一种包含钥孔血蓝蛋白片段的融合蛋白及其应用的制作方法

本发明涉及生物
技术领域：
，具体涉及一种包含钥孔血蓝蛋白片段的融合蛋白及其应用。
背景技术：
：抗体是高等动物特异性免疫应答反应所产生的免疫球蛋白，负责特异抗原的识别和清除。抗体不仅是机体抵抗病原体入侵的强大武器，也是基础科学研究中用于特异性分子识别的重要工具。随着基因组学研究深入，人们更加认识到基因组学研究的成果必须在蛋白水平得到进一步的研究和验证。一直以来，基于亲和(affinity)相互作用的研究手段是研究蛋白质功能的主要手段之一，而基于抗体-抗原亲和相互作用研究手段又是其中最主要的应用方法，在科研与生产应用中有多种衍生技术应用，包括elisa、免疫印记、免疫荧光、抗体芯片等。因此，制备高质量的抗体成为人们在科研、诊断以及治疗领域中一项核心技术。在抗体制备过程中，一般都用人工合成的多肽或者细菌表达的重组蛋白质作为抗原。人工合成多肽首先需对该蛋白含有的表位信息进行准确预测，由于合成的片段较短，因此还需要选择合适的免疫原性载体蛋白偶联以形成免疫原，否则用多肽刺激动物产生的抗体对相应的天然蛋白质抗原亲和力低或完全不能识别。而用细菌表达的重组蛋白质经常会遇到一些蛋白难以表达等情况。另外，有些蛋白质免疫原性较低，很难在机体刺激产生特异性抗体。因此，在制备针对各种不同的蛋白质特异性抗体时，首先需要在对抗原制备技术进行革新。研究发现，将特异性抗原蛋白与某一免疫原性增强蛋白构建成融合蛋白，可明显促进免疫动物对特异性靶蛋白的免疫应答。如suzue等发现结核分枝杆菌hsp70可加强小鼠对于与之相连的蛋白产生特异性的抗体或细胞免疫应答(suzue，k.&young,r.a.:j.immunol.156:873-9,1996)。专利cn201310437663公开结核分枝杆菌细胞色素c氧化酶亚基ⅱ蛋白和特异性抗原蛋白组成融合蛋白能显著增强后者的免疫原性。专利cn201210181392公开白喉毒素无毒突变体crm197或其片段在融合蛋白中作为分子内佐剂增强与其融合的目的蛋白的免疫原性。钥孔血蓝蛋白(keyholelimpethemocyanin，klh)广泛作为载体蛋白交联于半抗原和其他抗原，使它们具有更强的免疫原性以用于抗体的制备。但钥孔血蓝蛋白的氨基酸序列较长，其中的哪个片段与其他抗原连接能够显著增加免疫原性，迄今为止现有技术从未报道过。技术实现要素：本发明的目的在于提供一种包含钥孔血蓝蛋白片段的融合蛋白，用以增强与之融合的目的蛋白的免疫原性。为实现上述目的，本发明通过以下方案予以实现：一种融合蛋白，包含钥孔血蓝蛋白片段(sklh片段)及目的蛋白。优选地，所述的钥孔血蓝蛋白片段包含钥孔血蓝蛋白第1-245位氨基酸残基。优选地，所述的钥孔血蓝蛋白片段包含seqidno:1所示的第1-245位氨基酸残基。我们对klh蛋白序列研究发现，该蛋白n端含有多段t-细胞和b-细胞表位。动物实验表明，上述融合蛋白skhl片段能有效刺激针对与之相连蛋白的特异性免疫应答。优选地，所述的目的蛋白为植物线粒体膜蛋白(nad4)或其免疫原性片段。优选地，所述的目的蛋白的序列如seqidno:5所示。优选地，所述的钥孔血蓝蛋白片段连接至所述目的蛋白的n端和/或c端。在本发明中，将两种或更多种蛋白融合表达以形成融合蛋白的技术是本领域公知的(参见例如，j.sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989)。通常，通过使用重组dna技术将编码两种或更多种蛋白的dna片段符合读框地连接在一起，并进行蛋白质表达来获得融合蛋白。一种编码上述的融合蛋白的核苷酸。优选地，所述的核苷酸，包含seqidno:3所述的核苷酸序列。一种上述融合蛋白在制备针对特异性目的蛋白多克隆抗体中的应用。一种上述融合蛋白在作为疫苗或药物的应用。本发明方法具有如下优点：在将sklh片段与目的蛋白融合表达后，sklh片段显著增强了目的蛋白的免疫原性。实施例试验数据证明，sklh片段与目的蛋白nad4融合后，其作为抗原免疫获得的多克隆抗体特异性显著优于单独使用nad4蛋白制备的抗体。由于本发明的融合蛋白展示了比单独的目的蛋白更强的免疫原性，因此，通过融合sklh片段可用于制备针对特异性目的蛋白多克隆抗体。附图说明图1为sklh片段表达载体pe-sklh质粒图。图2为nad4蛋白表达载体pe-nad4质粒图。图3为融合蛋白表达载体pe-sklh-nad4质粒图。图4为nad4蛋白表达、纯化sds-page图。图5为sklh-nad4融合蛋白表达、纯化sds-page图。图6为westernblot鉴定兔抗血清质量效果图。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。实施例1sklh片段基因合成及nad4基因克隆sklh片段基因合成根据genbank数据库中钥孔血蓝蛋白蛋白基因编码序列(accessionnumber:aj698341.2，其核苷酸序列如seqidno:2所示)，同时依据e.coli遗传密码偏好性将sklh片段(1-245位氨基酸)基因序列进行了密码子优化，由南京金斯瑞生物科技有限公司合成并构建puc57-sklh质粒，经密码子优化并加入酶切位点后的核酸序列如seqidno:3所示。nad4基因克隆拟南芥叶片组织经液氮研磨并经trizol(invitrogen)提取获得rna，再经iii反转录酶处理获得cdna。以cdna为模板，高保真pcr获得nad4基因核苷酸序列(如seqidno:4所示)。扩增引物设计如表1所示：表1.nad4基因扩增引物表seqidno:引物名称引物序列(5’-3’)6nad4_ecori_ftccgaattcggtgttttatatgacc7nad4_hindiii_rgcaagcttatgaaatttgccatgttg实施例2蛋白表达载体pe-sklh-nad4质粒的构建pe-sklh质粒的构建质粒puc57-sklh(实施例1制备得到)经ncoi和bamhi双酶切(neb)，胶回收酶切含sklh片段基因(～740bp)，与同样经ncoi和bamhi双酶切胶回收后的pet-28a质粒连接，转化dh5α感受态细菌，卡拉霉素抗性平板筛选阳性克隆，t7引物测序鉴定pe-sklh质粒。pe-nad4质粒的构建高保真pcr获得的nad4基因核苷酸序列(实施例1制备得到)经ecori和hindiii双酶切(neb)，胶回收酶切含nad4核酸片段(～430bp)，与同样经ecori和hindiii双酶切胶回收后的pet-28a质粒连接，转化dh5α感受态细菌，卡拉霉素抗性平板筛选阳性克隆，t7引物测序鉴定pe-nad4质粒。pe-sklh-nad4质粒的构建质粒pe-nad4经ecori和hindiii双酶切(neb)，胶回收酶切含nad4核酸片段(～430bp)，与同样经ecori和hindiii双酶切胶回收后的pe-sklh质粒连接，转化dh5α感受态细菌，卡拉霉素抗性平板筛选阳性克隆，t7引物测序鉴定pe-sklh-nad4质粒。实施例3sklh-nad4融合蛋白表达及纯化从含卡那霉素lb平板上挑取含有pe-nad4及pe-sklh-nad4质粒的单菌落，分别接种于10ml含卡那霉素的液体lb培养基中，在37℃180rpm下振荡培养过夜，取5ml再转接到500ml含卡那霉素的lb培养液中，在37℃180rpm下振荡培养4-5小时。当od600达0.6左右时，加入iptg至终浓度0.5mm，在37℃下振荡诱导4个小时。诱导后，以8000g离心10min收集菌体，然后按1g菌体对应20ml裂解液(20mmtris-hcl，ph7.6，300mmnacl，5mmedta)悬浮，高压均质破碎2次，600bar。12000g离心30min，弃上清保留沉淀(即包涵体)。再用重悬液(20mmtris-hclph8.0，300mmnacl)重悬沉淀，高压均质破碎1次，600bar。离心弃上清，沉淀用含8m尿素上样缓冲液(20mmnapo3，ph8.0，300mmnacl，8murea)重悬，20000g离心30min后，取上清进行ni-nti(qiagen)亲和层析。nad4蛋白及sklh-nad4融合蛋白的表达、纯化情况如图5所示。实施例4兔多克隆抗血清的制备用纯化的抗原nad4蛋白及sklh-nad4融合蛋白分别对4月龄的健康新西兰大白兔进行免疫，初免剂量为0.3mg/只，二免、三免剂量为0.2mg/只。初免抗原为蛋白抗原与等体积弗氏完全佐剂混匀乳化，二免、三免、抗原蛋白与等体积弗氏不完全佐剂混匀乳化。每次免疫间隔时间为2周，三免2周后取采血，3000g离心15分钟后得到抗血清。弗氏完全佐剂与弗氏不完全佐剂均购自sigma。实施例5westernblot鉴定兔抗血清质量拟南芥叶片蛋白裂解提取后，进行sds-page电泳分离，并转移到pvdf膜上，用于westernblot鉴定。pvdf膜用含5％脱脂牛奶tbst溶液(50mmtris-hcl，ph7.6，150mmnacl，0.05％tween-20)封闭1h，然后加入按1:2500稀释，反应1h；用tbst溶液洗膜3次，每次15min；然后加入羊抗兔lgg偶联(辣根过氧化物酶)二抗孵育1h；继续用tbst溶液洗膜3次，每次15min；然后加入ecl反应液至膜上反应2min，胶片曝光10s-5min。分别用sklh-nad4融合蛋白和nad4蛋白免疫得到的多克隆抗体进行的westernblot检测结果示于图6。结果显示，使用融合蛋白sklh-nad融合蛋白作为抗原免疫获得的多克隆抗体特异性显著优于单独使用nad4蛋白制备的抗体。虽然，上文中已经用一般性说明及具体实施例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。sequencelisting<110>攸归（上海）生物科技有限公司<120>一种包含钥孔血蓝蛋白片段的融合蛋白及其应用<130><160>7<170>patentinversion3.5<210>1<211>3408<212>prt<213>钥孔血蓝蛋白（klh,keyholelimpethemocyanin）<400>1metleuservalargleuleuilevalvalleualaleualaasnala151015gluasnleuvalarglysservalgluhisleuthrglnglugluthr202530leuaspleuglnalaalaleuarggluleuglnmetaspserserser354045ileglypheglnlysilealaalaalahisglyalaproalasercys505560valhislysaspthrserilealacyscysilehisglymetprothr65707580pheprohistrphisargalatyrvalvalhismetgluargalaleu859095glnthrlysargargthrserglyleuprotyrtrpasptrpthrglu100105110proilethrglnleuproserleualaalaaspprovaltyrileasp115120125serglnglyglylysalahisthrasntyrtrptyrargglyasnile130135140asppheleuasplyslysthrasnargalavalaspaspargleuphe145150155160glulysvallysproglyglnhisthrhisleumetgluservalleu165170175aspalaleugluglnaspgluphecyslysphegluileglnpheglu180185190leualahisasnalailehistyrleuvalglyglylyshisasptyr195200205sermetalaasnleuglutyrthralatyrasppr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