CN1467222A - 人白细胞介素-17受体样分子融合蛋白及其编码基因与表达细胞系 - Google Patents
人白细胞介素-17受体样分子融合蛋白及其编码基因与表达细胞系 Download PDFInfo
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Abstract
本发明公开了一种人白细胞介素-17受体样分子融合蛋白及其编码基因与表达细胞系。本发明的目的是提供一种人白细胞介素-17受体样蛋白与可溶性Fc受体的融合蛋白及其编码基因。一种人白细胞介素-17受体样蛋白与可溶性Fc受体的融合蛋白,它具有序列表中序列2的氨基酸残基序列或将序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。本发明的融合蛋白由531个氨基酸残基组成。人白细胞介素-17受体样蛋白的编码基因是序列表中序列1的DNA序列。本发明的融合蛋白可望在治疗男性生殖不育、隐睾、睾丸癌、前列腺癌及某些炎症性疾病中得到广泛应用。
Description
技术领域
本发明涉及一种融合蛋白及其编码基因,该融合蛋白的表达细胞系与应用;特别是涉及一种人白细胞介素-17受体样分子融合蛋白及其编码基因,该融合蛋白的表达细胞系与应用。
背景技术
白细胞介素是一类重要的细胞因子,可以通过与靶细胞膜表面的特异受体结合而介导广泛的生物学作用,诸如:细胞增殖、分化、造血调节、免疫及炎症应答等。白细胞介素17(IL-17/IL-17A)是IL-17家族第一个被克隆的T细胞来源的促炎症细胞因子。人IL-17通常以一个二硫键连接组成的同二聚体分泌性糖蛋白形式存在,分子质量为30~35kD。尽管IL-17只在局限的组织和细胞系表达,但却有广泛的生物学功能,尤其是在促炎症和造血功能方面较为突出。另外,IL-17还能刺激多种细胞因子的产生,如:来源于巨嗜细胞的肿瘤坏死因子α,来源于成纤维细胞的IL-1β、IL-6、IL-8、ICAM-1以及来源于滑膜细胞的G-CSF、PGE2等。
尽管目前至少已克隆了7种IL-17家族成员,但有关该家族成员的受体研究却知之甚少。本发明的发明人经研究,发现了一种人白细胞介素-17受体样蛋白,并将其命名为hIL-17RLM-L(专利申请号:02123447.7)。该蛋白是一种I型单跨膜细胞因子受体,氮末端含有一个信号肽,一个跨膜结构域和一个较长的胞浆结构域,其较长的胞浆结构域还含有一个潜在的SH3相互作用结构域和众多的酪氨酸磷酸化部位,在胞浆邻膜区含有一个Toll/IL-1受体样结构域-TIR。hIL-17RLM-L基因在肾及睾丸具有较多的表达,少量在心、脑、脾、子宫表达。研究表明,hIL-17RLM-L在生精过程中具有细胞表达特异性,主要在睾丸组织的生精细胞、支持细胞表达,而在曲细精管基底膜部位的成纤维细胞和睾丸支持组织部位的间质细胞染色阴性。在隐睾组织,生精过程中止,但是hIL-17RLM-L在曲细精管的生精细胞却有极强的染色。hIL-17RLM-L可以使Stat5因子活化,同时还具有介导细胞增殖的作用。
可溶性Fc-受体融合蛋白经常被用来体外研究配体与受体之间的相互作用,及特异阻断IL-17RLM的潜在配体所介导的信号通路和生物学效应。为了寻找IL-17RLM-L的潜在偶联配体,构建并表达纯化可溶性Fc受体与hIL-17RLM-L的融合蛋白是一种行之有效的手段。
发明内容
本发明的目的是提供一种人白细胞介素-17受体样蛋白与可溶性Fc受体的融合蛋白及其编码基因。
一种人白细胞介素-17受体样蛋白与可溶性Fc受体的融合蛋白,它具有序列表中序列2的氨基酸残基序列或将序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。
本发明的融合蛋白由531个氨基酸残基组成
一种人白细胞介素-17受体样蛋白与可溶性Fc受体融合蛋白的编码基因,它是下列核苷酸序列之一:
1)序列表中序列1的DNA序列;
2)与序列表中序列1限定的DNA序列具有95%以上同源性,且编码相同功能蛋白质的DNA序列。
本发明编码人白细胞介素-17受体样蛋白与可溶性Fc受体融合蛋白的基因是由1620个核苷酸组成的DNA序列。
本发明的第二个目的是提供一株能够稳定分泌人白细胞介素-17受体样蛋白与可溶性Fc受体融合蛋白的细胞系。
本发明提供的细胞系为可溶性人白细胞介素-17受体样分子稳定表达细胞系X-931 CGMCC №0755及其突变体或变异体。
细胞系X931,已于2002年06月27日保藏在中国微生物菌种保藏管理委员会普通微生物中心,其简称为CGMCC,保藏编号为CGMCC №0755。
本发明巧妙地将人IgGl.Fc的编码序列克隆于pcDNA3真核表达载体,然后再将人白细胞介素-17受体样蛋白的胞外区亚克隆于pcDNA3-IgGl.Fc真核表达载体的N-末端,并使其编码阅读框一致,转染293T细胞,收集被转染细胞培养上清中的融合蛋白。SDS-PAGE显示所纯化的融合蛋白具有较高的纯度。
本发明的融合蛋白可望在治疗男性生殖不育、隐睾、睾丸癌、前列腺癌及某些炎症性疾病(如类风湿性关节炎等)中得到广泛应用。
具体实施方式
实施例1、稳定分泌人白细胞介素-17受体样蛋白与可溶性Fc受体融合蛋白细胞系的建立
1、转染前一天,传代293T细胞至60mm培养皿(1X105),使细胞在转染时达到40-80%的细胞汇合率(conflucence);
2、在转染时,稀释5μg Fc融合蛋白表达质粒DNA于装有无血清和抗生素的细胞培养基的转染管。瞬时离心几秒钟。添加20μlSuperfection转染试剂于DNA溶液,瞬时离心10秒钟;
3、室温(15-25℃)孵育样品5-10分钟,允许转染复合体的形成;
4、在转染复合体形成时,轻柔吸弃培养盘中的培养基,PBS洗涤;
5、添加1000μl细胞生长培养基(含血清和抗生素)于转染复合体,轻柔混匀两次后,小心转移至培养盘中的细胞表面;
6、在正常细胞培养条件下,孵育转染复合体和细胞2-3小时;
7、吸取培养基(含转染复合体),PBS洗涤一次,添加4ml新鲜的细胞生长培养基,培养36-48小时;
8、1∶10或1∶15传代细胞于含有1.2mg/ml G418的选择性培养基的6孔培养盘。每隔3天后,更新选择性培养基,直至15天左右,出现细胞克隆为止。(增强绿色荧光蛋白表达质粒作为转染对照,检测转染效率);
9、扩大培养单克隆细胞至适宜密度。采用Northern blot(也可以用RT-PCR或Western blot)鉴定阳性克隆X931;
实施例2、采用HiTrap FF亲和层析柱,纯化培养细胞上清中的人白细胞介素-17受体样蛋白与可溶性Fc受体融合蛋白,根据紫外分光光度计初步测定的蛋白浓度,制作洗脱曲线,纯化出融合蛋白。采用BCA法蛋白定量,结果表明:每100ml转染细胞培养上清中约含100-150μg的Fc-受体融合蛋白。具体做法是:
1、添加100μl 1M Tris-HCl(pH9.0)收集液于1ml的收集管。装填HiTrapFF亲和层析柱。用至少5倍体积的去离子水或结合缓冲液洗柱;
2、采用5倍体积的流出缓冲液再生柱。采用5-10倍柱床体积的结合缓冲液平衡柱;
3、缓慢添加样品于亲和层析柱的中央,连接10ml的一次性注射器,打开流出阀,缓慢挤压使其流速约为1ml/min;
4、使用5-10倍柱床体积的结合缓冲液洗柱,直到流出液无蛋白为止;
5、采用2-5倍体积的流出缓冲液洗柱,小心收集最初的流出液。紫外分光光度计初步检测流出液的蛋白浓度;
6、填充5倍体积的20%的乙醇于层析柱,预防微生物的生长。密封层析柱的流出口。于4℃贮存;
7、采用BCA法(pierce)定量融合蛋白的浓度;
8、10%SDS-PAGE电泳,检测分离的融合蛋白的纯度。
实施例3、采用RT-PCR方法对细胞系X931分泌蛋白进行鉴定分别使用受体胞外区编码序列的正义引物:5’……ATAAAGCTTATGGAATCTCAACCTTTCCTG…..3’和人IgG1.Fc编码序列的反义引物:5’…AATTCTAGATCATTTACCCGGAGACAGGG…..3’,以野生型293T细胞为对照,采用RT-PCR方法对细胞系分泌蛋白进行鉴定,结果表明,细胞系X931含有Fc-受体融合蛋白的序列。
实施例4、采用Western blot对细胞系X931分泌蛋白进行鉴定
1、搜集X931细胞培养上清液,0.45um滤膜过滤;
2、将滤过的上清液经Hitrap HF亲和层析柱纯化,采用Millipore浓缩柱浓缩,-20℃或-80℃分装保存;
3、添加20μl上样缓冲液溶解500mg纯化的可溶性本发明融合蛋白上样,行10%SDS-PAGE;
4、于4℃冷室,100伏,300毫安转膜1.5小时;
5、于丽春红染液约5分钟染色,标记蛋白分子量Marker;
6、于封闭液(10%脱脂奶粉,TBST配制),37℃孵育封闭45分钟;
7、采用TBST室温洗膜三遍,持续摇动,每次15分钟;
8、按照1∶5000-10000比例TBST稀释抗IgGl.Fc特异性抗兔血清,在37℃,一抗孵育膜45分钟,持续摇动;
9、采用TBST室温洗膜三遍,持续摇动,每次15分钟;
10、按照1∶1000比例TBST稀释二抗,在37℃孵育膜45分钟,持续摇动。采用TBST室温洗膜三遍,持续摇动,每次15分钟;
11、按照1∶5000比例TBST稀释三抗,在37℃三抗孵育膜45分钟,持续摇动;
12、采用TBST室温洗膜三遍,持续摇动,每次15分钟;
13、将膜放在滤纸上简短晾干,与ECF底物孵育2-10分钟后,phosphoimager(520nm)显影。
结果表明,细胞系X931含有预期表达的Fc-受体融合蛋白的编码序列,能够稳定表达预期融合蛋白。
序列表<160>2<210>1<211>1620<212>DNA<213>人工序列<220><223><400>1atggccccgt ggctgcagct ctgctccgtc ttctttacgg tcaacgcctg cctcaacggc 60tcgcagctgg ctgtagccgc tggcgggtcc ggccgcgcgc ggggcgccga cacctgtggc 120tggaggggag tggggccagc cagcagaaac agtgggctgt acaacatcac cttcaaatat 180gacaattgta ccacctactt gaatccagtg gggaagcatg tgattgctga cgcccagaat 240atcaccatca gccagtatgc ttgccatgac caagtggcag tcaccatcct ttggtcccca 300ggggccctcg gcatcgaatt cctgaaagga tttcgggtaa tactggagga gctgaagtcg 360gagggaagac agtgccaaca actgattcta aaggatccga agcagctcaa cagtagcttc 420aaaagaactg gaatggaatc tcaacctttc ctgaatatga aatttgaaac ggattatttc 480gtaaaggttg tcccttttcc ttccattaaa aacgaaagca attaccaccc tttcttcttt 540agaacccgag cctgtgacct gttgttacag ccggacaatc tagcttgtaa acccttctgg 600aagcctcgga acctgaacat cagccagcat ggctcggaca tgcaggtgtc cttcgaccac 660gcaccgcaca acttcggctt ccgtttcttc tatcttcact acaagctcaa gcacgaagga 720cctttcaagc gaaagacctg taagcagggg caaactacag agatgaccag ctgcctcctt 780caaaatgttt ctccagggga ttatataatt gagctggtgg atgacactaa cacaacaaga 840aaagtgatgc attatgcctt aaagccagtg cactccccgt gggccgggcc cgatatcgag 900tccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga attcgagggt 960gcaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggact 1020cctgaggtca catgcgtggt ggtggacgta agccacgaag accctgaggt caagttcaac 1080tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1140aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1200aaggagtaca agtgcaaggt ctccaacaaa gccctcccaa cccccatcga gaaaaccatc 1260tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1320gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tccaagcgac 1380atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1440gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1500tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1560acgcagaaga gcctctccct gtctccgggt aaatgactcg agcatgcatc tagagggccc 1620<210>2<211>531<212>PRT<213>人工序列<220><223><400>序列号Met Ala Pro Trp Leu Gln Leu Cys Ser Val Phe Phe Thr Val Asn1 5 10 15Ala Cys Leu Asn Gly Ser Gln Leu Ala Val Ala Ala Gly Gly Ser
20 25 30Gly Arg Ala Arg Gly Ala Asp Thr Cys Gly Trp Arg Gly Val Gly
35 40 45Pro Ala Ser Arg Asn Ser Gly Leu Tyr Asn Ile Thr Phe Lys Tyr
50 55 60Asp Ash Cys Thr Thr Tyr Leu Asn Pro Val Gly Lys His Val Ile
65 70 75Ala Asp Ala Gln Asn Ile Thr Ile Ser Gln Tyr Ala Cys His Asp
80 85 90Gln Val Ala Val Thr Ile Leu Trp Ser Pro Gly Ala Leu Gly Ile
95 100 105Glu Phe Leu Lys Gly Phe Arg Val Ile Leu Glu Glu Leu Lys Ser
110 115 120Glu Gly Arg Gln Cys Gln Gln Leu Ile Leu Lys Asp Pro Lys Gln
125 130 135Leu Asn Ser Ser Phe Lys Arg Thr G1y Met Glu Ser Gln Pro Phe
140 145 150Leu Asn Met Lys Phe Glu Thr Asp Tyr Phe Val Lys Val Val Pro
155 160 165Phe Pro Ser Ile Lys Asn Glu Ser Asn Tyr His Pro Phe Phe Phe
170 175 180Arg Thr Arg Ala Cys Asp Leu Leu Leu Gln Pro Asp Asn Leu Ala
185 190 195Cys Lys Pro Phe Trp Lys Pro Arg Asn Leu Asn Ile Ser Gln His
200 205 210Gly Ser Asp Met Gln Val Ser Phe Asp His Ala Pro His Asn Phe
215 220 225Gly Phe Arg Phe Phe Tyr Leu His Tyr Lys Leu Lys His Glu Gly
230 235 240Pro Phe Lys Arg Lys Thr Cys Lys Gln Gly Gln Thr Thr Glu Met
245 250 255Thr Ser Cys Leu Leu Gln Asn Val Ser Pro Gly Asp Tyr Ile Ile
260 265 270Glu Leu Val Asp Asp Thr Asn Thr Thr Arg Lys Val Met His Tyr
275 280 285Ala Leu Lys Pro Val His Ser Pro Trp Ala Gly Pro Asp Ile Glu
290 295 300Ser Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
305 310 315Pro Glu Phe Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys
320 325 330Pro Lys Asp Thr Leu Met lle Ser Arg Thr Pro Glu Val Thr Cys
335 340 345Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
350 355 360Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
365 370 375Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
380 385 390Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
395 400 405Lys Val Ser Asn Lys Ala Leu Pro Thr Pro Ile Glu Lys Thr Ile
410 415 420Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
425 430 435Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
440 445 450Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
455 460 465Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
470 475 480Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
485 490 495Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
500 505 510Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
515 520 525Ser Leu Ser Pro Gly Lys
530 531
Claims (8)
1、一种人白细胞介素-17受体样蛋白,它具有序列表中序列2的氨基酸残基序列或将序列2的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。
2、根据权利要求1所述的蛋白质,其特征在于:所述蛋白质具有序列表中序列2的氨基酸残基序列。
3、一种人白细胞介素-17受体样蛋白的编码基因,它是下列核苷酸序列之一:
1)序列表中序列1的DNA序列;
2)与序列表中序列1限定的DNA序列具有95%以上同源性,且编码相同功能蛋白质的DNA序列。
4、根据权利要求3所述的基因,其特征在于:所述人白细胞介素-17受体样蛋白的编码基因是序列表中序列1的DNA序列。
5、可溶性人白细胞介素-17受体样分子稳定表达细胞系X-931 CGMCC №0755及其突变体或变异体。
6、权利要求1所述的融合蛋白在制备治疗男性生殖不育、隐睾的药物中的应用。
7、权利要求1所述的融合蛋白在制备治疗睾丸癌、前列腺癌的药物中的应用。
8、权利要求1所述的融合蛋白在制备治疗类风湿性关节炎的药物中的应用。
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