本发明涉及蛋白表达技术领域,具体涉及一种增加人甲状腺过氧化物酶产量试剂和的表达方法。
背景技术:
甲状腺过氧化物酶(thyroidperoxidase,tpo)是一种含有血红素辅基的膜结合糖蛋白,整个分子分为膜内区,跨膜区和膜外区,cdna全长3kb,跨膜区的存在使tpo在表达时难以分泌出来。
自身免疫性甲状腺疾病(autoimmunethyroiddiseases,aitd)是由t细胞介导的器官特异自身免疫性疾病,患者血清内存在多种针对甲状腺抗原的自身抗体,甲状腺过氧化物酶抗体(thyroidperoxidaseantibody,tpo-ab)就是其中最重要的一种,普遍存在于自身免疫性甲状腺疾病中,血清中甲状腺过氧化物酶抗体水平的测定,是对自身免疫性甲状腺疾病高危人群筛查、诊断和治疗的有效手段。因此,tpo-ab水平的检测可用于对甲减、aitd等甲状腺疾病的诊断、病程进展、用药和愈后观察;且tpo-ab的测定还可作为graves病、桥本甲亢等诊断和鉴别诊断的首选指标,因此表达量高、性能优良的tpo蛋白,对于制作高效、低成本的甲状腺疾病检测试剂盒的开发显得尤为重要。
目前,国内外所用的抗原都来自进口,成本较高,且较难订货,现有技术多用传统杆状病毒制备重组tpo,再导入昆虫细胞中进行表达,但往往存在表达量低、折叠不充分的问题,会影响后续检测阳性率,难以用于临床检测。
技术实现要素:
为了解决tpo在昆虫细胞中表达产量低、折叠不充分,影响后续检测阳性率的问题,本文介绍一种可以满足市场需求的高产量、高质量和低成本的方案。
为了实现上述目的,本发明采用如下技术手段:
一种增加人甲状腺过氧化物酶表达产量的试剂,包括信号肽和分子伴侣。
优选地,所述信号肽包括pepidylglycinealphaamidatingmonooxygenase(pam,seqidno:1)、dbip(seqidno:2)、melittin(seqidno:3)和gp64(seqidno:4)。
优选地,所述分子伴侣为bip(seqidno:8)、pdi(seqidno:9)、calnexin(seqidno:10)或hsp70(seqidno:11)中的一种或几种。
更优选地,所述分子伴侣为calnexin。
本发明的另一方面提供了一种增加人甲状腺过氧化物酶表达产量的方法,包括
a、信号肽标记的tpo转入病毒细胞,制作tpo病毒;
b、分子伴侣转入病毒细胞,制作分子伴侣病毒;
c、将步骤a所述的tpo病毒和步骤b所述的分子伴侣病毒导入昆虫细胞中进行表达,得tpo蛋白。
可选地,所述步骤a之前可以包括信号肽的筛选过程,即将n端添加信号肽的tpo基因构建到载体中,转入dh10bac细菌中进行蓝白班筛选和pcr验证,提取正确细胞的bacmid质粒。所述信号肽筛选之后,将正确细胞的bacmid质粒,即信号肽标记的tpo,转入病毒细胞进行tpo病毒制作。
上述信号肽的筛选过程可以确保tpo蛋白顺利表达,并筛选表达量最高的信号肽进行步骤a。
优选地,所述步骤b分子伴侣病毒的制作具体过程为,合成分子伴侣序列并将其构建到载体中,转入dh10bac细菌中进行蓝白班筛选和pcr验证,提取正确细胞的bacmid质粒,转入病毒细胞进行分子伴侣病毒制作。
步骤a和步骤b无先后顺序,所述载体优选为pfastbac1载体,所述病毒细胞优选为sf9细胞,所述tpo病毒和所述分子伴侣病毒制作后,都需要利用qpcr进行病毒滴度定量,完成滴度测量之后,进行步骤c。
优选地,所述信号肽选自:pepidylglycinealphaamidatingmonooxygenase(pam,seqidno:1)、dbip(seqidno:2)、melittin(seqidno:3)或gp64(seqidno:4)中的一种或多种。
优选地,所述分子伴侣为bip(seqidno:8)、pdi(seqidno:9)、calnexin(seqidno:10)或hsp70(seqidno:11)中的一种或几种。
优选地,步骤c中所述昆虫细胞为h.five细胞
步骤c中tpo病毒和分子伴侣病毒共同的表达,可以帮助tpo蛋白正确折叠同时促进分泌,以此表达出高产量、高质量的tpo蛋白,满足下游检测的原料需求。
优选地,还包括步骤d,所述步骤d包括将步骤c中表达得到的tpo蛋白进行纯化。
优选地,所述纯化为柱层析纯化,所述柱层析纯化的洗脱液需要在10℃以内低温透析。更优选地为4℃透析,透析后对蛋白进行定量,优选地,所述定量利用bca法。
本发明还提供了信号肽和分子伴侣在人甲状腺过氧化物酶表达中的应用,所述信号肽包括pam(seqidno:1)、dbip(seqidno:2)、melittin(seqidno:3)或gp64(seqidno:4);所述分子伴侣包括bip(seqidno:8)、pdi(seqidno:9)、calnexin(seqidno:10)或hsp70(seqidno:11)。
本发明的有益效果在于:
(1)通过信号肽的筛选,可以确定最高分泌的信号肽,不仅可以提高产量,同时为分子伴侣提高产量打下基础。
(2)利用分子伴侣与tpo共表达,帮助tpo蛋白正确折叠并促进蛋白从细胞内分泌到细胞外,可以将产量在未使用分子伴侣的基础上提升5倍左右。
(3)表达蛋白的纯度大于95%,同时下游应用评价,对临床样品的评价结果较好,可以作为可靠优质诊断原料应用。
附图说明
图1是;添加信号肽标签的人tpo蛋白表达情况;
图2是;纯化后的共表达人tpo蛋白;
图3是:本方法生产tpo与市售天然tpo相关性实验。
具体实施例
为了使本发明的目的、技术方案和有点更加清楚,下面将结合附图对本发明的实施方式作进一步的详细描述。
实施例1:信号肽筛选
人tpo基因序列(seqidno:7)由上海捷瑞生物技术有限公司合成,n端添加不同信号肽(表1),然后构建到pfastbac1载体中,转入dh10bac细菌中进行蓝白班筛选和pcr验证(引物m13f5’-gtaaaacgacggccag-3’,m13r5’-gttttcccagtcacgac-3’,),确定正确后,提取bacmid质粒。转入sf9细胞进行病毒制作,扩大到p3代后,利用qpcr进行病毒滴度定量。最后在h.five细胞中进行表达验证,利用elisa检测试剂盒(throidperoxidasehumanelisakit,invitrogen)进行定量,结果如表2,其中pam、dbip、melittin和gp64信号肽标签的tpo表达量较高,适合后续实验,其中melittin效果最好;而azu1和serumalbuminpreproprotein虽然有活性,但表达量极低。
同时进行sds-page分析,结果如图1所示,其中泳道1-11分别代表:1:pam胞内2:dbip胞内3:melittin胞内4:gp64胞内5:负对照6:marker7:pam上清8:dbip上清9:melittin上清10:gp64上清11:负对照上清,发现胞内含有大量未分泌目的蛋白,因此尝试不同分子伴侣来增加目的蛋白的折叠和分泌,同时提高质量。
表1n端添加的信号肽的序列
表2添加不同信号肽标签的tpo表达量
实施例2:信号肽与分子伴侣共表达比较
a、制作tpo病毒
人tpo基因序列(seqidno:7)由上海捷瑞生物技术有限公司合成,n端添加不同信号肽(表1),然后构建到pfastbacl载体中,转入dh10bac细菌中进行蓝白班筛选和pcr验证(引物m13f5’-gtaaaacgacggccag-3’,m13r5’-gttttcccagtcacgac-3’,),确定正确后,提取bacmid质粒。转入sf9细胞进行病毒制作,扩大到p3代后,利用qpcr进行病毒滴度定量。
b、制作分子伴侣病毒
选择五种分子伴侣用于实验,分别为bindingimmunoglobulinprotein,bip(seqidno:8),proteindisulfideisomerase,pdi(seqidno:9),calnexin(seqidno:10),grp94(seqidno:12)、hsp70(seqidno:11),其中前4个都来自于老鼠,最后一种来自于人,基因序列委托上海捷瑞生物技术有限公司合成。构建到pfastbac1载体中,转入dh10bac细菌中进行蓝白班筛选和pcr验证(引物m13f5’-gtaaaacgacggccag-3’,m13r5’-gttttcccagtcacgac-3’),确定正确后,提取bacmid质粒。转入sf9细胞进行病毒制作,扩大到p3代后,利用qpcr进行病毒滴度定量。
c、将步骤a所述的tpo病毒和步骤b所述的分子伴侣病毒导入昆虫细胞进行共表达。
分子伴侣病毒和tpo病毒混合共转染,最后在h.five细胞中进行表达验证,利用elisa检测试剂盒(throidperoxidasehumanelisakit,invitrogen)进行定量,结果如表3,结果显示对于表达效果较好的信号肽标签pam、dbip、melittin、gp64,calnexin分子伴侣共表达的效果最好,比单纯的tpo+信号肽,表达量提升了5倍左右,最终产量达到19mg/l,可以用于临床实验。
挑选了五种分子伴侣,其中grp94对于表达没有提升作用,其余四种分子伴侣(bip、pdi、hsp70、calnexin)对表达都有明显提升作用,其中hsp70、calnexin效果最明显,但不同的分子伴侣对同一种信号肽的提升效果不同,同一分子伴侣对不同信号肽的提升效果也有区别,说明信号肽标签是分子伴侣共表达的基础,信号肽和分子伴侣之间的不同搭配,会产生不同的表达效果,根据实际情况可以选择需要的搭配。
表3分子伴侣共表达
实施例3:表达纯化
利用分子伴侣calnexin和tpo共表达模式摇瓶发酵1l,28℃,110rpm发酵72h收样,4500rpm4℃离心10min。离心后过niexcel柱进行纯化,洗脱液4℃透析到ph7.6的50mmtris、150mmnacl、0.1mm碘化钾的缓冲液中,bca法(piercetmbcaproteinassaykit,invitrogen,货号:23227)进行定量,共获得14mg蛋白,如图2所示,进行sds-page分析,结果表明纯化后的tpo蛋白纯度大于95%,可以用于临床试验。
实施例4:临床验证
阴阳符合率实验:通过对27例阳性样本和25例阴性样本进行评价,与市场天然tpo蛋白相比。结果如表4,结果表明本发明方法生产的tpo蛋白对阴性样本和阳性样本可以准确检测,不存在假阳性结果,也不存在假阴性结果。
相关性实验:利用本发明方法生产的tpo与市售天然tpo进行比较,在化学发光平台中,首先对tpo蛋白进行生物素标记,采用间接法反应模式对样本血清中anti-tpo进行测定,对27例阳性血清样本和25例阴性样本中anti-tpo进行测定,测定结果如图3所示,结果表明本发明方法制备的tpo蛋白与市场上售卖的天然tpo蛋白相关性达到0.9944,性能类似,所以通过此方案获得的蛋白,对于下游评价有明显优势,满足后续需求。
表4:阴阳符合率实验
应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
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545550555560
aspthrargasngluleuglusertyralatyrserleulysasngln
565570575
ileglyasplysglulysleuglyglylysleusersergluasplys
580585590
gluthrmetglulysalavalgluglulysileglutrpleugluser
595600605
hisglnaspalaaspilegluaspphelysalalyslyslysgluleu
610615620
glugluilevalglnproileileserlysleutyrglyserglygly
625630635640
proproprothrglyglugluaspthrserglulysaspgluleu
645650655
<210>9
<211>509
<212>prt
<213>mouse
<400>9
metleuserargalaleuleucysleualaleualatrpalaalaarg
151015
valglyalaaspalaleugluglugluaspasnvalleuvalleulys
202530
lysserasnphegluglualaleualaalahislystyrleuleuval
354045
gluphetyralaprotrpcysglyhiscyslysalaleualaproglu
505560
tyralalysalaalaalalysleulysalagluglysergluilearg
65707580
leualalysvalaspalathrglugluseraspleualaglnglntyr
859095
glyvalargglytyrprothrilelysphephelysasnglyaspthr
100105110
alaserprolysglutyrthralaglyargglualaaspaspileval
115120125
asntrpleulyslysargthrglyproalaalathrthrleuserasp
130135140
thralaalaalagluserleuvalaspsersergluvalthrvalile
145150155160
glyphephelysaspvalgluseraspseralalysglnpheleuleu
165170175
alaalaglualaileaspaspilepropheglyilethrserasnser
180185190
glyvalpheserlystyrglnleuasplysaspglyvalvalleuphe
195200205
lyslyspheaspgluglyargasnasnphegluglygluilethrlys
210215220
glulysleuleuasppheilelyshisasnglnleuproleuvalile
225230235240
gluphethrgluglnthralaprolysilepheglyglygluilelys
245250255
thrhisileleuleupheleuprolysservalserasptyraspgly
260265270
lysleuserserphelysargalaalagluglyphelysglylysile
275280285
leupheilepheileaspserasphisthraspasnglnargileleu
290295300
gluphepheglyleulyslysgluglucysproalavalargleuile
305310315320
thrleuglugluglumetthrlystyrlysprogluseraspgluleu
325330335
thralaglulysilethrgluphecyshisargpheleugluglylys
340345350
ilelysprohisleumetserglngluvalprogluasptrpasplys
355360365
glnprovallysvalleuvalglyalaasnpheglugluvalalaphe
370375380
aspglulyslysasnvalphevalgluphetyralaprotrpcysgly
385390395400
hiscyslysglnleualaproiletrpasplysleuglygluthrtyr
405410415
lysasphisgluasnileileilealalysmetaspserthralaasn
420425430
gluvalglualavallysvalhisserpheprothrleulysphephe
435440445
proalaseralaaspargthrvalileasptyrasnglygluargthr
450455460
leuaspglyphelyslyspheleugluserglyglyglnaspglyala
465470475480
glyaspaspgluaspleuaspleugluglualaleugluproaspmet
485490495
glugluaspaspaspglnlysalavallysaspgluleu
500505
<210>10
<211>586
<212>prt
<213>mouse
<400>10
metalalysalaalaalaileglyileaspleuglythrthrtyrser
151015
cysvalglyvalpheglnhisglylysvalgluileilealaasnasp
202530
glnglyasnargthrthrprosertyrvalalaphethraspthrglu
354045
argleuileglyaspalaalalysasnglnvalalaleuasnprogln
505560
asnthrvalpheaspalalysargleuileglyarglyspheglyasp
65707580
provalvalglnseraspmetlyshistrppropheglnvalileasn
859095
aspserglnargglnalathrlysaspalaglyvalilealaglyleu
100105110
asnvalleuargileileasngluprothralaalaalailealatyr
115120125
glyleuaspargthrglylysglygluargasnvalleuilepheasp
130135140
leuglyglyglythrpheaspvalserileleuthrileaspaspgly
145150155160
ilephegluvallysalathralaglyaspthrhisleuglyglyglu
165170175
asppheaspasnargleuvalasnhisphevalglugluphelysarg
180185190
lyshislyslysaspileserglnasnlysargalavalargargleu
195200205
argthralacysgluargalalysargthrleuserserserthrgln
210215220
alaserleugluileaspserleuphegluglyileaspphetyrthr
225230235240
serilethrargalaargpheglugluleucysseraspleuphearg
245250255
serthrleugluprovalglulysalaleuargaspalalysleuasp
260265270
lysalaglnilehisaspleuvalleuvalglyglyserthrargile
275280285
prolysvalglnlysleuleuglnaspphepheaspglyargaspleu
290295300
asnlysserileasnproaspglualavalalatyrglyalaalaval
305310315320
glnalaalaileleumetglyasplyssergluasnvalglnaspleu
325330335
leuleuleuaspvalalaproleuserleuglyleugluthralagly
340345350
glyvalmetthralaleuilelysargasnserthrileprothrlys
355360365
glnthrglnilephethrthrtyrseraspasnglnproglyvalleu
370375380
ileglnvaltyrgluglygluargalametthrlysaspasnasnleu
385390395400
leuglyargphegluleuserglyileproproalaproargglyval
405410415
proglnilegluvalthrpheaspileaspalaasnglyileleuasn
420425430
valthralathrasplysserthrglyasnalaasnlysilethrile
435440445
thrasnasplysglyargleuserlysglugluilegluargmetval
450455460
glnglualaglulystyrlysalagluaspgluvalglnarggluarg
465470475480
valseralalysasnalaleuglusertyralapheasnmetlysser
485490495
alavalgluaspgluglyleulysglylysileserglualaasplys
500505510
lyslysvalleuasplyscysglngluvalilesertrpleuaspala
515520525
asnthrleualaglulysaspgluphegluhislysarglysgluleu
530535540
gluglnvalcysasnproileileserglyleutyrglnglyalagly
545550555560
glyproglyproglyglypheglyalaglnglyprolysglyglyser
565570575
glyserglyprothrileglugluvalasp
580585
<210>11
<211>591
<212>prt
<213>human
<400>11
metgluglylystrpleuleucysleuleuleuvalleuglythrala
151015
alavalglualahisaspglyhisaspaspaspalaileaspileglu
202530
aspaspleuaspaspvalileglugluvalgluaspserlysserlys
354045
seraspalaserthrproproserprolysvalthrtyrlysalapro
505560
valprothrglygluvaltyrphealaaspserpheaspargglyser
65707580
leuserglytrpileleuserlysalalyslysaspaspthraspasp
859095
gluilealalystyraspglylystrpgluvalaspglumetlysglu
100105110
thrlysleuproglyasplysglyleuvalleumetserargalalys
115120125
hishisalaileseralalysleuasnlyspropheleupheaspthr
130135140
lysproleuilevalglntyrgluvalasnpheglnasnglyileglu
145150155160
cysglyglyalatyrvallysleuleuserlysthralagluleuser
165170175
leuaspglnphehisasplysthrprotyrthrilemetpheglypro
180185190
asplyscysglygluasptyrlysleuhispheilephearghislys
195200205
asnprolysthrglyvaltyrgluglulyshisalalysargproasp
210215220
alaaspleulysthrtyrphethrasplyslysthrhisleutyrthr
225230235240
leuileleuasnproaspasnserphegluileleuvalaspglnser
245250255
valvalasnserglyasnleuleuasnaspmetthrproprovalasn
260265270
proserarggluilegluaspprogluasparglysprogluasptrp
275280285
aspgluargprolysilealaaspproaspalavallysproaspasp
290295300
trpaspgluaspalaproserlysileproaspgluglualathrlys
305310315320
progluglytrpleuaspaspgluproglutyrileproaspproasp
325330335
alaglulysprogluasptrpaspgluaspmetaspglyglutrpglu
340345350
alaproglnilealaasnprolyscysgluseralaproglycysgly
355360365
valtrpglnargprometileaspasnproasntyrlysglylystrp
370375380
lysproprometileaspasnproasntyrglnglyiletrplyspro
385390395400
arglysileproasnproaspphephegluaspleugluprophelys
405410415
metthrpropheseralaileglyleugluleutrpsermetthrser
420425430
aspilephepheaspasnpheileileserglyaspargargvalval
435440445
aspasptrpalaasnaspglytrpglyleulyslysalaalaaspgly
450455460
alaalagluproglyvalvalleuglnmetleuglualaalagluglu
465470475480
argprotrpleutrpvalvaltyrileleuthrvalalaleuproval
485490495
pheleuvalileleuphecyscysserglylyslysglnserasnala
500505510
metglutyrlyslysthraspalaproglnproaspvallysaspglu
515520525
gluglylysglugluglulysasnlysargaspglugluglugluglu
530535540
glulysleugluglulysglnlysseraspalaglugluaspglyval
545550555560
thrglyserglnaspglugluaspserlysprolysalaglugluasp
565570575
gluileleuasnargserproargasnarglysproargargglu
580585590
<210>12
<211>802
<212>prt
<213>mouse
<400>12
metargvalleutrpvalleuglyleucyscysvalleuleuthrphe
151015
glyphevalargalaaspaspgluvalaspvalaspglythrvalglu
202530
gluaspleuglylysserarggluglyserargthraspaspgluval
354045
valglnargglugluglualaileglnleuaspglyleuasnalaser
505560
glnilearggluleuargglulysserglulysphealapheglnala
65707580
gluvalasnargmetmetlysleuileileasnserleutyrlysasn
859095
lysgluilepheleuarggluleuileserasnalaseraspalaleu
100105110
asplysileargleuileserleuthraspgluasnalaleualagly
115120125
asnglugluleuthrvallysilelyscysasplysglulysasnleu
130135140
leuhisvalthraspthrglyvalglymetthrargglugluleuval
145150155160
lysasnleuglythrilealalysserglythrserglupheleuasn
165170175
lysmetthrglualaglngluaspglyglnserthrsergluleuile
180185190
glyglnpheglyvalglyphetyrseralapheleuvalalaasplys
195200205
valilevalthrserlyshisasnasnaspthrglnhisiletrpglu
210215220
seraspserasnglupheservalilealaaspproargglyasnthr
225230235240
leuglyargglythrthrilethrleuvalleulysgluglualaser
245250255
asptyrleugluleuaspthrilelysasnleuvalarglystyrser
260265270
glnpheileasnpheproiletyrvaltrpserserlysthrgluthr
275280285
valglugluproleuglugluaspglualaalalysgluglulysglu
290295300
gluseraspaspglualaalavalgluglugluglugluglulyslys
305310315320
prolysthrlyslysvalglulysthrvaltrpasptrpgluleumet
325330335
asnaspilelysproiletrpglnargproserlysgluvalgluglu
340345350
aspglutyrlysalaphetyrlysserpheserlysgluseraspasp
355360365
prometalatyrilehisphethralagluglygluvalthrphelys
370375380
serileleuphevalprothrseralaproargglyleupheaspglu
385390395400
tyrglyserlyslysserasptyrilelysleutyrvalargargval
405410415
pheilethraspaspphehisaspmetmetprolystyrleuasnphe
420425430
vallysglyvalvalaspseraspaspleuproleuasnvalserarg
435440445
gluthrleuglnglnhislysleuleulysvalilearglyslysleu
450455460
valarglysthrleuaspmetilelyslysilealaaspglulystyr
465470475480
asnaspthrphetrplysglupheglythrasnilelysleuglyval
485490495
ilegluasphisserasnargthrargleualalysleuleuargphe
500505510
glnserserhishisserthraspilethrserleuaspglntyrval
515520525
gluargmetlysglulysglnasplysiletyrphemetalaglyser
530535540
serarglysglualagluserserprophevalgluargleuleulys
545550555560
lysglytyrgluvaliletyrleuthrgluprovalaspglutyrcys
565570575
ileglnalaleuproglupheaspglylysargpheglnasnvalala
580585590
lysgluglyvallyspheaspgluserglulysthrlysgluserarg
595600605
glualathrglulysgluphegluproleuleuasntrpmetlysasp
610615620
lysalaleulysasplysileglulysalavalvalserglnargleu
625630635640
thrgluserprocysalaleuvalalaserglntyrglytrpsergly
645650655
asnmetgluargilemetlysalaglnalatyrglnthrglylysasp
660665670
ileserthrasntyrtyralaserglnlyslysthrphegluileasn
675680685
proarghisproleuileargaspmetleuargargilelysgluasp
690695700
gluaspasplysthrvalmetaspleualavalvalleuphegluthr
705710715720
alathrleuargserglytyrleuleuproaspthrlysalatyrgly
725730735
aspargilegluargmetleuargleuserleuasnileaspproglu
740745750
alaglnvalgluglugluprogluglugluprogluaspthrserglu
755760765
aspalagluaspsergluglnaspgluglygluglumetaspalagly
770775780
thrglugluglugluglugluthrglulysgluserthrglulysasp
785790795800
gluleu