CN102174522A - 一种4-1bbl蛋白的制备方法 - Google Patents
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Abstract
本发明提供了一种4-1BBL蛋白的制备方法,所述方法是采用小鼠4-1BBL(TNFSF9,NP_033430.1)胞外段蛋白的cDNA以基因工程方法制备含206个氨基酸残基(Arg 104-Glu 309)的重组4-1BBL蛋白。采用本发明方法,可大量获得4-1BBL蛋白,大大提高了蛋白得率,获得的4-1BBL胞外段蛋白可以应用于制备对4-1BBL蛋白定量的临床前研究试剂盒或药用,应用前景广阔。
Description
(一)技术领域
本发明涉及一种4-1BBL蛋白的制备方法。
(二)背景技术
4-1BB/4-1BBL是CD28/B7之外的另一对重要的协同刺激信号分子。4-1BB(CD137)属肿瘤坏死因子受体(TNFR)超家族,主要分布在活化的T细胞、NK细胞和DC细胞等表面,其配体4-1BBL(CD137L)主要表达在专职APC细胞(如单核巨噬细胞、DC细胞、B细胞)、活化的T细胞及一些肿瘤细胞上。4-1BBL是II型跨膜糖蛋白,分子量为34KD,人和小鼠有36%的同源性。
4-1BB/4-1BBL信号对增强和维持免疫应答有重要作用。与CD28/B7信号传导通路比较,4-1BB/4-1BBL主要在免疫应答的后期,为CD8+T细胞克隆扩增、存活和记忆性杀伤提供信号,其刺激T细胞增殖和分泌细胞因子的作用在缺乏强大的CD28/B7信号时表现更明显。4-1BB的激活对CD4+和CD8+T细胞均有作用,但优先刺激CD8+T细胞的活化。除了通过4-1BB受体刺激T细胞外,4-1BBL也通过其反向信号传导通路刺激单核细胞,导致M-CSF表达及释放多种细胞因子,延长单核细胞存活期,促进细胞增殖和有丝***。
近年,4-1BB/4-1BBL信号传导通路已成为肿瘤免疫治疗设计的一个新靶点。体内实验表明,4-1BB单抗能预防活化诱导的细胞死亡(AICD),促进荷瘤宿主中淋巴细胞的增殖,并选择性促进I型细胞因子分泌,增强效应细胞的抗肿瘤作用。这种抗肿瘤作用对肿瘤的再次攻击有记忆性,证实有长期的抗肿瘤免疫反应存在。此外,转染4-1BBL的肿瘤细胞接种小鼠后无肿瘤生长,并存在抵御肿瘤再次攻击的免疫力。研究还发现,融合蛋白Ig-4-1BBL与抗-4-1BB抗体一样能有效诱导肿瘤持续的特异性CTLs,治疗小鼠肝结肠癌。
另一方面,在治疗性疫苗的研究中证实,表达4-1BBL的重组痘病毒明显增强HIV疫苗诱导小鼠产生以IFN-γ应答为特征的特异性CD8+T细胞应答,这种作用在免疫后2个月仍能检测到。在CEA-转基因小鼠模型中,4-1BBL重组痘病毒明显增强表达B7.1和CEA的重组痘病毒疫苗的治疗作用,该作用与CEA特异性CD4+和CD8+T细胞应答水平增高相关。最近有报道,以包括4-1BBL在内的几种共刺激分子作为DNA疫苗的分子佐剂能诱导强大的细胞应答和回忆反应。以上结果均展示了4-1BBL在人类肿瘤和感染性疾病免疫治疗中的应用前景。
现有技术对4-1BBL蛋白的制备方法是直接从组织细胞提取,得率低、成本高。不能大量应用于体内试验或临床。
(三)发明内容
为了提高4-1BBL蛋白的得率,降低成本,为制备临床前试验用药或诊断试剂提供大量的原材料,本发明提供了一种采用4-1BBL(TNFSF9,NP_033430.1)胞外段蛋白的cDNA以基因工程方法制备含206个氨基酸残基(Arg 104-Glu 309)的重组4-1BBL蛋白的方法。
本发明采用的技术方案是:
一种4-1BBL蛋白的制备方法,所述方法包括:
(1)取含4-1BBL蛋白的小鼠淋巴细胞,conA体外刺激培养后经trizol裂解,抽提得到mRNA;所用原料为常规含4-1BBL蛋白的小鼠淋巴细胞;
(2)将mRNA逆转录得到4-1BBL胞外段蛋白的cDNA;
(3)对步骤(2)所得cDNA进行PCR扩增,得到4-1BBL蛋白目的基因;PCR扩增引物为:上游引物:5’-CTC GAG AAA AGA AGAACC GAG CCA AGA CCT-3’,下游引物:5’-GAA TTC TTC CCATGG ATT ATC AGG-3’;
(4)将步骤(3)PCR产物用Xho I和EcoR I双酶切后定向***经同样双酶切的ppic9,获得重组质粒ppic9-4-1BBL;
(5)将重组质粒ppic9-4-1BBL转化至大肠杆菌DH5α,经培养扩增、抽提纯化、测序分析,获得测序正确的重组质粒;
(6)将测序正确的重组质粒转化至甲醇酵母菌,诱导表达重组蛋白;
(7)将表达重组蛋白的活化菌株进行诱导发酵培养,发酵液离心,取上清液分离纯化,得到纯化的4-1BBL重组蛋白。
步骤(5)重组质粒ppic9-4-1BBL的核苷酸序列如下: CTC GAG AAAAGA AGA ACC GAG CCA AGA CCT GCA TTG ACC ATT ACT ACT TCTCCA AAC TTG GGT ACA AGA GAA AAC A A C GCT GAC CAG GTCACT CCA GTT TCT CAC ATC GGA TGT CCA AAC ACC ACC CAG CAGGGA TCT CCT GTT TTT GCT AA G TTG TTG GCC AAA AAC CAG GCTTCT TTG TGT AAC ACT ACC TTG AAC TGG CAC TCT CAA GAC GGTGCC GGT TCT TCT TA C TTG TCT CAG GGT TTG AGA TAC GAG GAGGAC AAG AAG GAG TTG GTC GTC GAC TCT CCT GGA TTG TAC TACGTC TTT TTG GAG TTG AAG TTG TCT CCA ACA TTC ACC AAC ACTGGA CAC AAG GTT CAG GGA TGG GTT TCT TTG GTT TTG CAG G CTAAG CCA CAG GTT GAC GAT TTC GAC AAT TTG GCC TTG ACT GTCGAA TTG TTC CCT TGC TCT ATG GAA AAC A AA TTG GTC GAC AGATCT TGG TCT CAA TTG TTG TTG TTG AAG GCC GGA CAC AGA TTGTCT GTC GGA TTG AGA GCA TA C TTG C AT GGT GCA CAA GAC GCTTAC AGA GAC TGG GAG TTG TCT TAC CCT AAC ACC ACT TCT TTCGGT TTG TTC TTG
所制得重组蛋白的氨基酸序列如下:
RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE。
应用本发明方法获得的4-1BBL蛋白,是一个具有高灵敏度、高特异性的新指标,运用该发明技术获得的4-1BBL蛋白免疫动物制备抗体,可以制成诊断试剂盒,用于对上述免疫相关疾病动物模型的发病机制、诊断、鉴别诊断及预后与疗效的观察。运用本发明制成成品的4-1BBL蛋白可用于临床药物的前期制备和研究,如疫苗的佐剂应用和疗效。以上具体应用方式对本领域普通技术人员来说属于公知常识,因此不再赘述。
本发明的有益效果主要体现在:采用本发明方法,可大量获得4-1BBL蛋白,大大提高了蛋白得率,获得的4-1BBL胞外段蛋白可以应用于制备对4-1BBL蛋白定量的试剂盒或药用,应用前景广阔。
(四)附图说明
图1为4-1BBL胞外区蛋白的SDS-PAGE及Western blot分析;A:SDS-PAGE,1:protein marker;2:空质粒转化的酵母菌上清;3:4-1BBL重组质粒转化的酵母菌上清,目的蛋白为分子量约70KD的三聚体;B:Western blot;
图2为重组蛋白4-1BBL促T细胞增殖实验的分析(cpm);
图3为小鼠T细胞48h培养上清液的IL-2水平(ng/L);
图4为小鼠T细胞48h培养上清液的IFN-γ水平(ng/L)。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实施例1:4-1BBL蛋白的制备
(1)分离BALB/c小鼠脾细胞(无菌取BALB/c小鼠脾脏,玻璃针芯研磨后过150目筛网,获单细胞悬液),conA体外刺激培养(RPMI1640洗涤后,以含10%胎牛血清的RPMI1640(含conA5μg/ml)重悬并调整细胞浓度为1×106/ml,置37℃、5% CO2条件培养72h,收集细胞用于提取总RNA。)后经trizol裂解(0.1ml细胞/1ml trizol,42℃ 30~40min),抽提得到mRNA;
(2)将mRNA逆转录(Superscript II反转录试剂盒购自Invitrogen公司)得到4-1BBL胞外段蛋白的cDNA;
(3)以步骤(2)所得cDNA为模板进行PCR扩增,得到4-1BBL蛋白目的基因;
PCR扩增引物为:
上游引物:5’-CTC GAG AAA AGA AGA ACC GAG CCA AGA
CCT-3’
下游引物:5’-GAA TTC TTC CCA TGG ATT ATC AGG-3’;
PCR反应液组成(Buffer、dNTP、DNA Polymerase购自Invitrogen公司):
ddH2O 37.60μL
10×Buffer 5μL
dNTP mixture(2.5mM) 4μL
上游引物(20pmol/μL) 1μL
下游引物(20pmol/μL) 1μL
MgCl2(25mM) 0.5μL
cDNA模板(约104拷贝) 0.5μL
DNA Polymerase 0.4μL
总体积 50μL
PCR反应条件:
95℃变性处理3min后进入循环,94℃ 30S、56℃ 30S、72℃1min,30个循环后72℃延伸7min;
(4)将步骤(3)PCR产物用Xho I+EcoR I双酶切后定向***经同样双酶切的ppic9(NOVAGEN公司购得),获得重组质粒ppic9-4-1BBL;
(5)将重组质粒ppic9-4-1BBL转化至大肠杆菌DH5α,经培养扩增、抽提纯化、并用全自动测序仪进行序列分析,获得测序正确的重组质粒(序列见SEQ ID No.1);
(6)将测序正确的重组质粒转化至甲醇酵母菌(NOVAGEN公司购得),在28℃的条件下进行培养(2-YT培养基(含有kanamycin50μg/ml)96小时培养后分离上清液,用SDS-PAGE分离各蛋白条带,并用Western Blot检测;纯化产物经12%的SDS-PAGE分析显示,在分子量约70kD附近处有一条特异的蛋白质带(图1A),表明为4-1BBL自然形成的三聚体。Western blot结果表明,该蛋白质能与羊抗小鼠4-1BBL抗体(购自R&D公司)杂交,βME处理后形成分子量约23KD的单体,与预期结果相符(图1B)。
(7)将表达重组蛋白的菌株先划平板(酵母SD固体培养基)接种,然后再液体培养基(酵母SD液体培养基)中扩增,种子液按体积比5%的接种量接入到2-YT培养基(含有kanamycin 50μg/ml)中,28℃,300r/min,高溶氧培养至50OD以上。高溶氧条件下诱导8小时,放罐,离心,取上清液进行分离纯化操作:
a)、上清液采用0.45um滤膜过滤,加1/2体积H2O稀释至电导<5mS/cm,过阴离子交换柱,填料为Q XL(Amershame),平衡液(50mMTris+2.5mMEDTA+0.1mMPMSF,pH7.6),洗脱液为(50mMTris,0.3MNaCl+2.5mMEDTA,pH7.6),一步洗脱;
b)、收集样品准备过疏水层析,缓慢流加2倍体积平衡液(25mMPB+0.8M(NH4)2SO4,pH7.4)至样品中,同时搅拌,填料为Phenyl HP,洗脱液为25mMPB,pH7.4(A液为平衡液,B液为洗脱液,以A+B混合液进行淋洗和洗脱),step梯度洗脱,以洗脱液占60%(此为B液占A+B混合液的体积比),70%淋洗,90%,100%洗脱,换液为H2O为再洗脱一遍,每次洗脱前都待基线稳定。
c)、SDS-PAGE结果显示目标条带大小符合预期,在最后一步纯化中,杂蛋白大部分在流穿液中,经淋洗后洗脱可得纯度较好的样品。
d)、收集90%洗脱组分过Sephadex G25脱盐,换液为25mMPB,pH7.4。
e)、通过免疫测定或生物活性测定4-1BBL蛋白峰部分,得到纯化的4-1BBL胞外段蛋白(序列见SEQ ID No.2),冻干保存。
实施例2:4-1BBL蛋白的免疫学活性
采用本发明方法获得的4-1BBL胞外段蛋白,经刺激体外培养的小鼠T淋巴细胞,能有效增强CD3单抗活化的T淋巴细胞增殖(相对增强率为25.6%)及分泌IL-2(相对增强率为53.2%)、IFN-γ(相对增强率为65.5%)等细胞因子,显示具有显著的T细胞协同刺激作用(图2~图4)。
方法:小鼠脾细胞经尼龙毛柱过滤获得T淋巴细胞,将105/mL T细胞接种于96孔板,设立1个PBS对照组和3个实验组(4-1BBL,抗CD3+抗CD28,4-1BBL+抗CD3+抗CD28),其中4-1BBL浓度为1μg/mL,抗CD3和抗CD28(购自ebioscience公司)浓度均为10μg/mL,各组设3个复孔。在37℃、5%CO2条件下培养48h,收集细胞上清液,用ELISA试剂盒(购自Diaclone公司)分别检测IL-2和IFN-γ浓度。同样条件培养72h的T细胞经3H-TdR方法(试剂购自Sigma公司)检测T细胞增殖指数(cpm)。图中为三次独立实验的数据以“均值±标准差”表示。
SEQUENCE LISTING
<110> 杭州师范大学
<120> 一种4-1BBL蛋白的制备方法
<130>
<160> 4
<170> PatentIn version 3.4
<210> 1
<211> 636
<212> DNA
<213> Unknown
<220>
<223> 人工序列
<400> 1
ctcgagaaaa gaagaaccga gccaagacct gcattgacca ttactacttc tccaaacttg 60
ggtacaagag aaaacaacgc tgaccaggtc actccagttt ctcacatcgg atgtccaaac 120
accacccagc agggatctcc tgtttttgct aagttgttgg ccaaaaacca ggcttctttg 180
tgtaacacta ccttgaactg gcactctcaa gacggtgccg gttcttctta cttgtctcag 240
ggtttgagat acgaggagga caagaaggag ttggtcgtcg actctcctgg attgtactac 300
gtctttttgg agttgaagtt gtctccaaca ttcaccaaca ctggacacaa ggttcaggga 360
tgggtttctt tggttttgca ggctaagcca caggttgacg atttcgacaa tttggccttg 420
actgtcgaat tgttcccttg ctctatggaa aacaaattgg tcgacagatc ttggtctcaa 480
ttgttgttgt tgaaggccgg acacagattg tctgtcggat tgagagcata cttgcatggt 540
gcacaagacg cttacagaga ctgggagttg tcttacccta acaccacttc tttcggtttg 600
ttcttggtta aacctgataa tccatgggaa gaattc 636
<210> 2
<211> 206
<212> PRT
<213> Unknown
<220>
<223> 人工序列
<400> 2
Arg Thr Glu Pro Arg Pro Ala Leu Thr Ile Thr Thr Ser Pro Asn Leu
1 5 10 15
Gly Thr Arg Glu Asn Asn Ala Asp Gln Val Thr Pro Val Ser His Ile
20 25 30
Gly Cys Pro Asn Thr Thr Gln Gln Gly Ser Pro Val Phe Ala Lys Leu
35 40 45
Leu Ala Lys Asn Gln Ala Ser Leu Cys Asn Thr Thr Leu Asn Trp His
50 55 60
Ser Gln Asp Gly Ala Gly Ser Ser Tyr Leu Ser Gln Gly Leu Arg Tyr
65 70 75 80
Glu Glu Asp Lys Lys Glu Leu Val Val Asp Ser Pro Gly Leu Tyr Tyr
85 90 95
Val Phe Leu Glu Leu Lys Leu Ser Pro Thr Phe Thr Asn Thr Gly His
100 105 110
Lys Val Gln Gly Trp Val Ser Leu Val Leu Gln Ala Lys Pro Gln Val
115 120 125
Asp Asp Phe Asp Asn Leu Ala Leu Thr Val Glu Leu Phe Pro Cys Ser
130 135 140
Met Glu Asn Lys Leu Val Asp Arg Ser Trp Ser Gln Leu Leu Leu Leu
145 150 155 160
Lys Ala Gly His Arg Leu Ser Val Gly Leu Arg Ala Tyr Leu His Gly
165 170 175
Ala Gln Asp Ala Tyr Arg Asp Trp Glu Leu Ser Tyr Pro Asn Thr Thr
180 185 190
Ser Phe Gly Leu Phe Leu Val Lys Pro Asp Asn Pro Trp Glu
195 200 205
<210> 3
<211> 30
<212> DNA
<213> Unknown
<220>
<223> 人工序列
<400> 3
ctcgagaaaa gaagaaccga gccaagacct 30
<210> 4
<211> 24
<212> DNA
<213> Unknown
<220>
<223> 人工序列
<400> 4
gaattcttcc catggattat cagg 24
Claims (2)
1.一种4-1BBL蛋白的制备方法,所述方法包括:
(1)取含4-1BBL蛋白的小鼠淋巴细胞,conA体外刺激培养后经trizol裂解,抽提得到mRNA;
(2)将mRNA逆转录得到4-1BBL胞外段蛋白的cDNA;
(3)对步骤(2)所得cDNA进行PCR扩增,得到4-1BBL蛋白目的基因;PCR扩增引物为:上游引物:5’-CTC GAG AAA AGA AGAACC GAG CCAAGA CCT-3’,下游引物:5’-GAA TTC TTC CCATGG ATT ATC AGG-3’;
(4)将步骤(3)PCR产物用Xho I和EcoR I双酶切后定向***经同样双酶切的ppic9,获得重组质粒ppic9-4-1BBL;
(5)将重组质粒ppic9-4-1BBL转化至大肠杆菌DH5α,经培养扩增、抽提纯化、测序分析,获得测序正确的重组质粒;
(6)将测序正确的重组质粒转化至甲醇酵母菌,诱导表达重组蛋白;
(7)将表达重组蛋白的活化菌株进行诱导发酵培养,发酵液离心,取上清液分离纯化,得到纯化的4-1BBL重组蛋白。
2.如权利要求1所述的方法,其特征在于步骤(5)重组质粒ppic9-4-1BBL序列如下:CTC GAG AAA AGA AGA ACC GAG CCA AGA CCT GCATTG ACC ATT ACT ACT TCT CCA AAC TTG GGT ACA AGA GAAAAC AAC GCT GAC CAG GTC ACT CCA GTT TCT CAC ATC GGATGT CCA AAC ACC ACC CAG CAG GGA TCT CCT GTT TTT GCTAAG TTG TTG GCC AAA AAC CAG GCT TCT TTG TGT AAC ACTACC TTG AAC TGG CAC TCT CAA GAC GGT GCC GGT TCT TCTTAC TTG TCT CAG GGT TTG AGA TAC GAG GAG GAC AAG AAGGAG TTG GTC GTC GAC TCT CCT GGA TTG TAC TAC GTC TTTTTG GAG TTG AAG TTG TCT CCA ACA TTC ACC AAC ACT GGACAC AAG GTT CAG GGA TGG GTT TCT TTG GTT TTG CAG GCTAAG CCA CAG GTT GAC GAT TTC GAC AAT TTG GCC TTG ACTGTC GAA TTG TTC CCT TGC TCT ATG GAA AAC AAA TTG GTCGAC AGA TCT TGG TCT CAA TTG TTG TTG TTG AAG GCC GGACAC AGA TTG TCT GTC GGA TTG AGA GCA TAC TTG CAT GGTGCA CAA GAC GCT TAC AGA GAC TGG GAG TTG TCT TAC CCTAAC ACC ACT TCT TTC GGT TTG TTC TTG GTT AAA CCT GAT AATCCATGG GAA GAA TTC。
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CN103713125A (zh) * | 2014-01-08 | 2014-04-09 | 滨州医学院 | 一种用于检测恶性血液疾病cd137l突变的试剂盒及其应用 |
CN111607571A (zh) * | 2019-02-26 | 2020-09-01 | 南京大学 | 一种特异性激活免疫共刺激通路的复制型溶瘤腺病毒及其制备方法和应用 |
CN111606999A (zh) * | 2019-02-26 | 2020-09-01 | 南京大学 | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 |
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CN1844149A (zh) * | 2005-04-07 | 2006-10-11 | 苏州大学 | 抗人4-1bbl单克隆抗体制备及其应用 |
CN1976946A (zh) * | 2004-07-03 | 2007-06-06 | 财团法人牧岩生命工学研究所 | 诱导抗hcv有效ctl应答的超型表位、其编码寡核苷酸及其应用 |
CN101684456A (zh) * | 2008-09-28 | 2010-03-31 | 江门罗森生物制药有限公司 | 一种体外培养条件下扩增人nk细胞的方法 |
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CN1844149A (zh) * | 2005-04-07 | 2006-10-11 | 苏州大学 | 抗人4-1bbl单克隆抗体制备及其应用 |
CN101684456A (zh) * | 2008-09-28 | 2010-03-31 | 江门罗森生物制药有限公司 | 一种体外培养条件下扩增人nk细胞的方法 |
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CN103713125A (zh) * | 2014-01-08 | 2014-04-09 | 滨州医学院 | 一种用于检测恶性血液疾病cd137l突变的试剂盒及其应用 |
CN103713125B (zh) * | 2014-01-08 | 2015-12-30 | 滨州医学院 | 一种用于检测恶性血液疾病cd137l突变的试剂盒及其应用 |
CN111607571A (zh) * | 2019-02-26 | 2020-09-01 | 南京大学 | 一种特异性激活免疫共刺激通路的复制型溶瘤腺病毒及其制备方法和应用 |
CN111606999A (zh) * | 2019-02-26 | 2020-09-01 | 南京大学 | 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 |
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