CN101219214A - 近江牡蛎类立克次体内类外膜蛋白的全序列及其亚单位疫苗的制备 - Google Patents
近江牡蛎类立克次体内类外膜蛋白的全序列及其亚单位疫苗的制备 Download PDFInfo
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Abstract
本发明涉及近江牡蛎类立克次体中一种类ompH外膜蛋白的全序列,本发明的类ompH外膜蛋白可以用于制备类外膜蛋白亚单位疫苗,目的是用于预防近江牡蛎类立克次体病。本发明主要技术要点:1.以近江牡蛎类立克次体特有的基因组DNA为模板,设计扩增类ompH外膜蛋白基因的引物,获得编码类ompH外膜蛋白的基因全序列;2.利用生物技术手段将类ompH外膜蛋白的基因片段克隆到合适的pGEM-T easy载体中,转入相应的大肠杆菌TG I宿主细胞中,获得表达载体pET-28a(+)-ompH;3.类ompH外膜蛋白的体外表达,Western-blot,SDS-聚丙烯酰胺凝胶电泳等方法鉴定目的蛋白;4.利用亲和柱分离纯化重组类ompH外膜蛋白,制成亚单位疫苗。
Description
技术领域
本发明属于生物技术领域,涉及近江牡蛎类立克次体中一种类外膜蛋白(即类ompH外膜蛋白)的全序列及其亚单位疫苗的实验制备技术,本发明的类ompH外膜蛋白可以用于制备类外膜蛋白亚单位疫苗。
背景技术
牡蛎为重要的经济贝类和海水养殖品种。根据联合国粮农组织(FAO)的数字显示,我国每年牡蛎的产量具全世界首位,达到300万吨(湿重),占我国海洋水产养殖总产量(约1000万吨)的约三分之一。但是海洋环境的污染、恶化和海洋病害的频发危及到牡蛎产业的可持续发展。近江牡蛎体内寄生的类立克次体是引起近江牡蛎大规模死亡的重要病原微生物,目前还没有有效的治疗措施。类立克次体外膜蛋白具有抗原活性,是抗体的主要作用对象。但由于现有技术中未能获得ompH外膜蛋的全序列,没有表达载体,故难以制备出这种类立克次体中具有抗原性的类ompH外膜蛋白亚单位疫苗。
发明内容
本发明目的是利用基因工程技术制备近江牡蛎类立克次体中具有抗原性的类ompH外膜蛋白亚单位疫苗。以防治近江牡蛎体内寄生的类立克次体,预防近江牡蛎大规模死亡。
为实现上述目的,首先将近江牡蛎类立克次体的类ompH外膜蛋白的基因片段插入克隆和表达载体,然后转化相应的宿主细胞中,进行体外原核表达,重组蛋白纯化,以纯化的重组蛋白制备疫苗。
本发明关于近江牡蛎类立克次体内类外膜蛋白亚单位疫苗的制备方法,包括以下步骤:
1、以近江牡蛎类立克次体特有的基因组DNA为模板,本申请首次设计扩增类ompH外膜蛋白基因的引物,并首次获得编码类ompH外膜蛋白的基因全序列;
2、利用生物技术手段将类ompH外膜蛋白的基因片段克隆到合适的pGEM-T easy载体中,转入相应的大肠杆菌TG I宿主细胞中,获得表达载体pET-28a(+)-ompH;
3、类ompH外膜蛋白的体外表达,Western-blot,SDS-聚丙烯酰胺凝胶电泳等方法鉴定目的蛋白;
4、利用亲和柱分离纯化重组表达的蛋白制成亚单位疫苗。
本发明的显著优点
制备得到的这种类外膜蛋白亚单位疫苗具有特异性强、免疫原性好、高度安全性等特点。
具体实施方式
1.引物设计与合成
设计用于扩增类ompH外膜蛋白基因的寡聚核苷酸引物为:
SEQNO1
PF:5’GTGAAAAAGTGGTTATTAGCTG 3’
PR:5’TTATTTAACCTGTTTCAGTACG 3’
(此引物为本申请首次设计)
2.PCR扩增与表达载体的构建
以近江牡蛎类立克次体基因组DNA为模板扩增目的片段,PCR扩增的反应条件为:95℃5min;35个循环:94℃30s,55℃35s,72℃35s;72℃1min。PCR产物连入pGEM-T easy载体,转化大肠杆菌TG I,涂布LBA筛选平板,挑若干个克隆进行酶切和PCR鉴定,选一阳性克隆扩大培养,以碱法抽提质粒.将制备好的质粒以BamH I和Xho I限制性内切酶双酶切,连入经过同样双酶切的pET-28a(+)载体的相应位点上,转化大肠杆菌TG I。PCR筛选阳性克隆,经测序鉴定获得编码框正确的表达载体pET-28a(+)-ompH。经上述研究获得:
1、来自于近江牡蛎类立克次体内特有的类ompH外膜蛋白的核酸全序列如下
(在现有技术中未曾报导过):
SEQNO2
GTGAAAAAGT GGTTATTAGC TGCAGGTCTC GGTTTAGCAA TGGCTGCTTC
AGCTGGCGTT CAGGCAGCAG ATAAGATCGC TGTCGTTAAC GTCTCCAGCA
TTTTCCAACA GCTGCCGGCC CGCGAAGCGG TCGCTAAACA GCTGGAAAAC
GAGTTTAAAG GCCGTGCAAG CGAACTCCAG AACATGGAAC GTAGCCTGCA
GACCAAAATG CAACGTCTGC AGCGTGACGG CGCTACCATG AAAGCGAGCG
ACCGCAGCAA GTTGGAAAAA GACGTGATGG AGCAACGCGA GCAGTTCTCT
CAGAAAGCCC TGGCTTTCGA GCAGGACAAC CGCCGTCGCC AGATGGAAGA
ACGTAACAAG ATCCTGAGCC GCATTCAGGA CGCGGTGAAA TCTGTCGCCA
GCAAAGGCGG TTATGACGTG GTGATCCACG CCAACGCCGT GCCTATGCAG
ACTCTTCCAA AGATATCACT GCCGACGTAC TGAAACAGGT TAAATAA
1、来自于近江牡蛎类立克次体内特有的类ompH外膜蛋白的氨基酸序列如下(本申请首次获得):
SEQNO3
MAASAGVQAA DKIAVVNVSS IFQQLPAREA VAKQLENEFK GRASELQNME
RSLQTKMQRL QRDGATMKAS DRSKLEKDVM EQREQFSQKA LAFEQDNRRR
QMEERNKILS RIQDAVKSVA SKGGYDVVIH ANAVAYADSS KDITADVLKQ VK
3.重组蛋白的表达与鉴定
将上述阳性重组质粒pET-28a(+)-ompH转化表达宿主菌BL21感受态,涂布LBA平板,37℃培养过夜。挑取一个单克隆菌落,转入LBA培养液,37℃振摇培养过夜。取适量菌液,按1∶100扩大培养至OD600为0.4-0.5时,将菌液分为若干等份,不加或分别加入IPTG,使其终浓度在0-1.0mmol/L,继续培养6h,离心收集细菌。细菌经超声波裂解后,离心分离上清液和沉淀,分别上样,进行SDS-PAGE电泳。
4.重组蛋白的纯化与抗体制备
利用组氨酸亲和层析柱对表达产物进行分离纯化。以上述方法大量制备超声裂解的菌液,离心分离上清液,进行蛋白的纯化和洗脱操作,12%的SDS-PAGE电泳鉴定。
挑选体重约1.5kg的健康日本大耳白雄兔进行免疫。经过一次初始免疫和两次加强免疫后,颈动脉取血,分离血清,存储于-80℃。
5.蛋白质定量与抗体效价的检测
按Brad-ford法对蛋白进行定量(Bradford MM.A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principle of protein-dye binding.AnalBiochem.1976,72:248-254)。
免疫后的兔血清用ELISA方法检测效价,具体操作如下:
(1)包被与封闭:用包被缓冲液将蛋白稀释至1-10μg/ml。在每个聚苯乙烯酶标板的反应孔中加0.1ml,4℃过夜。弃去孔内溶液,5%的脱脂奶粉封闭2h,用PBST洗3次,每次3min
(2)加样:加指定稀释倍数的待检样品(待检测的抗血清稀释于PBST)0.1ml于上述已包被的反应孔中,置37℃孵育1h,PBST洗3次,每次3min(同时做空白孔,阴性对照孔及阳性对照孔)
(3)加酶标抗体:于各反应孔中,加入新鲜稀释的酶标抗体0.1ml,37℃1h,PBST洗3次,每次3min。
(4)加底物液显色:于各反应孔中加入新鲜配制的OPD底物溶液0.1ml,37℃显色10-30分钟。
(5)终止反应:于各反应孔中加入2M反应终止液0.05ml。
(6)结果分析:用酶标仪490nm测各孔OD值,大于规定的阴性对照OD值的2.1倍,即为阳性。计算抗体效价公式:(样本OD值-空白对照OD值)/(阴性对照OD值-空白对照OD值)。
6.Western蛋白印迹
Western蛋白印迹操作按文献(Sambrook J,Russell DW著.黄培堂等译.分子克隆实验指南,第3版.北京:科学出版社,2002.(Sambrook J,Russell D W.Molecular Cloning:A LaboratoryManual,3rd ed.New York:Cold Spring Harbor Laboratory Press,2001)),具体如下:
(1)蛋白质样品进行SDS-PAGE电泳
(2)转膜(电转):电泳结束后将胶条割至合适大小,浸入转膜缓冲液平衡20min。预先裁好与胶条同样大小的滤纸和PVDF膜,浸入转膜缓冲液中15min。转膜装置的搭建:按从下至上顺序依次为阴极塑料板、3层滤纸、PVDF膜、凝胶、3层滤纸、阳极塑料板,滤纸、凝胶、膜精确对齐,每一步去除气泡,吸干多余的液体。恒压38V,转移3h。
(3)免疫杂交反应:
a用PBST洗膜,5min×3次。
b加入封闭液,平稳摇动,室温2h。
c弃封闭液,用PBST洗膜,10min×3次。
d将膜转入一抗杂交液(抗血清按预定比例稀释于封闭液),室温杂交2h。阴性对照中以免疫前的血清取代一抗,其余步骤与实验组相同。
e弃杂交液,用PBST洗膜,10min×3次。
f将膜转入二抗杂交液(辣根过氧化物酶偶联的二抗按合适稀释比例稀释于PBST),平稳摇动,室温2h。
g弃杂交液,用PBST洗膜,10min×3次。
h加入显色液,避光显色至出现条带时放入双蒸水中终止反应。
本发明的实施例:
1、类ompH重组蛋白的表达与鉴定:
将阳性pET-28a(+)-ompH重组质粒转化表达宿主菌BL21感受态,涂布LBA平板,37℃培养过夜。挑取一个单克隆菌落,转入LBA培养液,37℃振摇培养过夜。取适量菌液,按1∶100扩大培养至OD600为0.4-0.5时,将菌液分为若干等份,不加或分别加入IPTG,使其终浓度在0-1.0mmol/L,继续培养6h,离心收集细菌。细菌经超声波裂解后,离心分离上清液和沉淀,分别上样,进行SDS-PAGE电泳。
2、利用亲和柱分离纯化重组表达的蛋白和抗类ompH重组蛋白抗体的制备:
(1)蛋白纯化
①表达His-ompH的菌液100ml,10000×g,离心10分钟;分别加入4mlPBS,超声破碎50次,每次3秒;加入PMSF之终浓度10mmol混匀,10000×g,离心10分钟;上清液经0.22μ的滤器过滤,至于冰上待用。
②每4ml的浓缩样品加入1ml Ni-NTA浆液,混合均匀装柱,收集流出液。
③用洗脱液洗脱结合的蛋白,每次500μl,洗4次,收集洗脱液。从亲和柱分离纯化来的重组表达的蛋白经免疫原性分析即可制成亚单位疫苗。
(2)免疫动物
将纯化的蛋白浓度调制2μg/μl,与等量的弗氏完全佐剂混合,乳化,注射免疫家兔。于两周后再以弗氏完全佐剂乳化纯化的蛋白,进行加强免疫。再两周后,耳静脉取2毫升血,检测抗体效价。
(3)抗体效价检测
用间接ELISA技术测定抗体效价及最佳工作浓度。
间接ELISA操作:包被液稀释抗原→抗原包备→洗板→加入不同稀释倍数的一抗→洗板→加入酶标二抗→洗板→加入底物显色液→终止反应→酶标仪检测。
序列表:
SEQNO1
<110>浙江大学
<120>近江牡蛎类产克次体内类外膜蛋白序列及其亚单位疫苗的制备
<160>3
<210>1
<211>45
<212>DNA
<213>近江牡蛎类立克次体内类外膜蛋白
<221>本发明设计寡聚核苷酸引物
<223>来自近江牡蛎类立克次体内类外膜蛋白
<400>1
GTGAAAAAGT GGTTATTAGC TG TTATTTA ACCTGTTTCA GTACG
SEQNO2
<211>489
<212>DNA
<213>近江牡蛎类立克次体内类外膜蛋白
<221>核酸全序列
<223>来自近江牡蛎类立克次体内类外膜蛋白
<400>2
GTGAAAAAGT GGTTATTAGC TGCAGGTCTC GGTTTAGCAA TGGCTGCTTC AGCTGGCGTT 60
CAGGCAGCAG ATAAGATCGC TGTCGTTAAC GTCTCCAGCA TTTTCCAACA GCTGCCGGCC 120
CGCGAAGCGG TCGCTAAACA GCTGGAAAAC GAGTTTAAAG GCCGTGCAAG CGAACTCCAG 180
AACATGGAAC GTAGCCTGCA GACCAAAATG CAACGTCTGC AGCGTGACGG CGCTACCATG 240
AAAGCGAGCG ACCGCAGCAA GTTGGAAAAA GACGTGATGG AGCAACGCGA GCAGTTCTCT 300
CAGAAAGCCC TGGCTTTCGA GCAGGACAAC CGCCGTCGCC AGATGGAAGA ACGTAACAAG 360
ATCCTGAGCC GCATTCAGGA CGCGGTGAAA TCTGTCGCCA GCAAAGGCGG TTATGACGTG 420
GTGATCCACG CCAACGCCGT GCCTATGCAG ACTCTTCCAA AGATATCACT GCCGACGTAC 480
TGAAACAGGT TAAATAA 497
SEQNO3
<211>152
<212>DNA
<213>近江牡蛎类立克次体内类外膜蛋白
<221>氨基酸序列
<223>来自近江牡蛎类立克次体内类外膜蛋白
<400>3
MAASAGVQAA DKIAVVNVSS IFQQLPAREA VAKQLENEFK GRASELQNME RSLQTKMQRL 60
QRDGATMKAS DRSKLEKDVM EQREQFSQKA LAFEQDNRRR QMEERNKILS RIQDAVKSVA 120
SKGGYDVVIH ANAVAYADSS KDITADVLKQ VK 152
Claims (4)
1.用于扩增近江牡蛎类立克次体内的类ompH外膜蛋白基因的寡聚核苷酸引物为SEQNO1
PF:5’GTGAAAAAGTGGTTATTAGCTG 3’
PR:5’TTATTTAACCTGTTTCAGTACG 3’
2.近江牡蛎类立克次体内的类ompH外膜蛋白的核酸全序列为SEQNO2。
3.近江牡蛎类立克次体内的类ompH外膜蛋白的氨基酸序列SEQNO3。
4.近江牡蛎类立克次体内的类外膜蛋白亚单位疫苗的制备方法,其特征包括以下内容:(1)以近江牡蛎类立克次体特有的基因组DNA为模板,设计扩增权利要求1所述的类ompH外膜蛋白基因的引物,获得权利要求2所述的编码类ompH外膜蛋白的基因全序列SEQNO1;(2)将类ompH外膜蛋白的基因片段克隆到合适的pGEM-T easy载体中,转入相应的大肠杆菌TG I宿主细胞中,获得表达载体pET-28a(+)-ompH;(3)类ompH外膜蛋白的体外表达,Western-blot,SDS-聚丙烯酰胺凝胶电泳等方法鉴定重组类ompH外膜蛋白;(4)利用亲和柱分离纯化重组类ompH外膜蛋白,制成亚单位疫苗。
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