本發(fā)明涉及生物
技術(shù)領(lǐng)域：
，具體涉及一種包含鑰孔血藍(lán)蛋白片段的融合蛋白及其應(yīng)用。
背景技術(shù)：
：抗體是高等動(dòng)物特異性免疫應(yīng)答反應(yīng)所產(chǎn)生的免疫球蛋白，負(fù)責(zé)特異抗原的識(shí)別和清除。抗體不僅是機(jī)體抵抗病原體入侵的強(qiáng)大武器，也是基礎(chǔ)科學(xué)研究中用于特異性分子識(shí)別的重要工具。隨著基因組學(xué)研究深入，人們更加認(rèn)識(shí)到基因組學(xué)研究的成果必須在蛋白水平得到進(jìn)一步的研究和驗(yàn)證。一直以來(lái)，基于親和(affinity)相互作用的研究手段是研究蛋白質(zhì)功能的主要手段之一，而基于抗體-抗原親和相互作用研究手段又是其中最主要的應(yīng)用方法，在科研與生產(chǎn)應(yīng)用中有多種衍生技術(shù)應(yīng)用，包括elisa、免疫印記、免疫熒光、抗體芯片等。因此，制備高質(zhì)量的抗體成為人們?cè)诳蒲小⒃\斷以及治療領(lǐng)域中一項(xiàng)核心技術(shù)。在抗體制備過(guò)程中，一般都用人工合成的多肽或者細(xì)菌表達(dá)的重組蛋白質(zhì)作為抗原。人工合成多肽首先需對(duì)該蛋白含有的表位信息進(jìn)行準(zhǔn)確預(yù)測(cè)，由于合成的片段較短，因此還需要選擇合適的免疫原性載體蛋白偶聯(lián)以形成免疫原，否則用多肽刺激動(dòng)物產(chǎn)生的抗體對(duì)相應(yīng)的天然蛋白質(zhì)抗原親和力低或完全不能識(shí)別。而用細(xì)菌表達(dá)的重組蛋白質(zhì)經(jīng)常會(huì)遇到一些蛋白難以表達(dá)等情況。另外，有些蛋白質(zhì)免疫原性較低，很難在機(jī)體刺激產(chǎn)生特異性抗體。因此，在制備針對(duì)各種不同的蛋白質(zhì)特異性抗體時(shí)，首先需要在對(duì)抗原制備技術(shù)進(jìn)行革新。研究發(fā)現(xiàn)，將特異性抗原蛋白與某一免疫原性增強(qiáng)蛋白構(gòu)建成融合蛋白，可明顯促進(jìn)免疫動(dòng)物對(duì)特異性靶蛋白的免疫應(yīng)答。如suzue等發(fā)現(xiàn)結(jié)核分枝桿菌hsp70可加強(qiáng)小鼠對(duì)于與之相連的蛋白產(chǎn)生特異性的抗體或細(xì)胞免疫應(yīng)答(suzue，k.&young,r.a.:j.immunol.156:873-9,1996)。專(zhuān)利cn201310437663公開(kāi)結(jié)核分枝桿菌細(xì)胞色素c氧化酶亞基ⅱ蛋白和特異性抗原蛋白組成融合蛋白能顯著增強(qiáng)后者的免疫原性。專(zhuān)利cn201210181392公開(kāi)白喉毒素?zé)o毒突變體crm197或其片段在融合蛋白中作為分子內(nèi)佐劑增強(qiáng)與其融合的目的蛋白的免疫原性。鑰孔血藍(lán)蛋白(keyholelimpethemocyanin，klh)廣泛作為載體蛋白交聯(lián)于半抗原和其他抗原，使它們具有更強(qiáng)的免疫原性以用于抗體的制備。但鑰孔血藍(lán)蛋白的氨基酸序列較長(zhǎng)，其中的哪個(gè)片段與其他抗原連接能夠顯著增加免疫原性，迄今為止現(xiàn)有技術(shù)從未報(bào)道過(guò)。技術(shù)實(shí)現(xiàn)要素：本發(fā)明的目的在于提供一種包含鑰孔血藍(lán)蛋白片段的融合蛋白，用以增強(qiáng)與之融合的目的蛋白的免疫原性。為實(shí)現(xiàn)上述目的，本發(fā)明通過(guò)以下方案予以實(shí)現(xiàn)：一種融合蛋白，包含鑰孔血藍(lán)蛋白片段(sklh片段)及目的蛋白。優(yōu)選地，所述的鑰孔血藍(lán)蛋白片段包含鑰孔血藍(lán)蛋白第1-245位氨基酸殘基。優(yōu)選地，所述的鑰孔血藍(lán)蛋白片段包含seqidno:1所示的第1-245位氨基酸殘基。我們對(duì)klh蛋白序列研究發(fā)現(xiàn)，該蛋白n端含有多段t-細(xì)胞和b-細(xì)胞表位。動(dòng)物實(shí)驗(yàn)表明，上述融合蛋白skhl片段能有效刺激針對(duì)與之相連蛋白的特異性免疫應(yīng)答。優(yōu)選地，所述的目的蛋白為植物線(xiàn)粒體膜蛋白(nad4)或其免疫原性片段。優(yōu)選地，所述的目的蛋白的序列如seqidno:5所示。優(yōu)選地，所述的鑰孔血藍(lán)蛋白片段連接至所述目的蛋白的n端和/或c端。在本發(fā)明中，將兩種或更多種蛋白融合表達(dá)以形成融合蛋白的技術(shù)是本領(lǐng)域公知的(參見(jiàn)例如，j.sambrook等人，分子克?。簩?shí)驗(yàn)室手冊(cè)，第2版，冷泉港實(shí)驗(yàn)室出版社，1989)。通常，通過(guò)使用重組dna技術(shù)將編碼兩種或更多種蛋白的dna片段符合讀框地連接在一起，并進(jìn)行蛋白質(zhì)表達(dá)來(lái)獲得融合蛋白。一種編碼上述的融合蛋白的核苷酸。優(yōu)選地，所述的核苷酸，包含seqidno:3所述的核苷酸序列。一種上述融合蛋白在制備針對(duì)特異性目的蛋白多克隆抗體中的應(yīng)用。一種上述融合蛋白在作為疫苗或藥物的應(yīng)用。本發(fā)明方法具有如下優(yōu)點(diǎn)：在將sklh片段與目的蛋白融合表達(dá)后，sklh片段顯著增強(qiáng)了目的蛋白的免疫原性。實(shí)施例試驗(yàn)數(shù)據(jù)證明，sklh片段與目的蛋白nad4融合后，其作為抗原免疫獲得的多克隆抗體特異性顯著優(yōu)于單獨(dú)使用nad4蛋白制備的抗體。由于本發(fā)明的融合蛋白展示了比單獨(dú)的目的蛋白更強(qiáng)的免疫原性，因此，通過(guò)融合sklh片段可用于制備針對(duì)特異性目的蛋白多克隆抗體。附圖說(shuō)明圖1為sklh片段表達(dá)載體pe-sklh質(zhì)粒圖。圖2為nad4蛋白表達(dá)載體pe-nad4質(zhì)粒圖。圖3為融合蛋白表達(dá)載體pe-sklh-nad4質(zhì)粒圖。圖4為nad4蛋白表達(dá)、純化sds-page圖。圖5為sklh-nad4融合蛋白表達(dá)、純化sds-page圖。圖6為westernblot鑒定兔抗血清質(zhì)量效果圖。具體實(shí)施方式以下實(shí)施例用于說(shuō)明本發(fā)明，但不用來(lái)限制本發(fā)明的范圍。實(shí)施例1sklh片段基因合成及nad4基因克隆sklh片段基因合成根據(jù)genbank數(shù)據(jù)庫(kù)中鑰孔血藍(lán)蛋白蛋白基因編碼序列(accessionnumber:aj698341.2，其核苷酸序列如seqidno:2所示)，同時(shí)依據(jù)e.coli遺傳密碼偏好性將sklh片段(1-245位氨基酸)基因序列進(jìn)行了密碼子優(yōu)化，由南京金斯瑞生物科技有限公司合成并構(gòu)建puc57-sklh質(zhì)粒，經(jīng)密碼子優(yōu)化并加入酶切位點(diǎn)后的核酸序列如seqidno:3所示。nad4基因克隆擬南芥葉片組織經(jīng)液氮研磨并經(jīng)trizol(invitrogen)提取獲得rna，再經(jīng)iii反轉(zhuǎn)錄酶處理獲得cdna。以cdna為模板，高保真pcr獲得nad4基因核苷酸序列(如seqidno:4所示)。擴(kuò)增引物設(shè)計(jì)如表1所示：表1.nad4基因擴(kuò)增引物表seqidno:引物名稱(chēng)引物序列(5’-3’)6nad4_ecori_ftccgaattcggtgttttatatgacc7nad4_hindiii_rgcaagcttatgaaatttgccatgttg實(shí)施例2蛋白表達(dá)載體pe-sklh-nad4質(zhì)粒的構(gòu)建pe-sklh質(zhì)粒的構(gòu)建質(zhì)粒puc57-sklh(實(shí)施例1制備得到)經(jīng)ncoi和bamhi雙酶切(neb)，膠回收酶切含sklh片段基因(～740bp)，與同樣經(jīng)ncoi和bamhi雙酶切膠回收后的pet-28a質(zhì)粒連接，轉(zhuǎn)化dh5α感受態(tài)細(xì)菌，卡拉霉素抗性平板篩選陽(yáng)性克隆，t7引物測(cè)序鑒定pe-sklh質(zhì)粒。pe-nad4質(zhì)粒的構(gòu)建高保真pcr獲得的nad4基因核苷酸序列(實(shí)施例1制備得到)經(jīng)ecori和hindiii雙酶切(neb)，膠回收酶切含nad4核酸片段(～430bp)，與同樣經(jīng)ecori和hindiii雙酶切膠回收后的pet-28a質(zhì)粒連接，轉(zhuǎn)化dh5α感受態(tài)細(xì)菌，卡拉霉素抗性平板篩選陽(yáng)性克隆，t7引物測(cè)序鑒定pe-nad4質(zhì)粒。pe-sklh-nad4質(zhì)粒的構(gòu)建質(zhì)粒pe-nad4經(jīng)ecori和hindiii雙酶切(neb)，膠回收酶切含nad4核酸片段(～430bp)，與同樣經(jīng)ecori和hindiii雙酶切膠回收后的pe-sklh質(zhì)粒連接，轉(zhuǎn)化dh5α感受態(tài)細(xì)菌，卡拉霉素抗性平板篩選陽(yáng)性克隆，t7引物測(cè)序鑒定pe-sklh-nad4質(zhì)粒。實(shí)施例3sklh-nad4融合蛋白表達(dá)及純化從含卡那霉素lb平板上挑取含有pe-nad4及pe-sklh-nad4質(zhì)粒的單菌落，分別接種于10ml含卡那霉素的液體lb培養(yǎng)基中，在37℃180rpm下振蕩培養(yǎng)過(guò)夜，取5ml再轉(zhuǎn)接到500ml含卡那霉素的lb培養(yǎng)液中，在37℃180rpm下振蕩培養(yǎng)4-5小時(shí)。當(dāng)od600達(dá)0.6左右時(shí)，加入iptg至終濃度0.5mm，在37℃下振蕩誘導(dǎo)4個(gè)小時(shí)。誘導(dǎo)后，以8000g離心10min收集菌體，然后按1g菌體對(duì)應(yīng)20ml裂解液(20mmtris-hcl，ph7.6，300mmnacl，5mmedta)懸浮，高壓均質(zhì)破碎2次，600bar。12000g離心30min，棄上清保留沉淀(即包涵體)。再用重懸液(20mmtris-hclph8.0，300mmnacl)重懸沉淀，高壓均質(zhì)破碎1次，600bar。離心棄上清，沉淀用含8m尿素上樣緩沖液(20mmnapo3，ph8.0，300mmnacl，8murea)重懸，20000g離心30min后，取上清進(jìn)行ni-nti(qiagen)親和層析。nad4蛋白及sklh-nad4融合蛋白的表達(dá)、純化情況如圖5所示。實(shí)施例4兔多克隆抗血清的制備用純化的抗原nad4蛋白及sklh-nad4融合蛋白分別對(duì)4月齡的健康新西蘭大白兔進(jìn)行免疫，初免劑量為0.3mg/只，二免、三免劑量為0.2mg/只。初免抗原為蛋白抗原與等體積弗氏完全佐劑混勻乳化，二免、三免、抗原蛋白與等體積弗氏不完全佐劑混勻乳化。每次免疫間隔時(shí)間為2周，三免2周后取采血，3000g離心15分鐘后得到抗血清。弗氏完全佐劑與弗氏不完全佐劑均購(gòu)自sigma。實(shí)施例5westernblot鑒定兔抗血清質(zhì)量擬南芥葉片蛋白裂解提取后，進(jìn)行sds-page電泳分離，并轉(zhuǎn)移到pvdf膜上，用于westernblot鑒定。pvdf膜用含5％脫脂牛奶tbst溶液(50mmtris-hcl，ph7.6，150mmnacl，0.05％tween-20)封閉1h，然后加入按1:2500稀釋?zhuān)磻?yīng)1h；用tbst溶液洗膜3次，每次15min；然后加入羊抗兔lgg偶聯(lián)(辣根過(guò)氧化物酶)二抗孵育1h；繼續(xù)用tbst溶液洗膜3次，每次15min；然后加入ecl反應(yīng)液至膜上反應(yīng)2min，膠片曝光10s-5min。分別用sklh-nad4融合蛋白和nad4蛋白免疫得到的多克隆抗體進(jìn)行的westernblot檢測(cè)結(jié)果示于圖6。結(jié)果顯示，使用融合蛋白sklh-nad融合蛋白作為抗原免疫獲得的多克隆抗體特異性顯著優(yōu)于單獨(dú)使用nad4蛋白制備的抗體。雖然，上文中已經(jīng)用一般性說(shuō)明及具體實(shí)施例對(duì)本發(fā)明作了詳盡的描述，但在本發(fā)明基礎(chǔ)上，可以對(duì)之作一些修改或改進(jìn)，這對(duì)本領(lǐng)域技術(shù)人員而言是顯而易見(jiàn)的。因此，在不偏離本發(fā)明精神的基礎(chǔ)上所做的這些修改或改進(jìn)，均屬于本發(fā)明要求保護(hù)的范圍。sequencelisting<110>攸歸（上海）生物科技有限公司<120>一種包含鑰孔血藍(lán)蛋白片段的融合蛋白及其應(yīng)用<130><160>7<170>patentinversion3.5<210>1<211>3408<212>prt<213>鑰孔血藍(lán)蛋白（klh,keyholelimpethemocyanin）<400>1metleuservalargleuleuilevalvalleualaleualaasnala151015gluasnleuvalarglysservalgluhisleuthrglnglugluthr202530leuaspleuglnalaalaleuarggluleuglnmetaspserserser354045ileglypheglnlysilealaalaalahisglyalaproalasercys505560valhislysaspthrserilealacyscysilehisglymetprothr65707580pheprohistrphisargalatyrvalvalhismetgluargalaleu859095glnthrlysargargthrserglyleuprotyrtrpasptrpthrglu100105110proilethrglnleuproserleualaalaaspprovaltyrileasp115120125serglnglyglylysalahisthrasntyrtrptyrargglyasnile130135140asppheleuasplyslysthrasnargalavalaspaspargleuphe145150155160glulysvallysproglyglnhisthrhisleumetgluservalleu165170175aspalaleugluglnaspgluphecyslysphegluileglnpheglu180185190leualahisasnalailehistyrleuvalglyglylyshisasptyr195200205sermetalaasnleuglutyrthralatyraspproilephepheleu210215220hishisserasnvalaspargilephealailetrpglnargleugln225230235240gluleuargasnlysaspprolysalametaspcysalaglngluleu245250255leuhisglnlysmetgluprophesertrpgluaspasnaspilepro260265270leuthrasnasptyraspthrleuasnleuasnglymetthrproglu275280285gluleulysthrtyrleuaspgluargserserargalaargalaphe290295300alaserpheargleulysglypheglyglyseralaasnvalpheval305310315320tyrvalcysileproaspaspasnaspargasnaspasphiscysglu325330335lysalaglyaspphephevalleuglyglyproserglumetlystrp340345350glnphetyrargprotyrleupheaspleuseraspthrvalhislys355360365metglymetlysleuaspglyhistyrthrvallysalagluleuphe370375380servalasnglythralaleuproaspaspleuleuprohisproval385390395400valvalhishisproglulysglyphethraspproprovallyshis405410415hisglnseralaasnleuleuvalarglysasnileasnaspleuthr420425430argglugluvalleuasnleuargglualaphehislyspheglnglu435440445aspargservalaspglytyrglnalathralaglutyrhisglyleu450455460proalaargcysproargproaspalalysaspargtyralacyscys465470475480valhisglymetproilepheprohistrphisargleuphevalthr485490495glnvalgluaspalaleuvalglyargglyalathrileglyilepro500505510tyrtrpglyleuprotyrtrpasptrpthrgluprometthrhisile515520525proglyleualaglyasnlysthrtyrvalaspserhisglyalaser530535540histhrasnprophehisserservalilealapheglugluasnala545550555560prohisthrlysargglnileaspglnargleuphelysproalathr565570575pheglyhishisthraspleupheasnglnileleutyralapheglu580585590glngluasptyrcysaspphegluvalglnphegluilethrhisasn595600605thrilehisalatrpthrglyglysergluhisphesermetserser610615620leuhistyrthralapheaspproleuphetyrphehishisserasn625630635640valaspargleutrpalavaltrpglnalaleuglnmetargarghis645650655lysprotyrargalahiscysalaileserleugluhismethisleu660665670lysprophealapheserserproleuasnasnasnglulysthrhis675680685alaasnalametproasnlysiletyrasptyrgluasnvalleuhis690695700tyrthrtyrgluaspleuthrpheglyglyileserleugluasnile705710715720glulysmetilehisgluasnglnglngluaspargiletyralagly725730735pheleuleualaglyileargthrseralaasnvalaspilepheile740745750lysthrthraspservalglnhislysalaglythrphealavalleu755760765glyglyserlysglumetlystrpglypheaspargvalphelysphe770775780aspilethrhisvalleulysaspleuaspleuthralaaspglyasp785790795800phegluvalthrvalaspilethrgluvalaspglythrlysleuala805810815serserleuileprohisalaservalilearggluhisalaarggly820825830lysleuasnargvallyspheasplysvalproargserargleuile835840845arglysasnvalaspargleuserprogluglumetasngluleuarg850855860lysalaleualaleuleulysgluasplysseralaglyglyphegln865870875880glnleuglyalaphehisglygluprolystrpcysproserproglu885890895alaserlyslysphealacyscysvalhisglymetservalphepro900905910histrphisargleuleuthrvalglnsergluasnalaleuargarg915920925hisglytyraspglyalaleuprotyrtrpasptrpthrserproleu930935940asnhisleuprogluleualaasphisglulystyrvalaspproglu945950955960aspglyvalglulyshisasnprotrppheaspglyhisileaspthr965970975valasplysthrthrthrargservalglnasnlysleupheglugln980985990proglupheglyhistyrthrserilealalysglnvalleuleuala99510001005leugluglnaspasnphecysaspphegluileglntyrgluile101010151020alahisasntyrilehisalaleuvalglyglyalaglnprotyr102510301035glymetalaserleuargtyrthralapheaspproleuphetyr104010451050leuhishisserasnthraspargiletrpalailetrpglnala105510601065leuglnlystyrargglylysprotyrasnvalalaasncysala107010751080valthrsermetarggluproleuglnpropheglyleuserala108510901095asnileasnthrasphisvalthrlysgluhisservalprophe110011051110asnvalpheasptyrlysthrasnpheasntyrglutyraspthr111511201125leuglupheasnglyleuserileserglnleuasnlyslysleu113011351140glualailelysserglnaspargphephea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