CN111454350A - 一种重组纤连蛋白突变体及其应用 - Google Patents
一种重组纤连蛋白突变体及其应用 Download PDFInfo
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- CN111454350A CN111454350A CN202010486557.6A CN202010486557A CN111454350A CN 111454350 A CN111454350 A CN 111454350A CN 202010486557 A CN202010486557 A CN 202010486557A CN 111454350 A CN111454350 A CN 111454350A
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Abstract
本发明属于生物学领域,具体涉及一种重组纤连蛋白突变体的核苷酸序列及其应用。本发明提供的重组纤连蛋白突变体,选择纤连蛋白上促细胞粘附、促细胞增殖结构域,并利用翻译暂停理论设计编码重组纤连蛋白突变体的核苷酸序列,在不改变重组纤连蛋白氨基酸序列的情况下,将重组纤连蛋白最后20个密码子中翻译速度较快的密码子替换成翻译速度较慢的密码子获得的。本发明提供的重组纤连蛋白突变体纯化过程简单,可以促进细胞增殖活性及粘附活性。
Description
技术领域
本发明属于生物学领域,具体涉及一种重组纤连蛋白突变体及其应用。
背景技术
纤连蛋白(Fibronectin,FN)是一种细胞外基质中的大分子糖蛋白,它广泛存在于血浆、多种细胞表面及细胞基质中,是细胞外基质中重要的粘附分子之一,纤连蛋白与细胞膜上的整合素受体结合,在细胞与细胞之间、细胞与基质之间的相互作用中发挥极其重要的功能,在调节细胞粘附、迁移、增殖等过程中发挥重要作用,在伤口修复和愈合中起重要作用(Klein,R.M.,et al.(2003)."Stimulation of extracellular matrix remodelingby the first type III repeat in fibronectin."J Cell Sci 116(Pt 22):4663-4674.)。
纤连蛋白是创面修复中一种关键的蛋白质。经研究发现,创伤修复由参与修复的细胞和细胞外基质相互作用共同完成。纤连蛋白是细胞外基质的重要成份,可通过介导分子间或细胞间粘附来参与修复,它有助于固定细胞分化和指导细胞运动;对创伤修复过程均有重要的调节作用。因此,临床上纤连蛋白主要应用于创伤修复、烧烫伤、角膜修复、牙周修复等方面(Kubow,K.E.,et al.(2015)."Mechanical forces regulate theinteractions of fibronectin and collagen I in extracellular matrix."Naturecommunications 6:8026.)。
纤连蛋白是一种多功能、高活性、纯天然的生物蛋白,分子量450KD。纤连蛋白具有两个相似的亚基,每个亚基都具有六个功能区和一个RGD序列。它的六个功能区每一区能与特殊的配体相结合:由氨基端起,第一区可与肝素、原纤维、肌动蛋白、细菌、凝血因子XIIIa相结合;第二区结合胶原和明胶;第三区结合纤维蛋白原;第四区结合细菌;第五区结合肝素;第六区与原纤维相结合。它的RGD序列能和11种整合素相结合,进而发挥强大的生物学功能(Xu,J.and D.Mosher(2011).Fibronectin and other adhesive glycoproteins.Theextracellular matrix:an overview,Springer:41-75.)。纤连蛋白有多个功能区,每个功能区通过与细胞表面的受体结合,从而发挥相应的生物学功能。研究表明纤连蛋白第三区对于细胞粘附至关重要,重组表达的纤连蛋白片段能购显著促进细胞的粘附。FN通过与细胞表面α4β7受体结合,促进成纤维细胞的分化,FN中的EDA结构域起关键作用,此外含有EDA结构域的FN能够促进TGF-β1的表达。纤连蛋白FN或者含有EDA结构域的纤连蛋白能够激活Erk1/2信号通路(Kohan,M.,et al.(2010)."EDA-containing cellular fibronectininduces fibroblast differentiation through binding toα4β7integrin receptorand MAPK/Erk 1/2-dependent signaling."The FASEB Journal 24(11):4503-4512.)。
重组纤连蛋白片段由于具有多种生物学功能,是目前纤连蛋白研究的热点之一,另外,由于重组纤连蛋白片段稳定性好,易于制备,能够更好的推广应用。纤连蛋白在伤口愈合中起着至关重要的作用,纤连蛋白在损伤部位沉积,形成血凝块,止血并保护皮下组织。纤连蛋白形成细胞运动及定位的基质,介导成纤维细胞、巨噬细胞参与损伤修复。纤连蛋白对创面愈合有深远影响,包括在肉芽组织的发育和组织过程中,细胞的迁移和生长,以及结缔组织基质的重塑和再合成。纤连蛋白促进成纤维细胞粘附血浆纤维蛋白,以及促进胶原蛋白在伤口处沉积等。
纤连蛋白在医学、美容、化妆品领域有广泛的应用前景,但是从人体或者动物血液、组织中提取的天然纤连蛋白产量极为有限,成本高,此外产品纯度较低。DNA重组技术能够解决纤连蛋白制备的难题,但是由于纤连蛋白单体分子量较大,利用DNA重组技术表达全长的纤连蛋白的会存在一定困难,蛋白往往稳定性差,活性低。如何利用重组DNA技术表达纤连蛋白的功能结构域,得到稳定性高、活性好的纤连蛋白是解决纤连蛋白应用瓶颈的重要途径。
传统生化理论“安芬森原则”认为,蛋白质的折叠信息由其氨基酸序列在一定环境下唯一决定。但是,最新的研究表明:不同的DNA虽然可以翻译成同样的氨基酸,蛋白质生成的速度(翻译速度)并非恒定,在某些区段上会比较缓慢,这种现象称为翻译暂停(translational pausing或translational attenuation)翻译暂停位点与蛋白质折叠高度相关,若翻译暂停位点不正确,该慢的地方快了,或者该快的地方慢了,都将导致蛋白质错误折叠聚集,无法得到有功能的可溶性蛋白。也就是说,蛋白质空间构象不仅由氨基酸的序列决定,也由核苷酸序列决定。
发明内容
针对现有技术普遍存在的缺点,本发明提供了一种重组纤连蛋白突变体及其应用。本发明提供的重组纤连蛋白突变体纯化过程简单,可以有效促进细胞增殖活性、粘附活性。
为了达到上述目的,本发明采用的技术方案为:
一种重组纤连蛋白突变体,其核苷酸序列如SEQ ID NO.1所示。
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTACATGATGATATGGAAAGCCAGCCGCTAATAGGCACACAAAGCACAGCG(SEQ ID NO.1);
优选地,所述的重组纤连蛋白突变体以可溶性的形式表达。
优选地,所述的重组纤连蛋白突变体,是将纤连蛋白突变体进行翻译暂停优化获得的。
优选地,所述纤连蛋白突变体的核苷酸序列如SEQ ID NO.2所示。
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTGCATGATGATATGGAAAGCCAGCCGCTGATCGGCACCCAAAGCACCGCG(SEQ ID NO.2);
优选地,所述翻译暂停优化过程为:将纤连蛋白突变体最后20个密码子中翻译较快的密码子替换成翻译较慢的密码子。
优选地,所述翻译较快的密码子包括ATC、ACC、CTG;所述翻译速度较慢的密码子包括ATA、ACA、CTA;是通过制造翻译暂停位点软件计算确定的。
优选地,所述核苷酸序列编码的氨基酸序列如SEQ ID NO.3所示。
TGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDGGGGSNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA(SEQ ID NO.3);
本发明还提供了一种所述重组纤连蛋白突变体在促进细胞增殖活性、粘附活性中的应用。
与现有技术相比,本发明提供的重组纤连蛋白突变体具有如下优势:
(1)本发明提供的重组纤连蛋白突变体,可以有效促进细胞增殖活性,提高细胞粘附活性;
(2)本发明提供的重组纤连蛋白突变体,制备过程简单,纯化方便;
(3)本发明提供的重组纤连蛋白,是一种新型的重组蛋白突变体,提供了一种新的获得突变体的方式。
附图说明
图1为未优化的纤连蛋白突变体诱导表达分析结果;
图2为重组纤连蛋白突变体优化前后的翻译曲线对比结果图;
图3为重组纤连蛋白突变体可溶性表达组分纯化层析图;
图4为优化后的重组纤连蛋白突变体表达分析及可溶性组分的纯化结果图;
图5为重组纤连蛋白突变体包涵体组分纯化层析图;
图6为重组纤连蛋白突变体包涵体组分纯化的SDS-PAGE电泳图;
图7为重组纤连蛋白突变体促细胞增值结果图;
图8为重组纤连蛋白促细胞粘附结果图;
图9为重组纤连蛋白促细胞粘附率结果图。
具体实施方式
下面结合具体实施例对本发明作进一步解释,但是应当注意的是,以下实施例仅用以解释本发明,而不能用来限制本发明,所有与本发明相同或相近的技术方案均在本发明的保护范围之内。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料为市售商品。
本发明实施例涉及的主要材料如下:宿主菌大肠杆菌BL21(DE3)(Merck)、质粒pET-28a(Merck)、预染蛋白Marker购自Fermentas公司;Ni SepharoseTM6Fast Flow购自GE公司;CCK-8试剂盒购自美国SIGMA公司;其它试剂均为分析纯试剂。NTA-0缓冲液(20mmol/LTris-HCl、pH 8.0+0.15mol/L NaCl),NTA-40缓冲液(20mmol/L Tris-HCl、pH 8.0+0.15mol/L NaCl+40mmol/L咪唑),NTA-80缓冲液(20mmol/L Tris-HCl、pH 8.0+0.15mol/LNaCl+80mmol/L咪唑),NTA-250缓冲液(20mmol/L Tris-HCl、pH 8.0+0.15mol/L NaCl+250mmol/L咪唑)。
实施例1重组纤连蛋白载体构建
我们通过生物大分子模拟选择了纤连蛋白促细胞增殖及粘附的功能结构域,设计了新的纤连蛋白突变体。根据大肠杆菌密码子偏好性,以及通过翻译暂停理论,对纤连蛋白突变体的核苷酸序列进行优化。并委托苏州泓迅生物科技股份有限公司全基因合成纤连蛋白突变体DNA。
未优化的重组纤连蛋白的核苷酸序列如下:
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTGCATGATGATATGGAAAGCCAGCCGCTGATCGGCACCCAAAGCACCGCG;
纤连蛋白突变体核苷酸序列未优化的表达菌株接种到1L、卡那霉素含量为50μg/mL的LB培养基中,按前述的表达条件进行摇瓶发酵并诱导。4℃、6000×g、10min离心收集菌体,然后将菌体沉淀按体积比1:10比例重新悬浮于NTA-10缓冲液,高压(1000bar)均质破碎细胞。4℃、25000×g、30min离心收集上清,离心沉淀用和上清等体积的含8M尿素的NTA-0缓冲液溶解,12%SDS-PAGE凝胶电泳分析重组纤连蛋白突变体的表达情况。结果如图1,由此可知,核苷酸序列未优化的重组纤连蛋白突变体在大肠杆菌中主要以包涵体的形式表达;而在实际应用中,多采用纤连蛋白突变体作为细胞增殖活性的促进剂,为了满足实际需要,需要对纤连蛋白突变体的核苷酸序列进行优化。
通过翻译暂停理论设计编码重组纤连蛋白突变体的核苷酸序列,在不改变重组纤连蛋白氨基酸序列的情况下,将重组纤连蛋白最后20个密码子中翻译速度较快的密码子(ATC、ACC、CTG)替换成翻译速度较慢的密码子(ATA、ACA、CTA),以制造翻译暂停位点(RiboTempo软件计算确定,http://bioinformatics.jnu.edu.cn/software/ribotempo),突变体经RiboTempo软件计算得到的翻译暂停曲线分别如图2所示,图中,A代表未优化前翻译曲线,B代表翻译暂停理论优化后的翻译曲线。翻译暂停曲线红线在预定的区域形成了翻译暂停位点,符合要求。设计得到的编码重组纤连蛋白突变体的核苷酸序列如下:
翻译暂停优化后的序列:
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTACATGATGATATGGAAAGCCAGCCGCTAATAGGCACACAAAGCACAGCG
重组纤连蛋白氨基酸序列:
TGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDGGGGSNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIHELFPAPDGEEDTAELQGLRPGSEYTVSVVALHDDMESQPLIGTQSTA。
实施例2重组纤连蛋白突变体的表达、纯化
(1)纤连蛋白表达菌株的制备:
所述纤连蛋白表达菌株的制备过程为:
①大肠杆菌BL21(DE3)感受态细胞的制备:制备过程详见《分子克隆实验指南》第三版;[美]J.莎姆布鲁克著,黄培堂译。
②将表达载体pET-28a-Fibronectin转化至大肠杆菌BL21(DE3)感受态细胞:转化过程详见《分子克隆实验指南》第三版;[美]J.莎姆布鲁克著,黄培堂译。
(2)纤连蛋白突变体诱导表达和可溶性分析
具体操作过程如下:
将步骤(1)得到的表达菌株pET-28a-Fibronectin接种到10mL含50μg/mL卡那霉素含量的LB培养基中,37℃、180rpm培养,当OD600=0.8时,加IPTG,终浓度为1mM,37℃诱导表达4h后,5000g、4℃离心10min收集菌体。菌体用20mmol/L Tris-HCl(pH 8.0,0.15mol/LNaCl)缓冲液重悬,高压(800bar)均质破碎细胞,18000×g、4℃离心30min,上清和沉淀分别留样待后续的SDS-PAGE电泳(5%浓缩胶、12%分离胶)及Western blot分析。
(3)纤连蛋白突变体摇瓶发酵和可溶性蛋白的纯化
将步骤(1)得到的纤连蛋白突变体表达菌株分别接种到1L、卡那霉素含量为50μg/mL的LB培养基中,按前述的表达条件进行摇瓶发酵并诱导。4℃、6000×g、10min离心收集菌体,然后将菌体沉淀按体积比1:10比例重新悬浮于NTA-10缓冲液,高压(1000bar)均质破碎细胞。4℃、25000×g、30min离心收集上清。
上清上样至柱床体积为20mL的Ni-NTA亲和层析柱中,流速0.6mL/min,NTA-0缓冲液洗回基线,流速为1mL/min,NTA-40缓冲液洗杂蛋白,NTA-80缓冲液洗杂蛋白,NTA-250缓冲液洗脱目的蛋白;纯化后的纤连蛋白突变体蛋白经Sephadex G-25分子筛脱咪唑以及3KDa的超滤管浓缩,SDS-PAGE电泳鉴定纯化的纤连蛋白突变体的纯度。
如图1所示,pET-28a-Fibonection/BL21工程菌经1mM IPTG诱导表达后,离心收集菌体,菌体经高压均质破碎离心,12%SDS-PAGE电泳分析纤连蛋白的表达情况,结果表明纤连蛋白主要存在菌体破碎离心的沉淀中,说明未优化的重组纤连蛋白突变体主要以包涵体的形式表达,图3中,1代表穿透峰,2代表40mM咪唑洗脱峰,3代表80mM咪唑洗脱峰,4代表250mM咪唑洗脱峰,而由图4可知,通过翻译暂停理论优化后的重组纤连蛋白突变体主要存在菌体破碎离心上清中,说明其主要以可溶性的形式表达。
(4)纤连蛋白突变体不可溶部分的纯化
将步骤(2)得到纤连蛋白表达菌体高压均质破碎、离心得到的沉淀,再经过以下步骤:
①0.4%脱氧胆酸钠洗涤包涵体,10000rpm,4℃离心,得到离心沉淀;
②沉淀用含有8M尿素的NTA-0缓冲液溶解。10000rpm,4℃离心,得到溶解上清;
③溶解上清上样至柱床体积为20mL的Ni-NTA亲和层析柱中,流速0.6mL/min,含有8M尿素的NTA-0缓冲液洗回基线,流速为1mL/min,含有8M尿素的NTA-40缓冲液洗杂蛋白;
④再通过柱上原位复性方法,对包涵体蛋白进行复性,最终通过NTA-250缓冲液洗脱目的蛋白;纯化后的纤连蛋白突变体蛋白经Sephadex G-25分子筛脱咪唑,SDS-PAGE电泳鉴定纯化的纤连蛋白突变体的纯度。
具体试验结果如图5、图6所示,SDS-PAGE电泳结果表明,纯化之后,可溶性表达和包涵体表达的重组纤连蛋白突变体纯度大于95%,由此可知,本发明提供的重组纤连蛋白纯度极高。
实施例3重组纤连蛋白突变体促细胞增殖活性测定
具体测定过程为:
(1)BALB/c 3T3细胞接种于96孔细胞培养板中(5000个细胞/孔),37℃,5%CO2细胞培养箱培养24h;
(2)换用DMEM无血清培养基继续培养12h;
(3)分别加入重组纤连蛋白突变体(可溶性表达纯化组分、包涵体表达纯化组分)以及PBS(阴性对照组),继续培养48~72h;
(4)每孔加入10μL CCK-8试剂,37℃,5%CO2细胞培养箱孵育2h后取出;
(5)用酶标仪读取96孔板在450nm和630nm的吸光值,以630nm为参比波长,在450nm处测定吸光度,记录测定结果。
相对促细胞增殖率=(实验组450nm吸光值-阴性对照组450nm吸光值)/阴性对照组450nm吸光值×100%。
具体试验结果如图7所示,在大肠杆菌中,可溶性表达和包涵体表达的重组纤连蛋白突变体均有较好的促细胞增殖效果,本发明对其核苷酸序列进行优化后,可溶性表达的重组纤连蛋白突变体促细胞增殖效果优于包涵体表达的重组纤连蛋白突变体。
实施例4重组纤连蛋白突变体促细胞粘附活性测定
所述细胞粘附活性测定的具体过程为:
(1)重组纤连蛋白突变体蛋白溶液浓度为20μg/ml,96孔细胞培养板每孔加入50μL样品溶液,37℃放置2h,对照孔加50μL PBS。
(2)BALB/3T3细胞用胰酶消化,计数,每孔加入5×104个细胞。37℃CO2培养箱培养2h;
(3)PBS洗3遍,洗去未粘附的细胞,再加入200μLDMEM培养基;
(4)每孔加入10μL CCK-8试剂,37℃,5%CO2细胞培养箱孵育2h后取出。
(5)用酶标仪读取96孔板在450nm和630nm的吸光值,以630nm为参比波长,在450nm处测定吸光度,记录测定结果。
(6)促细胞粘附率=(实验组450nm吸光值-阴性对照组450nm吸光值)/阴性对照组450nm吸光值×100%。
具体试验结果如图8、图9所示,细胞粘附实验结果表明,可溶性表达和包涵体表达的重组纤连蛋白突变体都有较好的促细胞粘附效果,而本发明对其核苷酸序列进行优化后,重组纤连蛋白突变体主要以可溶性形式表达,最终具有更强的促细胞粘附效果。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明做了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
序列表
<110> 广东丸美生物技术股份有限公司
<120> 一种重组纤连蛋白突变体及其应用
<130> 2020.4.9
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 552
<212> DNA
<213> 重组纤连蛋白突变体的核苷酸序列(Nucleotide sequence of recombinantfibronectin mutant)
<400> 1
acgggcatcg acttcagcga tatcaccgcg aacagcttca ccgttcactg gatcgcgcca 60
cgtgcgacga tcaccggcta tcgcatccgc catcacccgg aacactttag cggtcgtcca 120
cgcgaagatc gcgttccgca tagccgcaat agcatcacgc tgaccaatct gaccccgggc 180
accgaatatg ttgtgagcat cgtggcgctg aacggccgcg aagaaagccc actgctgatt 240
ggccagcaga gcaccgtgag tgatggtggc ggtggcagca atattgatcg cccgaaaggt 300
ctggccttca cggatgtgga cgtggacagc atcaaaatcg cgtgggaaag cccacaaggc 360
caagttagcc gctaccgcgt gacctatagc agcccggaag atggcatcca cgaactgttt 420
ccggcgccgg atggtgaaga agataccgcg gaactgcaag gtctgcgtcc gggcagcgaa 480
tacacggtta gcgtggtggc gctacatgat gatatggaaa gccagccgct aataggcaca 540
caaagcacag cg 552
<210> 2
<211> 552
<212> DNA
<213> 纤连蛋白突变体的核苷酸序列(Nucleotide sequence of fibronectinmutant)
<400> 2
acgggcatcg acttcagcga tatcaccgcg aacagcttca ccgttcactg gatcgcgcca 60
cgtgcgacga tcaccggcta tcgcatccgc catcacccgg aacactttag cggtcgtcca 120
cgcgaagatc gcgttccgca tagccgcaat agcatcacgc tgaccaatct gaccccgggc 180
accgaatatg ttgtgagcat cgtggcgctg aacggccgcg aagaaagccc actgctgatt 240
ggccagcaga gcaccgtgag tgatggtggc ggtggcagca atattgatcg cccgaaaggt 300
ctggccttca cggatgtgga cgtggacagc atcaaaatcg cgtgggaaag cccacaaggc 360
caagttagcc gctaccgcgt gacctatagc agcccggaag atggcatcca cgaactgttt 420
ccggcgccgg atggtgaaga agataccgcg gaactgcaag gtctgcgtcc gggcagcgaa 480
tacacggtta gcgtggtggc gctgcatgat gatatggaaa gccagccgct gatcggcacc 540
caaagcaccg cg 552
<210> 3
<211> 184
<212> PRT
<213> 重组纤连蛋白突变体的氨基酸序列(Amino acid sequence of recombinantfibronectin mutant)
<400> 3
Thr Gly Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His
1 5 10 15
Trp Ile Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His
20 25 30
Pro Glu His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser
35 40 45
Arg Asn Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val
50 55 60
Val Ser Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile
65 70 75 80
Gly Gln Gln Ser Thr Val Ser Asp Gly Gly Gly Gly Ser Asn Ile Asp
85 90 95
Arg Pro Lys Gly Leu Ala Phe Thr Asp Val Asp Val Asp Ser Ile Lys
100 105 110
Ile Ala Trp Glu Ser Pro Gln Gly Gln Val Ser Arg Tyr Arg Val Thr
115 120 125
Tyr Ser Ser Pro Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp
130 135 140
Gly Glu Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu
145 150 155 160
Tyr Thr Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser Gln Pro
165 170 175
Leu Ile Gly Thr Gln Ser Thr Ala
180
Claims (8)
1.一种重组纤连蛋白突变体,其特征在于,核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的重组纤连蛋白突变体,其特征在于,以可溶性的形式表达。
3.如权利要求1所述的重组纤连蛋白突变体,其特征在于,是将纤连蛋白突变体进行翻译暂停优化获得的。
4.如权利要求3所述的重组纤连蛋白突变体,其特征在于,所述纤连蛋白突变体的核苷酸序列如SEQ ID NO.2所示。
5.如权利要求3所述的重组纤连蛋白突变体,其特征在于,所述翻译暂停优化过程为:将纤连蛋白突变体最后20个密码子中翻译较快的密码子替换成翻译较慢的密码子。
6.如权利要求5所述的重组纤连蛋白突变体,其特征在于,所述翻译较快的密码子包括ATC、ACC、CTG;所述翻译速度较慢的密码子包括ATA、ACA、CTA;是通过制造翻译暂停位点软件计算确定的。
7.如权利要求1或4所述的重组纤连蛋白突变体,其特征在于,所述核苷酸序列编码的氨基酸序列如SEQ ID NO.3所示。
8.一种如权利要求1-7任一项所述的重组纤连蛋白突变体在促进细胞增殖活性、粘附活性中的应用。
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CN112941081A (zh) * | 2021-04-16 | 2021-06-11 | 广州启点生物科技有限公司 | 表达量高和活性强的纤连蛋白突变体的编码序列及其应用 |
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CN116063459A (zh) * | 2022-09-19 | 2023-05-05 | 暨南大学 | 一种高表达和高活性纤连蛋白突变体及其应用 |
CN117143224A (zh) * | 2023-10-26 | 2023-12-01 | 广州暨南大学医药生物技术研究开发中心有限公司 | 一种重组人纤连蛋白截短肽及其制备方法与应用 |
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CN113527523A (zh) * | 2021-09-14 | 2021-10-22 | 美慕(北京)科技有限公司 | 一种重组蛋白及其构建方法和用途 |
CN115976031A (zh) * | 2022-07-18 | 2023-04-18 | 烟台市华昕生物医药科技有限公司 | 一种重组纤连蛋白及其应用 |
CN116063459A (zh) * | 2022-09-19 | 2023-05-05 | 暨南大学 | 一种高表达和高活性纤连蛋白突变体及其应用 |
CN116063459B (zh) * | 2022-09-19 | 2023-10-13 | 暨南大学 | 一种高表达和高活性纤连蛋白突变体及其应用 |
CN117143224A (zh) * | 2023-10-26 | 2023-12-01 | 广州暨南大学医药生物技术研究开发中心有限公司 | 一种重组人纤连蛋白截短肽及其制备方法与应用 |
CN117143224B (zh) * | 2023-10-26 | 2024-01-30 | 广州暨南大学医药生物技术研究开发中心有限公司 | 一种重组人纤连蛋白截短肽及其制备方法与应用 |
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