CN105566461B - DNA改组后的细菌外膜蛋白ompAs-19及其作为免疫调节剂的应用 - Google Patents
DNA改组后的细菌外膜蛋白ompAs-19及其作为免疫调节剂的应用 Download PDFInfo
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- CN105566461B CN105566461B CN201511009125.1A CN201511009125A CN105566461B CN 105566461 B CN105566461 B CN 105566461B CN 201511009125 A CN201511009125 A CN 201511009125A CN 105566461 B CN105566461 B CN 105566461B
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- outer membrane
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Abstract
本发明属于DNA改组技术领域,具体公开了DNA改组后的细菌外膜蛋白ompAs‑19及其作为免疫调节剂的应用。发明首次采用DNA改组技术结合免疫学研究,以溶藻弧菌、副溶血弧菌、迟缓爱德华氏菌以及大肠杆菌的外膜蛋白OmpA为研究对象,通过DNA改组技术获得DNA改组后的细菌外膜蛋白ompAs‑19,其氨基酸序列如如SEQ ID NO:2所示。DNA改组后的细菌外膜蛋白ompAs‑19对溶藻弧菌具有较高的免疫保护作用,其相对免疫保护率(RPS)为100%。此外,还显示出对迟缓爱德华氏菌的交叉免疫原性,其免疫保护率(RPS)达到85.71%。这些结果说明19号改组OmpA(ompAs‑19)可作为疫苗组分。
Description
技术领域
本发明涉及DNA改组技术领域,具体涉及DNA改组后的细菌外膜蛋白ompAs-19及其作为免疫调节剂的应用。
背景技术
在水产动物养殖中,往往使用各种化学药物和抗生素来控制有关疾病, 但长期用药, 不仅导致病原菌的抗药性越来越明显, 而且使药物残留的危害也日益显现,严重地影响水产品安全,所以通过疫苗进行免疫预防更受到高度重视。目前我国水产养殖业的主要病原菌是弧菌和迟钝爱德华氏菌,而目前无针对多种细菌的多价高效疫苗,因此,研制以具有免疫保护作用的高效多价疫苗具有广阔的应用前景。
研究已经发现细菌外膜蛋白OmpA具有良好的免疫原性,不仅可以刺激体液免疫,而且对细胞免疫亦有刺激作用。但是外膜蛋白OmpA仅仅对同种细菌具有良好的免疫调节功能,对其他细菌的免疫调节功能较低。如溶藻弧菌OmpA针对溶藻弧菌感染时保护率达到87.5%,而副溶血弧菌OmpA对溶藻弧菌感染时保护率仅为35.6%;副溶血弧菌和迟缓爱德华氏菌的OmpA在针对自身细菌感染时保护率仅为40%左右。我国养殖鱼类个体偏小,从经济效率和实际操作来考虑,也难以对常见病原菌逐一进行疫苗免疫。
DNA改组技术可以在分子水平上对基因进行体外有性重组,由此改变单个基因或基因家族原有的核苷酸序列,创造新基因并赋予表达产物新功能,推动了生物工程的诸多领域突飞猛进地向前发展。但目前尚无用该技术获得多效多价疫苗的研制的报道。
发明内容
本发明的目的是为了克服现有技术的上述不足,提供一种DNA改组后的细菌外膜蛋白ompAs-19。
本发明的另一个目的是提供DNA改组后的细菌外膜蛋白ompAs-19作为免疫调节剂的应用。
为了实现上述目的,本发明是通过以下技术方案予以实现的:
DNA改组后的细菌外膜蛋白ompAs-19,编码该蛋白的核苷酸序列如SEQ ID NO:1所示。
DNA改组后的细菌外膜蛋白ompAs-19,其氨基酸序列如如SEQ ID NO:2所示。
SEQ ID NO:2所述的DNA改组后的细菌外膜蛋白ompAs-19在制备动物免疫调节剂中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明首次采用DNA改组技术结合免疫学研究,以溶藻弧菌、副溶血弧菌、迟缓爱德华氏菌以及大肠杆菌的外膜蛋白OmpA为研究对象,通过DNA改组技术获得改组OmpA基因,进一步构建了原核表达的改组OmpA质粒库;通过SDS-PAGE鉴定获得43个能正确表达的改组OmpA基因;将所有这些改组OmpA基因构建真核表达的改组OmpA质粒库,即DNA疫苗;最后用斑马鱼作为模式动物深入研究其免疫保护性。免疫和攻毒实验结果表明19号改组OmpA基因(ompAs-19)对溶藻弧菌具有较高的免疫保护作用,其相对免疫保护率(RPS)为100%。此外,还显示出对迟缓爱德华氏菌的交叉免疫原性,其免疫保护率(RPS)达到85.71%。这些结果说明19号改组OmpA(ompAs-19)可作为疫苗组分。
附图说明
图1为6个Ompa基因用DNAman软件进行序列比对分析结果。
图2为OmpA基因的PCR扩增图谱;1: DNA分子量标准,2,3:E.coli ompA的2个片段,4,5:ETAE_1267的2个片段,6:VP0764,7:VA0764,8:vpa1186,9:VPA1186。
图3为OmpA基因改组电泳图谱;A:膜板基因的DNaseI酶切;B:无引物PCR扩增产物;C:用特异的VA 0764引物PCR扩增,M:DNA分子量标准。
图4为改组OmpA 基因原核表达质粒库的筛选,A:PCR扩增,M: DNA分子量标准; B:改组OmpA原核表达质粒库表达,M:蛋白质分子标准; VA: V. Alginolyticus;1~43:ompAs表达; C:改组OmpA表达的Western-blotting鉴定。
图5为改组OmpA真核质粒的双酶切电泳图谱,M: DNA分子量标准。
图6为改组OmpA基因DNA疫苗的主动免疫保护评估,A, DNA疫苗免疫后斑马鱼体液Western-blotting. 1, 免疫VA0764; 2, 免疫载体pcDNA3.1; 3-12, 免疫改组DNA疫苗.B和C, 改组DNA疫苗对溶藻弧菌和迟缓爱德华氏菌的主动免疫保护作用评估**p < 0.01。
图7为改组ompAs-19 (上面)与VA0764(下面)基因序列对比结果。
图8为改组ompAs-19 (上面)与VA0764(下面)氨基酸序列对比结果。
具体实施方式
下面结合说明书附图和具体实施例对本发明作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
细菌外膜蛋白OmpA基因改组模板:选取6个细菌外膜蛋白OmpA基因作为改组对象,其分别来源于溶藻弧菌、副溶血弧菌、迟缓爱德华氏菌和大肠杆菌等4种细菌。这6个OmpA基因分别是溶藻弧菌0764(VA0764)、1186(vpa1186)、副溶血弧菌0764(VP0764)、1186(VPA1186)、迟缓爱德华氏菌ompA(ETAE_1267)、大肠杆菌ompA(E.coliompA),基因长度在960bp~1056bp之间。对这6个OmpA基因用DNAman软件进行序列比对分析,发现其同源性为65.59%,结果见图1,可用于进行基因改组研究。
实施例2细菌外膜蛋白OmpA改组基因的获得
改组模板的PCR扩增:根据NCBI已经公布的6个OmpA基因序列设计引物,引物序列见表1。通过分析6个OmpA基因序列,发迟缓爱德华氏菌ompA(ETAE_1267)760 bp处和大肠杆菌ompA(E.coliompA )745bp处都存在一个BamHI酶切位点。因后面使用的原核表达载体要使用这个酶切位点,所以这两个基因以BamHI酶切位点为分界点,各自设计两对引物,将E.coli ompA和ETAE_1267基因分别扩增出为两个片段。
表1用于改组ompA模板扩增的引物序列
E.coli ompA-1是从1~745 bp,E.coli ompA-2是从746~1041 bp,E. tardaompA-1是从1~760 bp,E. tardaompA -2是从761~1056 bp。
以已经构建好的6种OmpA重组质粒为模板,以各自引物扩增出各自全长OmpA基因,得到了相应的8个基因片段,长度与预期一致,结果见图2。
改组OmpA基因的获得:回收PCR产物,等量混合8个OmpA基因片段,然后用DNaseI进行消化,结果见图3A。从图中可以看出,DNaseI将OmpA混合片段酶切成大约50~100bp的小片段DNA。然后以回收的小片段DNA(50bp~100bp)为模板,先不加引物进行PCR扩增,得到了弥散型条带(图3B),再以无引物PCR获得的产物为模板,加入溶藻弧菌外膜蛋白OmpA基因(VA0764)特定引物(上游引物:5’-GCCGGATCCATGAAAAAACTAGCAGCGG-3’,下游引物:5’-GGGCTCGAGTTATTGCTGAACTTGG-3’,下划线所示为酶切位点,上游引物引入BamH I位点,下游引物引入XhoI位点),继续进行PCR扩增,结果见图3C。由图中看出,通过弧菌特异引物PCR得到了大约1000 bp的特异性条带,表明得到了OmpA改组基因,命名为ompAs。
实施例3改组OmpA基因的原核质粒库构建
将获得的OmpA改组基因(ompAs)产物回收后,用BamHI和XhoI进行双酶切,并用同样的内切酶对原核表达载体pET32a也进行双酶切,连接后转化至大肠埃希菌BL21感受态细胞中,涂布于含60微克/毫升Amp抗生素的LB平板上,获得1542个重组子。
快速法筛选阳性重组子:随机挑取改组重组子,以载体pET32a为阴性对照,通过比较质粒大小来进行阳性重组子的筛选。凡是比阴性对照分子量大的重组子,可以初步鉴定为阳性重组子。结果共筛选出43个阳性重组子,即构建了改组OmpA基因(ompAs)针对弧菌的原核质粒库,命名为PompAs-SV。
对原核质粒库中的43个阳性重组子,进一步用上述合成的弧菌特异性引物进行PCR扩增鉴定重组子插入片段大小,结果见图4A,由图中可知,经PCR扩增得到的改组OmpA基因的分子量接近1000bp,与模板OmpA基因的分子量大小在960bp~1056bp一致,说明这43个阳性重组子都含有改组OmpA基因。
接着对这些阳性重组子进行IPTG诱导,通过SDS-PAGE电泳检测改组OmpA基因是否能够表达,结果见图4B。由SDS-AGE电泳图谱可以看到,这43个改组OmpA基因经诱导后都有表达,其蛋白质分子量在50-60 kDa。根据改组模板OmpA基因长度推算OmpA蛋白分子量在34kDa-38kDa之间,加上所用载体pET32a的融合蛋白(大小为20kD),改组OmpA重组蛋白理论值应为54~58kDa。但25~29, 35和43号改组重组子的蛋白质分子量略低。
最后,为验证这些嵌合体表达的重组蛋白质确实是OmpA,随机选择了10个重组子,用溶藻弧菌OmpA即VA0764的抗体作为一抗进行了Western-blotting确认,结果见图4C,所有的重组子表达的重组蛋白质都有显色,表明这些表达蛋白质确实为OmpA蛋白质。
实施例4改组OmpA基因的疫苗库构建
分别提取原核质粒库中的43个阳性重组子的质粒,用HindIII和XhoI内切酶进行双酶切,经过琼脂糖电泳后回收改组OmpA基因片段;同时用HindIII和XhoI内切酶酶切真核载体pCDNA3.1。将每个改组OmpA基因片段分别与真核载体连接后转化DH5α感受态细胞,得到43个改组基因的疫苗库,命名为EompAs-SV。
为验证构建真核质粒库中的每一个重组子是否含有改组OmpA基因,分别提取质粒用HindIII和XhoI内切酶进行双酶切鉴定,结果见图5。由图可以看出,所有真核质粒均可酶切出约1000bp大小的基因片段,说明成功构建了真核质粒库,即得到了43个候选DNA疫苗。
按照同样的方法,构建了溶藻弧菌OmpA基因(VA0764)和迟缓爱德华氏菌OmpA基因(ETAE_1267)的真核质粒,用于随后DNA疫苗的保护性试验的对照DNA疫苗。
实施例5 DNA疫苗免疫保护性的研究
为研究构建的改组OmpA基因的DNA疫苗的免疫保护作用,以斑马鱼为模式动物进行了免疫保护性试验。
质粒提取:用常规方法提取所有质粒,包括43个改组OmpA质粒、溶藻弧菌OmpA基因(VA0764)和迟缓爱德华氏菌OmpA基因(ETAE_1267)的真核质粒,以及cDNA31.质粒。由于内毒素的存在对宿主是有毒性的,可引起机体发热、内毒素休克以及凝血等,在提取质粒时对质粒进行了无内毒素处理。
斑马鱼免疫:将买回的斑马鱼在循环水系统养殖1周后,随机分为46组,每组25尾。43组作为试验站,注射改组OmpA质粒;3组作为对照组,注射载体pCNA3.1, VA0764和ETAE_1267的真核表达质粒。每尾注射1.5微克质粒。
改组OmpA基因在斑马鱼体内表达情况:真核表达质粒自身不能作为抗原,而是通过外源基因在动物体内表达蛋白,产生抗原激活机体的免疫应答系统,因此在测定DNA疫苗的保护率之前,先对改组OmpA基因是否在鱼体内表达进行了检验。具体做法如下:DNA疫苗免疫斑马鱼后,在攻毒前从不同实验组随机挑选10尾斑马鱼,在冰上切成4-5段,按照斑马鱼的体重加入蛋白浸提液(500μL蛋白浸提液/g斑马鱼),在冰上进行均浆。然后4℃孵育3小时后离心取上清;SDS-PAGE电泳分离蛋白质后,以VA0764抗体为一抗,进行westernblotting,结果见6A。图中显示所有免疫后斑马鱼体液中均有特异性条带出现,说明改组OmpA基因在斑马鱼体内能够表达OmpA蛋白质。
免疫保护作用评估:DNA疫苗4周后,用6×105/毫升的溶藻弧菌攻毒,1天后斑马鱼开始出现死亡,在3天后基本稳定。连续观察15天,统计每组死亡率。根据相对免疫保护率来比较分析这些免疫原性蛋白质的保护效果。结果发现,与对照VA0764(对溶藻弧菌相对保护率为78.57%)进行比较,EompAs-19 的相对保护率为100%,EompAs-25 和EompAs-29的相对保护率为78.57%和77.14%,与对照类似(图6B)。而对照ETAE_1267对溶藻弧菌的相对保护率仅为20.58%。
交叉疫苗免疫保护作用评估:将对溶藻弧菌感染的相对保护率达到77%以上的3个改组DNA疫苗,用上述类似方法进行免疫斑马鱼,用剂量为2×104CFU /毫升的的迟缓爱德华氏菌EIB202进行攻毒,攻毒结果见图6C。从图中可以看到,与对照ETAE_1267对迟缓爱德华氏菌EIB202的相对免疫保护率(39.39%)比较,3个改组基因的DNA疫苗,EompAs-19,EompAs-25, EompAs-29对EIB202感染具有很好的免疫保护作用,相对免疫保护率分别为85.71%, 74.79%和75.59%。而VA0764对迟缓爱德华氏菌EIB202的相对免疫保护率仅为21.57%。
从上述结果可知,细菌OmpA对自身细菌有一定的保护性,如弧菌OmpA对溶藻弧菌的相对保护率为78.57%,迟缓爱德华氏菌OmpA对迟缓爱德华氏菌相对保护率为39.39%,而对异种细菌的保护性较弱,如弧菌OmpA对迟缓爱德华氏菌的相对保护率仅为21.57%,迟缓爱德华氏菌OmpA对溶藻弧菌相对保护率仅为20.58%。而通过DNA改组,得到的EompAs-19对弧菌感染的相对保护率为100%,对爱德华氏菌的相对保护率达到85.71%,所以EompAs-19基因可作为高效多价DNA疫苗。
实施例6 OmpA改组基因的免疫保护性机制研究
为探讨EompAs-19多价疫苗的保护机制,对EompAs-19序列进行了基因测序,其序列如SEQ ID NO:1所示,对于的氨基酸序列如SEQ ID NO:2所示。然后将其核苷酸序列与VA0764进行分析比对(见图7),发现有22个核苷酸发生了变化。进一步氨基酸分析对比(见图8),发现有3个氨基酸发生变化,分别是10位,11位和309位氨基酸。
SEQUENCE LISTING
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caggcttgtg acaaagacga ctctactctt ggcgcttttg ttggttacga aatgaataaa 180
tacttcgcag tagaagcagg tttcgacaac atcggtgatt ttaaccaaac ttctttcagt 240
ggccacgtag aagcaatcac tcttgcacct aaatttagcc taccaatcac tgaagacatc 300
gcactttacg gtaaagtggg tggcgcttac gtaatgtttg atggcaaaga tgattactct 360
tacctaggcg cagctggtct tgaattcaac ctaagccaaa acgtaacagc tcgtgcggaa 420
taccaaacac tgactgacat cagcaacgat gtaactcgtg cgacaggtaa cactgcaaca 480
ctgggtgttt ctttcaaatt cggcggcaac gatgagccag taatcgtaga agagccagtt 540
gttgttgaag aagtagttgt agaagaagtc gtagaagagc cagtagttgt aacgaaaaca 600
ttcgaaactc aaacaatcgg cactggtagc ttcgatctaa acagcacaac tctaaaacca 660
gagagcgctg caaaacttga taacctagtt gctttcctaa acgagcaccc acaagcgaac 720
gttgaagttg taggttacac agatacgtct ggcccagcag cttacaacct aaaagtttct 780
gagaaacgcg ctgaatctgt agctaacgca cttgttgaaa aaggtattga ttcatcacgt 840
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Gly Ser Phe Asp Leu Asn Ser Thr Thr Leu Lys Pro Glu Ser Ala Ala
210 215 220
Lys Leu Asp Asn Leu Val Ala Phe Leu Asn Glu His Pro Gln Ala Asn
225 230 235 240
Val Glu Val Val Gly Tyr Thr Asp Thr Ser Gly Pro Ala Ala Tyr Asn
245 250 255
Leu Lys Val Ser Glu Lys Arg Ala Glu Ser Val Ala Asn Ala Leu Val
260 265 270
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275 280 285
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Claims (3)
1.DNA改组后的细菌外膜蛋白ompAs-19,其特征在于,编码该蛋白的核苷酸序列如SEQID NO:1所示。
2.DNA改组后的细菌外膜蛋白ompAs-19,其特征在于,其氨基酸序列如如SEQ ID NO:2所示。
3.权利要求2所述的 DNA改组后的细菌外膜蛋白ompAs-19在制备针对脊椎动物的溶藻弧菌和迟缓爱德华氏菌EIB202疫苗中的应用。
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