有开发成通用型疫苗的潜力。
发明内容
本发明旨在提供一种大肠杆菌重组外膜蛋白rBamD的制备方法，及其在免疫功能方面的应用。
为实现上述目的，本发明采用的技术方案为：
一种大肠杆菌重组蛋白疫苗，重组蛋白疫苗为含有列表SEQ ID NO.1的原核重组表达质粒pET-28a-rBamD。
大肠杆菌重组蛋白疫苗的制备方法，所述质粒pET-28a-rBamD，以鸭疫里默氏杆菌ATCC 11845为模板，采用BamD F-BamHⅠ/BamD R-NotⅠ引物进行PCR扩增，PCR产物与载体pET-28a(+)连接，连接产物转化大肠杆菌DH5α，筛选转化子提取质粒，即为质粒pET-28a-rBamD。
所述引物为BamD F-BamHⅠ：5’-GGATCCTACGACCTAGCAATGAAA-3’，BamD R-NotⅠ：5’-GCGGCCGCCTGTTCTACTAACTTTTCT-3’。
大肠杆菌重组蛋白疫苗的应用，及所述重组蛋白疫苗在预防和治疗鸭疫里默氏杆菌中的应用。
附图说明
图1为重组质粒pET-28a-rBamD构建图。
图2为重组外膜蛋白rBamD诱导表达SDS电泳图。
图3为外膜蛋白纯化SDS电泳图。
图4为外膜蛋白rBamD免疫鸭疫里默氏杆菌后攻毒存活率图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，本发明实施例中所用的实验材料、试剂和仪器等均可市售获得，若未具体指明，实施例中所用的技术手段均为本领域技术人员所熟知的常规手段。以下实施例中的定量试验，均设置三次或三次以上重复实验，结果取平均值。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，如Sambrook等分子克隆实验手册(New York:Gold Spring Harbor Laboratory Press,1989)。
实施例1.pET-28a-rBamD载体构建
以大肠杆菌的基因组DNA为模板，进行PCR扩增，扩增产物为1089bp，其引物序列如下：BamD F-BamHⅠ：5’-GGATCCTACGACCTAGCAATGAAA-3’，BamD R-NotⅠ：5’-GCGGCCGCCTGTTCTACTAACTTTTCT-3’。
利用天根琼脂凝胶回收试剂盒(DP130419)对PCR产物切胶回收。将回收的PCR产物与pMD-19T simple vector连接，转化大肠杆菌DH5α，提取重组质粒测序。
对测序正确的质粒及pET28a载体进行BamHⅠ和NotⅠ双酶切。酶切体系如下：
37℃水浴4h，1.5％琼脂糖凝胶电泳，切胶回收。
将回收的BamD及双酶切片段与pET 28a载体进行连接，连接体系如下：
16℃连接2h.
实施例2.目标蛋白的表达
挑取重组菌1～2个菌落，在LB培养基中培养至对数中期。按1％比例转接至50mL自诱导培养基中，250rpm，37℃振荡培养24h。取1mL菌液，8000rmp，4℃离心2min后弃去上清，收集菌体；用200μL PBS重悬菌体后加入40μL 6×SDS-PAGE上样缓冲液，100℃煮沸10min，室温12 000rpm离心5min；取上清至新离心管中，4℃放置待分析空菌株对照和空载体对照组无目的蛋白表达，重组菌株均有目的蛋白表达。BamD蛋白占细胞总蛋白比例在45％～55％之间。
实施例3.菌体裂解及初步分离
1)菌液于4℃，7000rpm离心20min，用8mL/g裂解缓冲液悬浮菌体后，冰上超声裂解菌体(5s on，10s off)。
2)4℃，14000g离心20min，去掉上清。
3)每克包涵体溶于50mL 50mM Tris-HCl缓冲液(1.5％(v/v)LDAO，pH 7.9)中，20℃震荡孵育1h。
4)4℃，16000g离心30min沉淀包涵体。
5)每克包涵体重悬于20mL 50mM Tris-HCl，pH 7.9缓冲液中，4℃，16000g离心30min，弃上清。
6)每克包涵体加入80mL变性缓冲液，14000g离心20min，保留上清。
7)按1:1体积比例逐步加入重折叠缓冲液，同时快速搅拌。全部加入后20℃持续搅拌1h。
8)加入4L透析缓冲液，4℃透析6-8h；相同条件下透析2次。
实施例4.纯化与折叠重组蛋白
1)用10倍体积的缓冲液A平衡HISTrap柱。
2)加入透析的包涵体溶液。
3)用10倍体积的缓冲液A冲洗，收集穿透峰。
4)用8倍体积的缓冲液B梯度洗脱蛋白，收集洗脱峰。
5)样品经SDS-PAGE电泳分析检测。
6)用缓冲液C平衡HiPrepTM26/10脱盐柱，将含有目的蛋白的收集液过HiPrepTM26/10脱盐柱。
7)收集的样品进行SDS-PAGE电泳检测，测定蛋白浓度。并将收集的样品进行冻干，-20℃保存备用。
经纯化后，rBamD蛋白纯度为92.3％。
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
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<120> 针对鸭疫里默氏杆菌感染引起的通用型亚单位疫苗
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<210> 1
<211> 762
<212> DNA
<213> 鸭疫里默氏杆菌(Riemerella anatipestifer)
<400> 1
tacgacctag caatgaaaag tgctgataaa gatctgattc taaaaacggc taatgaaatg 60
tacaccaaaa agaaatggaa agaagctcta agtttatacg aaagagttca gaatctaatt 120
tctggtacag atgaagcttc cgatatttta ttcaaatctg cctacgctaa ctattacgac 180
aagcaataca gaattgcagg acatcaattt aaaaagtttt cggtaaatag tgctttggct 240
accgacccta gaaaggaaga agccgcttat atgtctgcga tatgttacta ccaaggctct 300
atggactaca acctagacca aaaagataca gaactagcaa taaacgaatt acaaagcttc 360
ttaaataact accctaattc cgaaagagct aaaaacatca acgagcttat tgatgaactt 420
tcttacaagc tagaatttaa ggcttatgaa aatgctcgtc aatactacaa aatgctagaa 480
ctaaaatcag ctattattag ttttgaaaat gtattggatg atttcccatc tacaaaactt 540
cgtccaaaaa tagaaaccat gcttatggac gcaaaagcca aactagcgat agactctaaa 600
tttgaactta aaagagaaag gttagagcat gcggtagctt acacccatct tatggaaaaa 660
aattatcctg ataccgatat tgctaaaaca gccgtaaccc taagaaaaaa gctagatgct 720
gaactagaaa actttgccaa gttagaaaag ttagtagaac ag 762
<210> 2
<211> 254
<212> PRT
<213> 鸭疫里默氏杆菌(Riemerella anatipestifer)
<400> 2
Tyr Asp Leu Ala Met Lys Ser Ala Asp Lys Asp Leu Ile Leu Lys Thr
1 5 10 15
Ala Asn Glu Met Tyr Thr Lys Lys Lys Trp Lys Glu Ala Leu Ser Leu
20 25 30
Tyr Glu Arg Val Gln Asn Leu Ile Ser Gly Thr Asp Glu Ala Ser Asp
35 40 45
Ile Leu Phe Lys Ser Ala Tyr Ala Asn Tyr Tyr Asp Lys Gln Tyr Arg
50 55 60
Ile Ala Gly His Gln Phe Lys Lys Phe Ser Val Asn Ser Ala Leu Ala
65 70 75 80
Thr Asp Pro Arg Lys Glu Glu Ala Ala Tyr Met Ser Ala Ile Cys Tyr
85 90 95
Tyr Gln Gly Ser Met Asp Tyr Asn Leu Asp Gln Lys Asp Thr Glu Leu
100 105 110
Ala Ile Asn Glu Leu Gln Ser Phe Leu Asn Asn Tyr Pro Asn Ser Glu
115 120 125
Arg Ala Lys Asn Ile Asn Glu Leu Ile Asp Glu Leu Ser Tyr Lys Leu
130 135 140
Glu Phe Lys Ala Tyr Glu Asn Ala Arg Gln Tyr Tyr Lys Met Leu Glu
145 150 155 160
Leu Lys Ser Ala Ile Ile Ser Phe Glu Asn Val Leu Asp Asp Phe Pro
165 170 175
Ser Thr Lys Leu Arg Pro Lys Ile Glu Thr Met Leu Met Asp Ala Lys
180 185 190
Ala Lys Leu Ala Ile Asp Ser Lys Phe Glu Leu Lys Arg Glu Arg Leu
195 200 205
Glu His Ala Val Ala Tyr Thr His Leu Met Glu Lys Asn Tyr Pro Asp
210 215 220
Thr Asp Ile Ala Lys Thr Ala Val Thr Leu Arg Lys Lys Leu Asp Ala
225 230 235 240
Glu Leu Glu Asn Phe Ala Lys Leu Glu Lys Leu Val Glu Gln
245 250
<210> 3
<211> 24
<212> DNA
<213> 鸭疫里默氏杆菌(Riemerell anatipestifer)
<400> 3
ggatcctacg acctagcaat gaaa 24
<210> 4
<211> 27
<212> DNA
<213> 鸭疫里默氏杆菌(Riemerell anatipestifer)
<400> 4
gcggccgcct gttctactaa cttttct 27
Claims (4)
1.一种抗鸭疫里默氏杆菌感染的基因工程疫苗的制备方法，其特征在于首先提取鸭疫里默氏杆菌基因组，扩增其外膜蛋白BamD表面抗原区域基因，连接重组表达载体pET-28a，转化大肠杆菌BL21。通过IPTG诱导大肠杆菌表达的包涵体利用6mol/L盐酸胍和LDAO进行复性，并通过镍柱进行纯化。
2.根据权利要求1所述的制备方法，其特征在于由外膜蛋白BamD的第21～274位氨基酸组成。
3.根据权利要求1所述的制备方法，其特征在于扩增rBamD的引物为BamD F-BamHⅠ：5’-GGATCCTACGACCTAGCAATGAAA-3’，BamD R-NotⅠ：5’-GCGGCCGCCTGTTCTACTAACTTTTCT-3’，扩增产物大小为882bp。
4.根据权利要求1所述的一种大肠杆菌重组外膜蛋白rBamD作为疫苗组分的应用。
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| CN114099660A (zh) * | 2021-11-11 | 2022-03-01 | 扬州优邦生物药品有限公司 | 一种预防鸭传染性浆膜炎三价基因工程亚单位疫苗组合物及其制备方法 |
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| CN114099660A (zh) * | 2021-11-11 | 2022-03-01 | 扬州优邦生物药品有限公司 | 一种预防鸭传染性浆膜炎三价基因工程亚单位疫苗组合物及其制备方法 |
| CN114099660B (zh) * | 2021-11-11 | 2022-07-19 | 扬州优邦生物药品有限公司 | 一种预防鸭传染性浆膜炎三价基因工程亚单位疫苗组合物及其制备方法 |
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