CN111217903A - 一种重组人纤连蛋白ⅲ1-c及其制备方法和应用 - Google Patents
一种重组人纤连蛋白ⅲ1-c及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种重组人纤连蛋白Ⅲ1‑C蛋白及其制备方法和应用。根据密码子表达偏好性先优化了该目的蛋白的目的基因片段,再将所得的基因片段插入pET‑32a中建重组质粒;将该重组质粒转化导入表达载体BL21(DE3)中,经筛选得到了能高效可溶性表达的阳性克隆菌;再将阳性克隆菌经放大发酵培养后诱导表达,再经破碎、离心得到的上清液经His‑tag亲和层析柱洗脱、透析、过滤一次性步骤纯化后,即可得到蛋白浓度高达1.0mg/mL以上,纯度大于90%的rhFNⅢ1‑C优质蛋白溶液。通过促细胞粘附性试验检测观察,显示rhFNⅢ1‑C具有明显促进MDBK细胞和Balb/c/3T3细胞的贴壁、粘附和细胞快速分裂生长的性能,表明该蛋白具有作为药妆医美护肤原料和成品的巨大潜力。
Description
技术领域
本发明属于生物技术领域,具体涉及一种重组人纤连蛋白Ⅲ1-C蛋白及其制备方法和应用。
背景技术
人皮肤由表皮层、真皮层、皮下组织及皮肤附属器官组成,具有保护、调节体温、新陈代谢和感觉等功能。其中表皮层和真皮层的状态与皮肤状态紧密相关,由细胞和胞外基质组成的细胞环境稳态与皮肤状态相关。纤连蛋白(fibronectin,FN)是一种高分子量(约440kDa)的细胞外基质的二聚体糖蛋白,它与整合素跨膜受体蛋白结合,由两个几乎相同的单体通过一对二硫键连接而成,其基因位于人类2号染色体上,由Ⅰ型、Ⅱ型、Ⅲ型3种同源性序列组成,在结构上该蛋白分为3个区域,即N端的明胶结合区,中间部分的细胞结合区和C端的Ⅱ型肝素结合区。Ⅰ型和Ⅱ型都含有两个二硫键,而Ⅲ型则没有二硫键,RGD序列在细胞的黏附及FN与其他蛋白的相互作用中具有重要作用,该序列位于细胞结合区的第10个Ⅲ型重复中,FN可通过RGD与受体相连,从而发挥调节作用。与整合素相似的是,纤连蛋白结合了细胞外基质成分,如胶原蛋白、纤维蛋白和肝素硫酸盐蛋白聚糖(如多配体聚糖)。FN广泛参与细胞迁移、黏附、增殖、止血及组织修复等过程,调动单核吞噬细胞系统清除损伤组织处有害物质,具有生长因子的作用。FN作为细胞培养的基质,可提高多种细胞的贴壁率、汇合率,缩短细胞汇合时间,使细胞形态结构良好,代谢率增强,DNA、RNA及蛋白质合成速度显著提高;细胞的集落率升高,原代培养成活率提高。FN作为一种胞外基质蛋白能够促进细胞增殖、粘附及其他胞外基质蛋白如胶原蛋白的形成,参与细胞迁移、黏附、增殖、止血及组织修复等过程,能促进单核吞噬细胞系统清除损伤组织处有害物质,具有生长因子的作用。现已知,纤连蛋白除已被用于创伤修复等临床应用外,还是一个重要的美容因子。
1999年,冯作化教授等在大肠埃希菌中表达了一种抗肿瘤转移多肽-人纤连蛋白Cell I-Hep Ⅱ-Cell Ⅱ三结构域重组多肽(CH82),即重组人纤连蛋白Ⅲ1-C蛋白(recombinant human FNⅢ1-C,rhFNⅢ1-C),其表达效率仅为21%。在37℃诱导下的该重组蛋白主要通过包涵体的形式分泌表达,需采用传统方法进行制备,即先用尿素进行变性,而后在缓冲液中透析复性而获得。此制备过程技术较繁杂且时间长,蛋白的活性易受损导致相应功能丧失。
目前现有技术中,尚未见有公开过rhFNⅢ1-C的研制技术及其应用方法。
发明内容
为解决上述技术问题,本发明提供了一种重组人纤连蛋白Ⅲ1-C(rhFNⅢ1-C)及其制备方法和应用,即根据密码子表达偏好性优化了人纤连蛋白Ⅲ1-C目的基因片段;将所得的基因片段插入pET-32a中构建出重组质粒,再将重组质粒转化导入表达载体BL21(DE3)中经筛选得到阳性克隆菌;该菌经放大发酵培养后即可诱导表达出目的蛋白rhFNⅢ1-C。经破碎、离心收集上清液,即可上样His-tag亲和层析柱纯化过滤后,可得到纯度大于90%rhFNⅢ1-C。因其具有促进细胞增殖、粘附及其他胞外基质蛋白如胶原蛋白的形成等功能,并能促进单核吞噬细胞系统清除损伤组织处有害物质,具有促生长因子的作用。因此,本发明研制的这种重组蛋白具有作为药妆护肤品原料和成品的巨大市场潜力。
本发明采取的技术方案为:
本发明公开了一种rhFNⅢ1-C,所述的rhFNⅢ1-C基因序列如SEQUENCE LISTING400〈3〉所示。
所述的rhFNⅢ1-C氨基酸序列如SEQUENCE LISTING 400〈1〉所示。
本发明还提供了所述的rhFNⅢ1-C研制方法,所述研制方法包括以下步骤:
(1)将所述的rhFNⅢ1-C基因插入到pET-32a中构建重组质粒rhFNⅢ1-C/pET32-a;
(2)将重组质粒转化导入表达载体BL21(DE3)中,在含氨苄青霉素的LB平板上筛选阳性克隆菌;
(3)将阳性克隆菌接种于含氨苄青霉素的培养液中进行发酵,培养至对数生长期,然后进行IPTG诱导表达,离心收集菌体蛋白;
(4)在冰浴中破碎菌体,然后进行离心,收集上清液。将该上清液进行His-tag亲和层析纯化、透析、无菌过滤,即得到rhFNⅢ1-C目的蛋白。。
进一步地,所述步骤(2)之后还包括对阳性克隆菌进行PCR鉴定、双酶切鉴定的步骤。
所述步骤(3)为步骤(2)之后还包括对菌体表达目的蛋白rhFNⅢ1-C方式的鉴定。
所述步骤(3)具体包括以下步骤:将在含氨苄青霉素的LB平板上的阳性克隆菌接种于100mL含100μg/mL的氨苄青霉素的培养液中进行发酵,培养至对数生长期即OD600在0.6~0.8时,加入千分之一的IPTG进行诱导表达10h。发酵结束后,7000r/min离心20min收集菌体蛋白沉淀。
本发明还提供了所述的rhFNⅢ1-C在促MDBK细胞、Balb/c/3T3、ST细胞、PK-15细胞、Vero细胞、Hela细胞、HF细胞或MRC-5细胞贴壁、增殖和粘附作用中的应用。
本发明还提供了所述的重组人纤连蛋白Ⅲ1-C蛋白在药妆医美护肤品原料及成品中的应用,可制备成晒后修复精华、美白淡斑精华、保湿补水面膜、抗衰老祛皱精华;等等。具有作为药妆护肤品原料和成品的巨大市场潜力。所有含有所述的重组人纤连蛋白Ⅲ1-C蛋白的药妆医美护肤品原料及成品均在本发明的保护范围之内。
本发明通过原核表达技术选择rhFNⅢ1-C目的基因片段并导入大肠埃希菌表达目的蛋白;还提供了rhFNⅢ1-C重组菌能够进行大规模上罐发酵、纯化、蛋白检测、活性检测等的方法。
本发明首先选择纤连蛋白Ⅲ1-C目的基因片段、根据密码子表达偏好性优化改造目的基因并将合成的基因片段插入pET-32a中,进而将转化导入表达载体BL21(DE3)中在含氨苄青霉素的LB平板上筛选阳性克隆菌,并设计特异性引物进行PCR鉴定,双酶切鉴定;接着进行蛋白表达方式的鉴定和筛选;摇瓶发酵后破碎菌体,筛选可溶性高效表达的菌株。筛选得到的阳性克隆菌经发酵培养、诱导表达后收集的菌体沉淀经破碎、离心、His-tag亲和层析柱洗脱、透析等纯化步骤得到纯度大于90%rhFNⅢ1-C重组蛋白。将获得的精纯目的蛋白与鼠源抗纤连蛋白的一抗(特异性单抗,R&D公司产品)进行western-blot蛋白鉴定,鉴定结果为阳性。采用体外培养的MDBK细胞做系列细胞贴壁与促细胞生长试验等试验,证实了该rhFNⅢ1-C纤连蛋白具有促细胞粘附和贴壁的功能,并同时建立了对该rhFNⅢ1-C的生物学活性检测的方法。
与现有技术相比,本发明具有以下几点优势:
1.构建以重组大肠埃希菌BL21/pET32a-rhFNⅢ1-C作为可溶性高效表达菌株,具有生产周期短、易纯化、产量高、生产成本低等优势,适用于大规模的工业化生产;
2.采用本发明的技术研制的表达蛋白SDS-PAGE鉴定后,确定BL21(DE3)/rhFNⅢ1-C的表达量高达35%以上;
3.rhFNⅢ1-C利用考马斯亮蓝染色法测定纯化后蛋白浓度均高达1.0mg/mL以上,纯度均在90%以上,蛋白浓度和纯度均达到优质水平;
4.通过促细胞粘附性试验检测观察,显示rhFNⅢ1-C具有明显促进MDBK细胞的贴壁、粘附和细胞快速分裂生长的性能。
附图说明
图1为rhFNⅢ1-C/pET32-a的PCR鉴定结果图,泳道M为DL2000DNA Marker,1~5号泳道分别为44.7℃、55℃、59.7℃、65.0℃退火PCR产物;
图2为rhFNⅢ1-C/pET32-a经BamHⅠ及HindⅢ双酶切鉴定结果图,泳道M为DL2000DNA Marker,泳道1:单酶切体系,泳道2:单酶切体系,泳道3:双酶切体系,泳道4:无酶切体系;
图3为诱导表达后的菌体蛋白的SDS-PAGE的结果图,泳道M:26610Marker,1号泳道:BL21/pET32a空载,2~7号泳道:诱导0~6h取样;
图4为诱导表达后的菌体蛋白表达方式鉴定结果,泳道M:26610Marker,1号泳道:破碎后上清(2μl),2号泳道:破碎后沉淀(2μl);
图5为诱导表达后的菌体蛋白纯化后SDS-PAGE鉴定结果图,泳道M:26610Marker,1号泳道:BL21/pET32a空载,2号泳道:纯化前样品,3、4、5号泳道:杂蛋白,6、7号泳道:目的蛋白收集样;
图6为纯化后的rhFNⅢ1-C的western-blot结果图,泳道M:26616Marker泳道1:阴性对照,泳道2:2μl蛋白上样,泳道3:1μl蛋白上样;
图7为rhFNⅢ1-C促MDBK细胞的粘附性实验结果A:细胞对照组,B:实施例1得到的rhFNⅢ1-C 10倍稀释物;C:实施例1得到的rhFNⅢ1-C 320倍稀释物;D:阳性对照纤连蛋白10倍稀释物;
图8为优化合成的基因及测序后的基因比对及峰图。
具体实施方式
下面结合实施例对本发明进行详细说明。
实施例1
rhFNⅢ1-C的研制方法,包括以下步骤:
(1)将如SEQUENCE LISTING 400〈1〉所示的氨基酸序列,如SEQUENCE LISTING 400〈2〉所示的基因序列的人纤连蛋白FNⅢ1-C基因片段,通过调整基因序列中的GC比例、调整人纤连蛋白在大肠埃希菌表达系统的偏好性密码子对人纤连蛋白FNⅢ1-C基因片段进行优化并合成,优化后的人纤连蛋白FNⅢ1-C基因序列如SEQUENCE LISTING 400〈3〉所示,合成优化后的rhFNⅢ1-C基因的上下游引物序列为:上游引物:5‘-GGATCCAACGCCCCTCAGCCG-3’,下游引物:5’-CGGCTGAGGGGCGTTGGATCC-3’;
(2)将优化后的rhFNⅢ1-C基因插入到pET-32a中构建重组质粒rhFNⅢ1-C/pET32-a;
(3)将重组质粒转化导入表达载体BL21(DE3)中,在含氨苄青霉素的LB平板上筛选阳性克隆菌;
(4)将在含氨苄青霉素的LB平板上的阳性克隆菌接种于100mL含100μg/mL的氨苄青霉素的培养液中进行发酵,培养至对数生长期即OD600在0.6~0.8时,然加入终浓度0.1mMIPTG进行诱导表达10h,发酵结束后,7000r/min离心20min收集菌体蛋白沉淀;并在培养的过程中提取培养至对数生长期的阳性克隆菌的质粒,用于测序、PCR鉴定、BamH Ⅰ及HindⅢ双酶切鉴定;并对诱导表达后的菌体蛋白进行SDS-PAGE鉴定及蛋白表达方式鉴定。
图8为优化合成的基因及测序后的基因的比对及峰图,从图中可以看出优化合成的基因已成功导入质粒并成功进行了转化。
PCR反应体系如表1所示:
表1
双酶切鉴定:
目的基因选取的酶切位点为BamH Ⅰ和HindⅢ,分别对质粒分别进行单酶切、双酶切和不加酶切的反应,双酶切反应体系如表2;
表2
PCR反应、双酶切反应结束后,对各反应体系进行琼脂糖凝胶电泳鉴定,根据目的基因大小配制了2%的琼脂糖凝胶,待凝固后按照10μL每孔上样,DL2000DNA Marker 5μL上样,110V电泳35min至条带分散开,在紫外光下显色观察拍照,PCR鉴定、双酶切鉴定的结果分别如图1、如图2所示,从图1中可以看出各退火温度下均有扩增出目的条带,从图2中可以看出单酶切和双酶切均有条带,说明目的基因已经成功导入宿主菌中。
对诱导表达后的菌体蛋白进行SDS-PAGE鉴定:
配制15%SDS-PAGE分离胶和5%SDS-PAGE浓缩胶,菌体蛋白样品按照2μL上样,60V电泳30min,110V电泳1.5h左右结束。电泳结束,切胶,用考马斯亮蓝染色约1h,弃去染液加入适量脱色液,摇晃脱色至蛋白条带清晰,观察在25~35kDa处有明显条带,结果如图3所示。
对诱导表达后的菌体蛋白进行蛋白表达方式鉴定:
将上述步骤(4)得到的菌体蛋白沉淀按照1g菌体蛋白沉淀:20mLPBS的比例分别加入约50mLPBS,用重悬仪重悬菌体至无明显沉淀;把重悬后的菌体悬浊液加至塑料烧杯中,冰浴破碎,取破碎菌液作革兰染色鉴定是否破碎完全;破碎完全后分别取上清样、沉淀样进行SDS-PAGE鉴定,结果如图4所示,从图中可以看出破碎后上清泳道中有目的蛋白条带且比沉淀泳道中条带明显,说明构建的重组菌株为可溶性表达rhFNⅢ1-C。
(5)在冰浴中,利用压力为600~800bar的均质机破碎菌体蛋白沉淀4~5次,然后进行离心,收集上清液,上清液进行His-tag亲和层析纯化,对层析镍柱按照超纯水平衡、上样、再平衡、150mM咪唑洗涤缓冲液洗涤杂蛋白、350mM咪唑浓度洗脱缓冲液洗脱目的蛋白、收集洗脱峰等到粗纯化蛋白,收集的洗脱液在10倍体积的TBS中进行透析换液,4℃透析2次,每次透析6小时以上。使用L-精氨酸保护剂母液与透析后的复合型硫氧化蛋白溶液按照1︰7体积比混合,使L-精氨酸终浓度为50mmol/L。再加入2%甘油轻轻混合均匀,避免产生气泡。最后无菌过滤,分装至盐水瓶中,即为重组纤连蛋白原液,放置-20℃冻存。将rhFNⅢ1-C进行SDS-PAGE鉴定和Western blot鉴定,结果分别如图5、6所示,从图中可见在25~35kDa处有明显的条带,说明成功制备得到了rhFNⅢ1-C;并从图5中可以看出rhFNⅢ1-C的表达量在35%以上。从图6中可以看出纯化后蛋白浓度均在1.0mg/mL以上,纯度约为90%以上。达到一次性高蛋白浓度并精纯的优质标准。
实施例2
rhFNⅢ1-C的促细胞粘附性试验,具体步骤如下:
(a)将实施例1得到的精制rhFNⅢ1-C和阳性对照纤连蛋白(购自上海纤连生物技术有限公司)分别以PBS缓冲液稀释10倍和100倍后,加入到96孔细胞板上按2倍梯度进行稀释;为了避免边缘效应,四周边缘孔不作实验,并设立阴性对照(阴性对照只加入100μL PBS缓冲液);4℃孵育过夜;
(b)每孔加入100μL PBS洗涤,共洗涤3次;洗涤结束后,每孔加入1%BSA封闭,即置37℃温箱孵育1h。孵育完成后,弃去板中液体;
(c)制备生长状态良好的MDBK单细胞悬液。将MDBK单细胞悬液密度调整为1.0×10/mL,每孔接种100μL,温箱孵育5h后,置显微镜下观察细胞贴壁和粘壁现象等。
(d)在倒置显微镜下观察细胞的贴壁情况,结果如图7所示。从图中可以看出,细胞对照组中仅有少量细胞贴壁,大部分细胞呈圆形悬浮状于板孔中;而采用实施例1得到的rhFNⅢ1-C处理过的板孔中,可见1:10稀释孔中有50%以上细胞贴壁,细胞形态已经发生改变,呈三角形和多角形。可见本发明研制的rhFNⅢ1-C较阳性对照组具有更好的促细胞贴壁和粘附的生物学活性。
(e)在显微镜视野范围内对贴壁细胞进行计数,计数结果如表3所示。
表3
稀释度(×10) | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 29 | 210 | C |
蛋白浓度(mg/mL) | 0.07 | 0.04 | 0.02 | 0.01 | 0.005 | 0.003 | 0.002 | 0.001 | 0.001 | 0 |
细胞数 | 117 | 129 | 60 | 42 | 27 | 12 | 15 | 9 | 9 | 6 |
并对表3中的数据进行拟合曲线,计算得到rhFNⅢ1-C的ED50=0.023mg/mL。
上述参照实施例对一种rhFNⅢ1-C及其制备方法和应用进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,均应属本发明的保护范围之内。
SEQUENCE LISTING
<110> 芜湖天明生物技术有限公司
<120> 一种重组人纤连蛋白Ⅲ1-C及其制备方法和应用
<130> 1
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 75
<212> PRT
<213> rhFNⅢ1-C氨基酸序列
<400> 1
Asn Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu Arg Trp Arg
1 5 10 15
Pro Lys Asn Ser Val Gly Arg Trp Lys Glu Ala Thr Ile Pro Gly His
20 25 30
Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys Pro Gly Val Val Tyr Glu
35 40 45
Gly Gln Leu Ile Ser Ile Gln Gln Tyr Gly His Gln Glu Val Thr Arg
50 55 60
Phe Asp Phe Thr Thr Thr Ser Thr Ser Thr Pro
65 70 75
<210> 2
<211> 237
<212> DNA
<213> 优化前FNⅢ1-C基因序列
<400> 2
ggatccaatg caccacagcc atctcacatt tccaagtaca ttctcaggtg gagacctaaa 60
aattctgtag gccgttggaa ggaagctacc ataccaggcc acttaaactc ttacaccatc 120
aaaggcctga agcctggtgt ggtatacgag ggccagctca tcagcatcca gcagtacggc 180
caccaagaag tgactcgctt tgacttcacc accaccagca ccagcacacc taagctt 237
<210> 3
<211> 237
<212> DNA
<213> 优化后FNⅢ1-C基因序列
<400> 3
ggatccaacg cccctcagcc gagtcatatt agtaaatata ttctgcgctg gcgcccgaaa 60
aatagcgtgg gtcgctggaa agaagccacc attccgggtc atctgaatag ctataccatt 120
aagggtctga aaccgggtgt ggtttatgaa ggtcagctga ttagtattca gcagtatggt 180
catcaggaag tgacccgctt tgattttacc accaccagta ccagtacccc gaagctt 237
Claims (9)
1.一种重组人纤连蛋白Ⅲ1-C蛋白(recombinant human FNⅢ1-C,rhFNⅢ1-C)其特征在于,所述rhFNⅢ1-C的基因序列如SEQUENCE LISTING 400〈3〉所示。
2.如权利要求1所述的重组人纤连蛋白Ⅲ1-C蛋白的制备方法,其特征在于,所述制备方法包括以下步骤:
(1)将所述rhFNⅢ1-C的基因插入到pET-32a中构建重组质粒rhFNⅢ1-C/pET32-a;
(2)将重组质粒转化导入表达载体BL21(DE3)中,在含氨苄青霉素的LB平板上筛选阳性克隆菌;
(3)将阳性克隆菌接种于含氨苄青霉素的培养液中进行发酵,培养至对数生长期,然后进行IPTG诱导表达,离心收集菌体蛋白;
(4)在冰浴中破碎菌体,然后进行离心,收集上清液,上清液经His-tag亲和层析纯化、透析、无菌过滤,即可得到高浓度高纯度的rhFNⅢ1-C目的蛋白。
3.根据权利要求2所述的制备方法,其特征在于,所述步骤(2)之后还包括对阳性克隆菌进行PCR鉴定、双酶切鉴定的步骤。
4.根据权利要求2所述的制备方法,其特征在于,所述步骤(3)之后还包括对菌体蛋白表达方式进行鉴定的步骤。
5.根据权利要求2-4任意一项所述的制备方法,其特征在于,所述步骤(3)具体包括以下步骤:将在含氨苄青霉素的LB平板上的阳性克隆菌接种于100mL含100μg/mL的氨苄青霉素的培养液中进行发酵,培养至对数生长期即OD600在0.6~0.8时,加入千分之一的IPTG进行诱导表达10h,发酵结束后,7000r/min离心20min收集菌体蛋白沉淀。
6.如权利要求1所述的重组人纤连蛋白Ⅲ1-C蛋白在促MDBK细胞、Balb/c/3T3、ST细胞、PK-15细胞、Vero细胞、Hela细胞、HF细胞或MRC-5细胞贴壁、增殖和粘附作用中的应用。
7.如权利要求1所述的重组人纤连蛋白Ⅲ1-C蛋白在护肤品原料及成品中的应用。
8.含有如权利要求1所述的重组人纤连蛋白Ⅲ1-C蛋白的药妆医美护肤品原料及成品。
9.如权利要求1所述的重组人纤连蛋白Ⅲ1-C蛋白在药妆医美护肤品原料及成品中的应用。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112876569A (zh) * | 2021-02-08 | 2021-06-01 | 安徽绿珏生物科技有限公司 | 一种rhTSG6-FNⅢ1-C融合蛋白及其在皮肤护理组合物中的用途和其制备方法 |
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 |
CN114702571A (zh) * | 2022-04-24 | 2022-07-05 | 安博威生物科技(厦门)有限公司 | 一种具有促进干细胞定植的纤连蛋白及其制备方法 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070015708A1 (en) * | 2000-12-04 | 2007-01-18 | Erkki Ruoslahti | Methods and compositions for inhibiting tumor growth and angiogenesis |
US20100292161A1 (en) * | 2007-06-14 | 2010-11-18 | Clark Richard A | Polypeptides and methods of use |
CN102834114A (zh) * | 2010-04-13 | 2012-12-19 | 米迪缪尼有限公司 | 基于纤连蛋白iii型结构域的多聚体支架 |
US20140057807A1 (en) * | 2010-07-30 | 2014-02-27 | The University Of Chicago | Fibronectin cradle molecules and libraries thereof |
CN109790536A (zh) * | 2016-07-29 | 2019-05-21 | 宝生物工程株式会社 | 用于干细胞产生的纤连蛋白片段 |
CN110577592A (zh) * | 2019-10-08 | 2019-12-17 | 广州暨南大学医药生物技术研究开发中心 | 一种重组人纤连蛋白肽 |
-
2020
- 2020-02-25 CN CN202010116877.2A patent/CN111217903B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070015708A1 (en) * | 2000-12-04 | 2007-01-18 | Erkki Ruoslahti | Methods and compositions for inhibiting tumor growth and angiogenesis |
US20100292161A1 (en) * | 2007-06-14 | 2010-11-18 | Clark Richard A | Polypeptides and methods of use |
CN102834114A (zh) * | 2010-04-13 | 2012-12-19 | 米迪缪尼有限公司 | 基于纤连蛋白iii型结构域的多聚体支架 |
US20140057807A1 (en) * | 2010-07-30 | 2014-02-27 | The University Of Chicago | Fibronectin cradle molecules and libraries thereof |
CN109790536A (zh) * | 2016-07-29 | 2019-05-21 | 宝生物工程株式会社 | 用于干细胞产生的纤连蛋白片段 |
CN110577592A (zh) * | 2019-10-08 | 2019-12-17 | 广州暨南大学医药生物技术研究开发中心 | 一种重组人纤连蛋白肽 |
Non-Patent Citations (5)
Title |
---|
R MATTHEW KLEIN等: "Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin", 《J CELL SCI》 * |
李明才等: "三结构域重组纤连蛋白多肽在大肠杆菌中表达及其产物的纯化与鉴定", 《同济医科大学学报》 * |
王峰: ""表皮调节素、纤联蛋白与去唾液酸糖蛋白受体介导HBV进入肝细胞机理初步研究"", 《中国知网硕士电子期刊》 * |
田德桥: "《生物技术发展知识图谱》", 30 June 2018, 科学技术文献出版社 * |
石耀华等: "《水产养殖学专业生物学基础课程实验》", 31 August 2011, 海洋出版社 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112876569A (zh) * | 2021-02-08 | 2021-06-01 | 安徽绿珏生物科技有限公司 | 一种rhTSG6-FNⅢ1-C融合蛋白及其在皮肤护理组合物中的用途和其制备方法 |
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