CN110498859A - 重组PLB-hbFGF融合蛋白及其应用 - Google Patents
重组PLB-hbFGF融合蛋白及其应用 Download PDFInfo
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- CN110498859A CN110498859A CN201910699141.XA CN201910699141A CN110498859A CN 110498859 A CN110498859 A CN 110498859A CN 201910699141 A CN201910699141 A CN 201910699141A CN 110498859 A CN110498859 A CN 110498859A
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- hbfgf
- fusion protein
- plb
- recombination
- albumen
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Abstract
本发明公开一种重组PLB‑hbFGF融合蛋白及其应用,所述重组PLB‑hbFGF融合蛋白包括由蛋白L的B结构域与人碱性成纤维细胞生长因子蛋白融合得到的融合蛋白,其中,所述蛋白L的B结构域包括如SEQ ID NO:1~5所示的PLB1~PLB5蛋白中的任意一个,所述人碱性成纤维细胞生长因子蛋白的氨基酸序列如SEQ ID NO:8所示。本发明提供的重组PLB‑hbFGF融合蛋白,在构建成为融合蛋白表达载体用于表达hbFGF时,提高了hbFGF的可溶性表达,节省了体外复性的耗时工艺,且显著提高了比活性,对工业化生产可溶性、高活性hbFGF具有重要价值。
Description
技术领域
本发明涉及基因工程和蛋白质表达技术领域,特别涉及重组人碱性成纤维细胞生长因子融合蛋白构建技术领域,具体涉及一种重组PLB-hbFGF融合蛋白及其应用。
背景技术
蛋白L(Protein L,PL)是1988年首次发现于人类大消化链球菌(Peptostreptococcus magnus)细胞壁上的一种蛋白,随后分离鉴定,因为能与抗体的L链(轻链)结合而得名“Protein L”。PL由719个氨基酸残基组成,分子量约为76kD,分子内不含任何二硫键,也不具有任何二硫键连接的亚基。它是一种酸性蛋白分子,等电点(pI)4.0。PL基因包含5个编码成分:1个由18AA组成的信号肽,1个N-端79AA组成的“A”区,5个72-76AA同源重复的“B”区,1个C-端由2个52AA重复的“C”区。在C区之后有一段亲水性富含脯氨酸的“W”区,推测其为细菌细胞壁的“跨壁区”,最后一段为疏水性的“M”区,应当是细菌细胞膜的“锚定区”。蛋白L的B结构域被发现能与Ig的L链相互作用。
人碱性成纤维细胞生长因子(Human basic fibroblast growth factor,hbFGF)是人体的一种极其重要的细胞生长因子。人bFGF是含155个氨基酸的促有丝分裂的碱性蛋白质,其氨基酸序列与aFGF有55%的同源性,但其生物学活性比aFGF强30~100倍。bFGF分子量为16~18.5KD。
bFGF成熟肽的羧基端较氨基端更稳定,将氨基端截去24个氨基酸时,尚不影响其生物学活性。但如果将6xHis-Tag纯化标签置于人bFGF(hbFGF)C-端,镍柱通常挂不上,说明hbFGF的羧基端可能相互遮盖或缩进。bFGF在生物进化上具有很强的保守性,人和牛的bFGF蛋白同源性达98.7%。
bFGF作为细胞分裂原,主要作用在起源于中胚层和神经外胚层的骨骼肌细胞、成纤维细胞和骨细胞等,其受体也相应的分布于上述细胞表面。成纤维细胞生长因子(FGF)存在两类受体:一类是高亲和力受体,属跨膜性酪氨酸蛋白激酶类受体;另一类是低亲和力受体,即肝素样受体,为硫酸乙酰肝素蛋白多糖类物质。
现今资料表明,bFGF的生物学作用极其广泛,它在组织修复、促进创伤愈合与血管形成、促进组织再生和神经组织生长发育过程中起着十分重要的作用。bFGF的生物学效应分体内和体外两大部分。体外研究发现,bFGF对成纤维细胞、骨细胞、软骨细胞、血管内皮细胞、肾上腺皮质和髓质细胞、神经元和神经胶质细胞等有很强的促细胞增值活性。体外细胞培养中,能在低浓度(1ng·ml-1)发挥其作用。bFGF是重要的促有丝分裂因子,也是形态发生和分化的诱导因子。其主要生物学作用有:(1)作为血管生长因子;(2)促进创伤愈合与组织修复;(3)促进组织再生;(4)参与神经再生等。
bFGF在体内含量甚微,但分布广泛,生理功能复杂多样。bFGF生物活性的多效性以及神经营养的广谱性,为其从基础走向临床提供了依据。在皮肤衰老的修复和预防皮肤衰老的研究以及神经损伤的研究中,神经-体液因子的联合营养调控早已得到认证,如肢体神经损伤,肢体的皮肤、肌肉萎缩;皮肤神经末稍萎缩或功能异常,皮肤松弛,感觉迟钝,营养障碍,出现神经性皮炎、色素沉着、色斑、或老年斑。皮肤表面的临床使用证明,少量的bFGF透皮吸收(毛囊或汗腺口),经过一段时间后,皮肤质感丰实,皱纹减少、沟纹变浅。这些发现使得bFGF在护肤抗衰领域拓开了巨大的应用前景。
目前,国际日化名牌也有bFGF的添加,以联合添加表皮生长因子(EGF)的居多,bFGF更倾向于皮下建筑细胞——成纤维细胞——的活化与新生,促进胶原蛋白新生和更替,以修复深皱纹的基底组织;同时bFGF还对神经末梢有营养与保护功能,从而在皮肤的干细胞活力维持和皮肤组织神经-体液营养方面提供延缓皮肤衰老的作用。与EGF联合应用,将形成全方位皮肤抗衰调控,由内向外、由里及表的全层维持细胞与组织的优良状态,这将是皮肤生理学需要的全方位仿生生物学皮肤抗衰老内容。
随着hbFGF在医药和日化领域的应用市场扩大,对hbFGF的需求量快速上升,目前常见的原核细胞hbFGF基因表达工程获得的hbFGF因子,其在细菌细胞内的表达过程中,可溶性低,包涵体多,经常借助体外复性,造成产量低、活性低、工艺繁,不利于大工艺生产。因此,hbFGF原核表达基因工程和生产工艺的进一步探索、改进、优化仍然必要。
发明内容
本发明的主要目的是提出一种重组PLB1-hbFGF融合蛋白及其应用,旨在提高hbFGF的可溶性表达。
为实现上述目的,本发明提出一种重组PLB-hbFGF融合蛋白,包括由蛋白L的B结构域与hbFGF(人碱性成纤维细胞生长因子蛋白)融合得到的融合蛋白,其中,所述蛋白L的B结构域包括如SEQ ID NO:1~5所示的PLB1~PLB5蛋白中的任意一个,所述人碱性成纤维细胞生长因子蛋白的氨基酸序列如SEQ ID NO:8所示。
可选地,所述蛋白L的B结构域为如SEQ ID NO:1所示的PLB1蛋白。
可选地,所述PLB1蛋白的N端添加有翻译起始优化序列,而形成N端优化的PLB1蛋白,所述N端优化的PLB1蛋白的氨基酸序列如SEQ ID NO:7所示。
可选地,所述N端优化的PLB1蛋白的C端添加有柔性接头、内置组氨酸标签、蛋白酶识别区和优化的hbFGF编码区,其中,
所述柔性接头包括6~30个氨基酸;
所述内置组氨酸标签包含5~10个His;
所述蛋白酶识别区包括凝血因子Xa蛋白酶识别区、凝血酶识别区、肠激酶识别区和TEV酶识别区中的任意一种。
可选地,所述柔性接头包括7~20个氨基酸;
所述组氨酸标签包含9个His;
所述蛋白酶识别区为凝血因子Xa蛋白酶识别区。
可选地,所述柔性接头的序列如SEQ ID NO:14,15所示;
所述组氨酸标签的序列如SEQ ID NO:16,17所示;
所述凝血因子Xa蛋白酶识别区的序列如SEQ ID NO:18,19所示。
可选地,所述重组PLB1-hbFGF融合蛋白的氨基酸序列如SEQ ID NO:10所示。
本发明还提出一种如上所述的重组PLB-hbFGF融合蛋白的编码基因。
可选地,所述重组PLB-hbFGF融合蛋白的编码基因的核苷酸序列如SEQ ID NO:11所示。
本发明还进一步提出一种包含如上所述的重组PLB-hbFGF融合蛋白的编码基因的重组PLB-hbFGF融合蛋白表达载体或表达工程菌。
可选地,所述重组PLB-hbFGF融合蛋白表达载体是将上述重组PLB-hbFGF融合蛋白的编码基因插入到原核细胞表达质粒的NcoI和XhoI酶切位点之间得到。
可选地,所述原核细胞表达质粒选用pET28作为亲本表达载体。
本发明还提出如上所述的重组PLB-hbFGF融合蛋白表达载体在表达PLB-hbFGF融合蛋白中的应用。
本发明还提出一种重组PLB-hbFGF融合蛋白的制备方法,包括以下步骤:将如上所述的重组PLB-hbFGF融合蛋白表达载体转化感受态表达菌株获得表达工程菌,然后对所述表达工程菌进行培养,获得重组PLB-hbFGF融合蛋白。
此外,本发明还提出一种重组PLB-hbFGF融合蛋白表达工程菌的制备方法,包括以下步骤:
基于多聚酶链式反应的全基因人工合成方法,合成PLB与hbFGF的融合蛋白PLB-hbFGF的DNA编码序列,其中,所述融合蛋白PLB-hbFGF的序列如SEQ ID NO:10所示,所述DNA编码序列如SEQ ID NO:11所示;
将所述融合蛋白PLB-hbFGF的编码DNA经过NocI和XhoI DNA限制性内切酶消化和琼脂糖凝胶电泳分离纯化后,得到插入片段;
经过内切酶NocI和XhoI双酶切并琼脂糖凝胶电泳分离纯化原核细胞表达质粒,得到线性化空载体;
用T4DNA连接酶将所述线性化空载体与所述融合蛋白PLB-hbFGF的编码DNA片段相连接,然后转化感受态菌DH5α,经阳性克隆扩增、质粒制备及DNA测序后,获得序列正确的工程质粒;
将所述工程质粒转化BL21(DE3)感受态表达菌株,在卡那霉素存在下进行筛选与IPTG诱导表达测试,获得重组PLB-hbFGF融合蛋白表达质粒高的表达工程菌株。
本发明提供的技术方案中,将蛋白L的B结构域与人碱性成纤维细胞生长因子蛋白融合得到PLB-hbFGF融合蛋白,使用该PLB-hbFGF融合蛋白构建得到的融合蛋白表达载体表达hbFGF时,提高了hbFGF的可溶性表达,节省了体外复性的耗时工艺,且显著提高了比活性,对工业化生产可溶性、高活性hbFGF具有重要价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅为本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为本发明提供的融合蛋白表达载体的一实施例中PLB1-hbFGF融合蛋白表达框示意图;
图2为实施例1中制得的pET28-PLB1-hbFGF表达载体物理图;
图3为实施例1中制得的pET28-hbFGF对照表达载体物理图;
图4为实施例2中pET28-PLB1-hbFGF表达载体测试诱导表达的SDS-PAGE图;
图5为实施例2中pET28-hbFGF对照表达载体测试诱导表达的SDS-PAGE图。
本发明目的的实现、功能特点及优点将结合实施例,参照附图做进一步说明。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
随着hbFGF在医药和日化领域的应用市场扩大,对hbFGF的需求量快速上升,目前常见的原核细胞hbFGF基因表达工程获得的hbFGF因子,其在细菌细胞内的表达过程中,可溶性低,包涵体多,经常借助体外复性,造成产量低、活性低、工艺繁,不利于大工艺生产。因此,hbFGF原核表达基因工程和生产工艺的进一步探索、改进、优化仍然必要。
为解决上述技术问题,本发明在探索PL的重组表达时,发现去除N-端79个AA后,在大肠杆菌中B区结构域仍然具有很好的自折叠功能,甚至在去除B2、B4后,将B1、B3、B5编码区合并,仍然保持自折叠、全可溶性“融合”表达,而高度自折叠可溶性蛋白具有一定程度诱导其它蛋白折叠的效应,即“类伴侣样蛋白”效应。因此,本发明在研究过程中尝试了融合其它不易折叠蛋白的分子折叠诱导效应,无论是对多二硫键或无二硫键的下游融合蛋白的折叠诱导,都取得了明显效果。基于此,本发明提出一种重组PLB-hbFGF融合蛋白,包括由蛋白L的B结构域(PLB)与人碱性成纤维细胞生长因子蛋白融合得到的融合蛋白,其中,所述蛋白L的B结构域包括如SEQ ID NO:1~5所示的PLB1~PLB5蛋白中的任意一个,所述人碱性成纤维细胞生长因子蛋白的氨基酸序列如SEQ ID NO:8所示。
本发明提供的技术方案中,将蛋白L的B结构域与人碱性成纤维细胞生长因子蛋白(hbFGF)融合得到PLB-hbFGF融合蛋白,使用该PLB-hbFGF融合蛋白构建得到的融合蛋白表达载体表达hbFGF时,提高了hbFGF的可溶性表达,节省了体外复性的耗时工艺,且显著提高了比活性,对工业化生产可溶性、高活性hbFGF具有重要价值。
所述蛋白L的B结构域PLB1~PLB5中的任意一个均可用作上游融合蛋白,在本发明提供的重组PLB-hbFGF融合蛋白的一优选实施例中,所述蛋白L的B结构域为如SEQ ID NO:1所示的PLB1蛋白,对应得到的融合蛋白命名为重组PLB1-hbFGF融合蛋白。
在该实施例中,所述PLB1蛋白还经过优化,具体为所述PLB1蛋白的N端添加有翻译起始优化序列,而形成N端优化的PLB1蛋白,所述N端优化的PLB1蛋白的氨基酸序列如SEQID NO:7所示。
进一步地,所述N端优化的PLB1蛋白的C端添加有柔性接头、内置组氨酸标签、蛋白酶识别区和优化的hbFGF编码区,对应形成如图1所示的PLB1-FL-PRS-hbFGF融合蛋白。在图1中,RBS表示核糖体结合区,PLB1表示蛋白L的B1结构域,FL表示柔性接头,PRS表示蛋白酶识别区,hbFGF表示优化后的hbFGF编码区,9xHis-Tag表示9个组氨酸组成的内置标签。
更进一步地,所述柔性接头包括6~30个氨基酸,优选为7~20个氨基酸,更优选为所述柔性接头的序列如SEQ ID NO:14,15所示;所述组氨酸标签包含5~10个His,优选为包含9个His,更优选为所述组氨酸标签的序列如SEQ ID NO:16,17所示;所述蛋白酶识别区包括凝血因子Xa(FXa)蛋白酶识别区、凝血酶识别区、肠激酶识别区和TEV酶识别区中的任意一种,优选为FXa蛋白酶识别区,更优选为包含4个氨基酸残基,其序列如SEQ ID NO:18,19所示。
基于上述提供的所述柔性接头、FXa蛋白酶识别区以及组氨酸标签的序列,本发明提供的所述重组PLB1-hbFGF融合蛋白更优选为其氨基酸序列如SEQ ID NO:10所示。
本发明进一步提出一种如上所述的重组PLB-hbFGF融合蛋白的编码基因。
作为上述编码基因的一优选实施方案,所述重组PLB-hbFGF融合蛋白的编码基因的核苷酸序列如SEQ ID NO:11所示。
本发明还进一步提出一种包含如上所述的重组PLB-hbFGF融合蛋白的编码基因的重组PLB-hbFGF融合蛋白表达载体或表达工程菌,借助于所述重组PLB-hbFGF融合蛋白表达载体,可以实现自折叠可溶性重组hbFGF的发酵生产。
在本发明提供的重组PLB-hbFGF融合蛋白表达载体的一实施例中,所述重组PLB-hbFGF融合蛋白表达载体是将上述重组PLB-hbFGF融合蛋白的编码基因插入到原核细胞表达质粒的NcoI和XhoI酶切位点之间得到,其中,所述重组PLB-hbFGF融合蛋白的编码基因的核苷酸序列如SEQ ID NO:11所示,所述原核细胞表达质粒优选为pET系统表达载体,更优选为以pET28作为亲本表达载体。
如上所述的重组PLB-hbFGF融合蛋白表达载体可用于表达PLB1-hbFGF融合蛋白,且在表达目标蛋白时具有以下优点:(1)可溶性蛋白与包涵体蛋白的比值大于50%;(2)容易借助组氨酸标签(His-Tag)亲和纯化获得目标蛋白;(3)还可借助抗体Kappa链配体亲和色谱进一步纯化切除PLB。
基于上述提供的重组PLB-hbFGF融合蛋白表达载体,本发明还提出一种重组PLB-hbFGF融合蛋白的制备方法,包括以下步骤:将如上所述的重组PLB-hbFGF融合蛋白表达载体转化感受态表达菌株获得表达工程菌,然后对所述表达工程菌进行培养,获得重组PLB-hbFGF融合蛋白。
此外,基于上述提供的重组PLB-hbFGF融合蛋白表达工程菌本发明还提出一种重组PLB-hbFGF融合蛋白表达工程菌的制备方法,包括以下步骤:
步骤S10、基于多聚酶链式反应(PCR)的全基因人工合成方法,合成PLB与hbFGF的融合蛋白PLB-hbFGF的DNA编码序列,其中,所述融合蛋白PLB-hbFGF的序列如SEQ ID NO:10所示,所述DNA编码序列如SEQ ID NO:11所示;
步骤S20、将所述融合蛋白PLB-hbFGF的编码DNA经过NocI和XhoI DNA限制性内切酶消化和琼脂糖凝胶电泳分离纯化后,得到插入片段;
步骤S30、经过内切酶NocI和XhoI双酶切并琼脂糖凝胶电泳分离纯化原核细胞表达质粒,得到线性化空载体,作为亲本载体(Parents vector);
步骤S40、用T4DNA连接酶将所述线性化空载体与所述融合蛋白PLB-hbFGF的编码DNA片段相连接,然后转化感受态菌DH5α,经阳性克隆扩增、质粒制备及DNA测序后,获得序列正确的工程质粒;
步骤S50、将所述工程质粒转化BL21(DE3)感受态表达菌株,在卡那霉素存在下进行筛选与IPTG(异丙基硫代半乳糖苷,Isopropylβ-D-Thiogalactoside,一种作用极强的诱导剂)诱导表达测试,获得重组PLB-hbFGF融合蛋白表达质粒高的表达工程菌株。
在本发明提供的重组PLB-hbFGF融合蛋白表达工程菌的制备方法的一实施例中,所述原核细胞表达质粒可选用pET系统表达载体,更优选为pET28,使用T4DNA连接酶将载体pET28与融合蛋白PLB-hbFGF的编码DNA片段相连接,转化感受态菌DH5α,获得正确序列的阳性克隆pET28-PLB-hbFGF,作为所述工程质粒,其序列如SEQ ID NO:40所示;然后将所述pET28-PLB-hbFGF质粒转化BL21(DE3)感受态表达菌株,在卡那霉素存在下进行筛选和诱导表达测试,获得表达工程菌株pET28-PLB-hbFGF/BL21(DE3)。
以下结合具体实施例和附图对本发明的技术方案作进一步详细说明,应当理解,以下实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1重组PLB1-hbFGF融合蛋白表达载体的构建
步骤一、重组PLB1与hbFGF融合蛋白编码DNA的人工合成和优化,本实施例以PLB1为例(SEQ ID NO:1,6),hbFGF以155个氨基酸的成熟肽为例(SEQ ID NO:8):
(1)逆翻译:分别用DNAWorks软件优化PLB1和hbFGF编码DNA序列(SEQ ID NO:6,9)。
(2)插入子(Insert)表达框(ORF)组装:
将翻译起始区优化序列添加到密码子优化的PLB1的5’-端(SEQ ID NO:7,12,13),将柔性接头DNA编码序列(SEQ ID NO:14,5)、内置9 x His组氨酸标签序列(SEQ ID NO:16,17)、FXa识别区(PRS)DNA编码序列(SEQ ID NO:18,19)和hbFGF密码子优化序列(SEQ IDNO:9)按次序合并插入PLB1的3’-端,添加终止密码。
(3)ORF全合成引物设计:
①引物设计:将拼接完成的PLB1-hbFGF融合蛋白ORF编码序列(SEQ ID NO:11)输入NCBI在线软件DNAworks设计引物,如SEQ ID NO:20~39;
②插入子重组位点设计:在5’-端第一个引物的起始密码5’-端添加[5’-CC-3’]形成NcoI内切酶识别序列并在酶切位点5’-端添加四个保护碱基(SEQ ID NO:20);在最后一个引物的最末密码子的5’-端添加XhoI内切酶识别序列和四个保护碱基(SEQ ID NO:39)。
(4)重叠延伸PCR(Overlap PCR)法合成全长编码DNA序列:
①将引物用去菌去离子灭菌水分别稀释至5μMole浓度;
②从每管引物分别取1μL加入1支清洁200μL PCR管;
③然后依照Takara Pyrobest PCR试剂盒说明,依次加入10mM dNTP 4μL,10 xPyrobest PCR Buffer 5μL,无菌去离子水加至50μL;
④最后加入Pyrobest高保真DNA聚合酶0.25μL;
⑤PCR参数:预变性95℃/3min;变性94℃/30sec,退火58℃/30sec,延伸72℃/1min,18个循环;补全延伸72℃/5min。
(5)常规多聚酶链式反应(General PCR)法扩增全长编码DNA序列:
①取上一步PCR产物1μL作模板,加入到200μL清洁PCR管;
②分别加入起始引物和末端引物(SEQ ID NO:20,39)各4μL;
③然后依照Takara Pyrobest PCR试剂盒说明,依次加入10mM dNTP 4μL,10 xPybest Buffer 5μL,无菌去离子水加至50μL;
④最后加入Pyrobest高保真DNA聚合酶0.5μL;
⑤PCR参数:预变性95℃/3min,变性94℃/30sec,退火66℃/30sec,延伸72℃/2min,25个循环;补全延伸72℃/5min。
(6)PCR产物的纯化与酶切:PCR产物经微型硅胶柱纯化,NcoI和XhoI限制性内切酶双酶切过夜。
(7)酶切DNA产物的纯化:采用0.8%琼脂糖凝胶电泳,胶上DNA回收试剂盒回收获得两端分别是NcoI和XhoI的粘性接头插入子。
步骤二、原核表达载体pET28的制备:
(1)取pET28载体2μg,用限制性内切酶NcoI和XhoI双酶切。
(2)0.8%琼脂糖凝胶电泳分离、胶回收纯化线性化后的pET28载体。
步骤三、连接与转化
(1)用T4DNA连接酶将处理好的PLB1-hbFGF插入子和pET28载体连接。
(2)转化大肠杆菌感受态菌株DH5α,含卡那霉素(Kan)抗生素的琼脂培养皿37℃培养过夜,然后挑取单菌落,常规PCR鉴定阳性克隆。
(3)将阳性克隆送交DNA序列分析,保留序列正确的克隆,常规微型硅胶柱法制备DNA质粒。
获得的工程载体质粒命名为pET28-PLB1-hbFGF(该pET28-PLB1-hbFGF表达载体的物理图如图2所示,序列如SEQ ID NO:40所示)。
步骤四、对照载体pET28-hbFGF的构建
利用pET28载体的5’-端组氨酸编码序列和其下游的NdeI限制性内切酶位点,将hbFGF的PCR扩增片段插入到pET28载体的NdeI和XhoI双酶切定向克隆区间,形成pET28-hbFGF表达工程质粒。
(1)PCR引物设计:
上游引物5’-端ATG前添加了[5’-CAT-3’]碱基和4个T碱基保护NdeI酶切位点,下游引物在hbFGF ORF末端添加终止密码[5’-TAA-3’],并在终止密码的5’-端的添加[5’-CTCGAG-3’]形成XhoI酶切识别位点和4个T碱基保护XhoI酶切位点,其氨基酸和核苷酸序列如SEQ ID NO:41,42。
hbFGF上游引物[5’-TTTTCATATGGCTGCTGGTTCTATCACC-3’];hbFGF下游引物[5’-TTTTCTCGAGTTAAGATTTAGCAGACATCG-3’](如SEQ ID NO:43,44所示)。
(2)PCR法扩增带NdeI和XhoI重组位点的hbFGF插入子:
①以10ng pET28-PLB1-hbFGF质粒为DNA模板,分别加入2μL浓度为10μM的上下游PCR引物(SEQ ID NO:43,44)。
②然后依照Takara Pyrobest PCR试剂盒说明,依次加入10mM dNTP 4μL,10 xPyrobest PCR Buffer 5μL,无菌去离子水加至50μL;
③最后加入Pyrobest高保真DNA聚合酶0.25μL;
④PCR参数:预变性95℃/3min;变性94℃/30sec,退火58℃/30sec,延伸72℃/2min,18个循环;补全延伸72℃/5min。
(3)PCR产物和pET28空载体DNA分别用NdeI和XhoI双酶切处理过夜,0.8%琼脂糖凝胶电泳分离、切胶回收目标条带、胶回收试剂盒纯化。
(4)T4DNA连接酶连接载体与插入片段DNA,4℃冰箱过夜反应。
(5)连接产物转化感受态BL21(DE3)感受态菌株,Kan筛选单菌落,小量质粒制备送DNA序列分析,保留序列正确的克隆用作表达对照测试。
该步获得的对照载体是pET28-hbFGF(该pET28-hbFGF对照载体的物理图如图3所示,序列如SEQ ID NO:45所示),表达菌株是pET28-hbFGF/BL21(DE3)。
实施例2载体表达测试
步骤一、pET28-PLB1-hbFGF/BL21(DE3)表达菌株构建:
(1)将得到的pET28-PLB1-hbFGF表达载体DNA质粒转化大肠杆菌表达菌株BL21(DE3)感受态细胞,获得卡那霉素(Kan)抗性的单菌落。
(2)PCR鉴定阳性克隆,并送质粒测序再次肯定无误,保留正确的pET28-PLB1-hbFGF/BL21(DE3)表达菌株用于表达测试。
步骤二、
(1)将以上获得的pET28-PLB1-hbFGF/BL21(DE3)和对照质粒菌株pET28-hbFGF/BL21(DE3)分别1:100~500稀释,涂布含Kan琼脂平皿,再行筛选一次,尽量减少夹带生长的、不含表达载体的BL21(DE3)细胞,以保证表达菌株的表达效率,减少营养竞争。
(2)分别挑取单菌落数个,LB培养基培养,Kan提供筛选压,IPTG 0.5mM诱导12小时,收集菌液1mL,离心收集菌体,1×PBS 1mL洗涤一次,再悬浮于0.5mL 1×PBS,超声破碎菌体,离心取上清,保留沉淀,加入0.25mL 8M尿素溶液,震荡悬浮沉淀物,然后再加入1×PBS 0.25mL,混匀。
(3)各取20μL上清液和沉淀物悬浮液与SDS-PAGE上样缓冲液混合,95℃加热变性10分钟,离心收集至管底,置于冰上。
(4)各取10μL上样,12%SDS-PAGE电泳,考马斯亮蓝染色、脱色,观察细菌总蛋白质条带、可溶性PLB1-hbFGF融合蛋白条带(上清液)和包涵体PLB1-hbFGF融合蛋白条带的表达情况、以及它们之间的比例。图4所示为pET28-PLB1-hbFGF表达载体测试诱导表达的SDS-PAGE图,诱导条件为:0.5mM IPTG,25℃,200RPM,诱导12小时,所获得的菌体蛋白SDS-PAGE电泳图中可以看出,PLB1-hbFGF融合蛋白主要分布在上清液中;图5所示为pET30-hbFGF对照表达载体测试诱导表达的SDS-PAGE图,诱导条件为:0.5mM IPTG,25℃,200RPM,诱导12小时,所获得的菌体蛋白SDS-PAGE电泳图中可以看出,所表达的hbFGF蛋白主要分布在上沉淀物中。
测试结果为:重组PLB1-hbFGF,266aa(SEQ ID NO:41),理论分子量29kDa。根据SDS-PAGE电泳测算,PLB1-hbFGF融合蛋白表达量占菌体总蛋白的20%左右,可溶性蛋白占80~85%(如图4所示),包涵体占15~20%,目标蛋白的可溶性优于pET28-hbFGF的直接表达,后者几乎全部以包涵体形式表达(如图5所示)。
以上仅为本发明的优选实施例,并非因此限制本发明的专利范围,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包括在本发明的专利保护范围内。
SEQUENCE LISTING
<110> 因之彩生物科技(武汉)有限公司
<120> 一种重组PLB-hbFGF融合蛋白及其应用
<130> 20190517
<160> 45
<170> PatentIn version 3.5
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aaagcgacct ccgaagctta cgcctatgcg gacaccctca aaaaagacaa cggtgagtac 180
acggttgatg tcgcagacaa aggttacacc ctgaacatta agttcgcagg t 231
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Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
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Tyr Ser Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
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ctgcgtatcc acccggacgg tcgtgttgac ggtgttcgtg aaaaatctga cccgcacatc 180
aaactgcagc tgcaggctga agaacgtggt gttgtttcta tcaaaggtgt ttctgctaac 240
cgttacctgg ctatgaaaga agacggtcgt ctgctggctt ctaaaagcgt taccgacgaa 300
tctttcttct tcgaacgtct ggaatctaac aactacaaca cctaccgttc tcgtaaatac 360
acctcttggt acgttgctct gaaacgtacc ggtcagtaca aactgggttc taaaaccggt 420
ccgggtcaga aagctatcct gttcctgccg atgtctgcta aatct 465
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Ile Lys Ala Asn Leu Ile Phe Ala Asn Gly Ser Thr Gln Thr Ala Glu
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Phe Lys Gly Thr Phe Glu Lys Ala Thr Ser Glu Ala Tyr Ala Tyr Ala
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aaaggttaca ccctgaacat taagttcgca ggtaaagaga aaaccccgga agaacaccat 300
caccatcacc atcaccatca catcgaaggt cgtatggctg ctggttctat caccaccctg 360
ccggctctgc cggaagacgg tggttctggt gctttcccgc cgggtcactt caaagacccg 420
aaacgtctgt acagcaaaaa cggtggtttc ttcctgcgta tccacccgga cggtcgtgtt 480
gacggtgttc gtgaaaaatc tgacccgcac atcaaactgc agctgcaggc tgaagaacgt 540
ggtgttgttt ctatcaaagg tgtttctgct aaccgttacc tggctatgaa agaagacggt 600
cgtctgctgg cttctaaaag cgttaccgac gaatctttct tcttcgaacg tctggaatct 660
aacaactaca acacctaccg ttctcgtaaa tacacctctt ggtacgttgc tctgaaacgt 720
accggtcagt acaaactggg ttctaaaacc ggtccgggtc agaaagctat cctgttcctg 780
ccgatgtctg ctaaatctta a 801
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Ile Glu Gly Arg
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<210> 17
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His His His His His His His His His
1 5
<210> 19
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caccatcacc atcaccatca ccatcac 27
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<212> DNA
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ttttccatgg ctagcaccta caaactgatc ctgaacggta aaactagcaa caaagaaga 59
<210> 21
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<212> DNA
<213> 人工合成
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tcttcttcag agtcggtttc aggcgtttcc ggcgtctctt ctttgttgct agttttaccg 60
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<212> DNA
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ctgaaaccga ctctgaagaa gaagttacta ttaaagctaa cctgatcttt gcgaacggtt 60
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<212> DNA
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gctttctcga aagtgccttt aaactccgca gtctgagtag aaccgttcgc aaagatcagg 60
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<212> DNA
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<210> 25
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<212> DNA
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acctttgtct gcgacatcaa ccgtgtactc accgttgtct tttttgaggg tgtccgcata 60
<210> 26
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<212> DNA
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ttgatgtcgc agacaaaggt tacaccctga acattaagtt cgcaggtaaa gagaaaaccc 60
<210> 27
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<212> DNA
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cgatgtgatg gtgatggtga tggtgatggt gttcttccgg ggttttctct ttacctgcga 60
<210> 28
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<212> DNA
<213> 人工合成
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atcaccatca ccatcacatc gaaggtcgta tggctgctgg ttctatcacc accctgccgg 60
<210> 29
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<212> DNA
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tgacccggcg ggaaagcacc agaaccaccg tcttccggca gagccggcag ggtggtgata 60
<210> 30
<211> 60
<212> DNA
<213> 人工合成
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ctttcccgcc gggtcacttc aaagacccga aacgtctgta cagcaaaaac ggtggtttct 60
<210> 31
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<212> DNA
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cgaacaccgt caacacgacc gtccgggtgg atacgcagga agaaaccacc gtttttgctg 60
<210> 32
<211> 60
<212> DNA
<213> 人工合成
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tcgtgttgac ggtgttcgtg aaaaatctga cccgcacatc aaactgcagc tgcaggctga 60
<210> 33
<211> 60
<212> DNA
<213> 人工合成
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aacggttagc agaaacacct ttgatagaaa caacaccacg ttcttcagcc tgcagctgca 60
<210> 34
<211> 60
<212> DNA
<213> 人工合成
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aaggtgtttc tgctaaccgt tacctggcta tgaaagaaga cggtcgtctg ctggcttcta 60
<210> 35
<211> 60
<212> DNA
<213> 人工合成
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ttccagacgt tcgaagaaga aagattcgtc ggtaacgctt ttagaagcca gcagacgacc 60
<210> 36
<211> 60
<212> DNA
<213> 人工合成
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ttcttcttcg aacgtctgga atctaacaac tacaacacct accgttctcg taaatacacc 60
<210> 37
<211> 60
<212> DNA
<213> 人工合成
<400> 37
ttgtactgac cggtacgttt cagagcaacg taccaagagg tgtatttacg agaacggtag 60
<210> 38
<211> 60
<212> DNA
<213> 人工合成
<400> 38
gaaacgtacc ggtcagtaca aactgggttc taaaaccggt ccgggtcaga aagctatcct 60
<210> 39
<211> 58
<212> DNA
<213> 人工合成
<400> 39
ttttctcgag ttaagattta gcagacatcg gcaggaacag gatagctttc tgacccgg 58
<210> 40
<211> 6034
<212> DNA
<213> 人工合成
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cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatggcta gcacctacaa actgatcctg 300
aacggtaaaa ctagcaacaa agaagagacg ccggaaacgc ctgaaaccga ctctgaagaa 360
gaagttacta ttaaagctaa cctgatcttt gcgaacggtt ctactcagac tgcggagttt 420
aaaggcactt tcgagaaagc gacctccgaa gcttacgcct atgcggacac cctcaaaaaa 480
gacaacggtg agtacacggt tgatgtcgca gacaaaggtt acaccctgaa cattaagttc 540
gcaggtaaag agaaaacccc ggaagaacac catcaccatc accatcacca tcacatcgaa 600
ggtcgtatgg ctgctggttc tatcaccacc ctgccggctc tgccggaaga cggtggttct 660
ggtgctttcc cgccgggtca cttcaaagac ccgaaacgtc tgtacagcaa aaacggtggt 720
ttcttcctgc gtatccaccc ggacggtcgt gttgacggtg ttcgtgaaaa atctgacccg 780
cacatcaaac tgcagctgca ggctgaagaa cgtggtgttg tttctatcaa aggtgtttct 840
gctaaccgtt acctggctat gaaagaagac ggtcgtctgc tggcttctaa aagcgttacc 900
gacgaatctt tcttcttcga acgtctggaa tctaacaact acaacaccta ccgttctcgt 960
aaatacacct cttggtacgt tgctctgaaa cgtaccggtc agtacaaact gggttctaaa 1020
accggtccgg gtcagaaagc tatcctgttc ctgccgatgt ctgctaaatc ttaactcgag 1080
caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg 1140
gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg 1200
aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta 1260
gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 1320
gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 1380
ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 1440
acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 1500
agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 1560
aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 1620
cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 1680
acaaaatatt aacgtttaca atttcaggtg gcacttttcg gggaaatgtg cgcggaaccc 1740
ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag 1800
aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 1860
tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 1920
atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 1980
aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 2040
tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca 2100
ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 2160
tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 2220
aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct 2280
tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 2340
ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 2400
ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 2460
tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 2520
tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta 2580
gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 2640
gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 2700
gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 2760
ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 2820
gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 2880
ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 2940
cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 3000
cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 3060
ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 3120
tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 3180
cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 3240
ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 3300
aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 3360
ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 3420
tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 3480
gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg 3540
cggtatttca caccgcatat atggtgcact ctcagtacaa tctgctctga tgccgcatag 3600
ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc 3660
cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 3720
aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 3780
gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg 3840
cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga 3900
taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag 3960
ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac 4020
gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg 4080
tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata 4140
cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa 4200
tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca 4260
ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc 4320
gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca 4380
acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct 4440
tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga 4500
ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc 4560
cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag 4620
tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga 4680
aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa 4740
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 4800
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt 4860
ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc 4920
agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac 4980
ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca 5040
ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg 5100
gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa 5160
ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg 5220
agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct 5280
aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg 5340
tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac 5400
gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag 5460
ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct 5520
tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga 5580
gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg 5640
ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc 5700
agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg 5760
ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac 5820
gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata 5880
ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga 5940
ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag 6000
gaatggtgca tgcaaggaga tggcgcccaa cagt 6034
<210> 41
<211> 175
<212> PRT
<213> 人工合成
<400> 41
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu
20 25 30
Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp
35 40 45
Pro Lys Arg Leu Tyr Ser Lys Asn Gly Gly Phe Phe Leu Arg Ile His
50 55 60
Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile
65 70 75 80
Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly
85 90 95
Val Ser Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu
100 105 110
Ala Ser Lys Ser Val Thr Asp Glu Ser Phe Phe Phe Glu Arg Leu Glu
115 120 125
Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr
130 135 140
Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly
145 150 155 160
Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser
165 170 175
<210> 42
<211> 528
<212> DNA
<213> 人工合成
<400> 42
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggctgctg gttctatcac caccctgccg gctctgccgg aagacggtgg ttctggtgct 120
ttcccgccgg gtcacttcaa agacccgaaa cgtctgtaca gcaaaaacgg tggtttcttc 180
ctgcgtatcc acccggacgg tcgtgttgac ggtgttcgtg aaaaatctga cccgcacatc 240
aaactgcagc tgcaggctga agaacgtggt gttgtttcta tcaaaggtgt ttctgctaac 300
cgttacctgg ctatgaaaga agacggtcgt ctgctggctt ctaaaagcgt taccgacgaa 360
tctttcttct tcgaacgtct ggaatctaac aactacaaca cctaccgttc tcgtaaatac 420
acctcttggt acgttgctct gaaacgtacc ggtcagtaca aactgggttc taaaaccggt 480
ccgggtcaga aagctatcct gttcctgccg atgtctgcta aatcttaa 528
<210> 43
<211> 28
<212> DNA
<213> 人工合成
<400> 43
ttttcatatg gctgctggtt ctatcacc 28
<210> 44
<211> 30
<212> DNA
<213> 人工合成
<400> 44
ttttctcgag ttaagattta gcagacatcg 30
<210> 45
<211> 5761
<212> DNA
<213> 人工合成
<400> 45
cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag 60
tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct 120
gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg 180
cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa 240
taattttgtt taactttaag aaggagatat accatgggca gcagccatca tcatcatcat 300
cacagcagcg gcctggtgcc gcgcggcagc catatggctg ctggttctat caccaccctg 360
ccggctctgc cggaagacgg tggttctggt gctttcccgc cgggtcactt caaagacccg 420
aaacgtctgt acagcaaaaa cggtggtttc ttcctgcgta tccacccgga cggtcgtgtt 480
gacggtgttc gtgaaaaatc tgacccgcac atcaaactgc agctgcaggc tgaagaacgt 540
ggtgttgttt ctatcaaagg tgtttctgct aaccgttacc tggctatgaa agaagacggt 600
cgtctgctgg cttctaaaag cgttaccgac gaatctttct tcttcgaacg tctggaatct 660
aacaactaca acacctaccg ttctcgtaaa tacacctctt ggtacgttgc tctgaaacgt 720
accggtcagt acaaactggg ttctaaaacc ggtccgggtc agaaagctat cctgttcctg 780
ccgatgtctg ctaaatctta actcgagcac caccaccacc accactgaga tccggctgct 840
aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa 900
ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc 960
ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 1020
cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 1080
cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 1140
tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 1200
gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 1260
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 1320
attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 1380
tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca 1440
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1500
tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat 1560
ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga 1620
gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg 1680
actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt 1740
gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct 1800
ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc 1860
aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa 1920
ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca 1980
atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc 2040
gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga 2100
ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg 2160
ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag 2220
attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca 2280
tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata 2340
acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc 2400
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 2460
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 2520
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 2580
cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 2640
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 2700
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 2760
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 2820
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 2880
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 2940
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 3000
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 3060
cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 3120
gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 3180
ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 3240
gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc 3300
agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg 3360
actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 3420
gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 3480
agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt 3540
ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct 3600
ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct 3660
gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga 3720
tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg 3780
aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc 3840
agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc 3900
atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac 3960
tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc 4020
agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc 4080
aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg 4140
ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga 4200
cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg 4260
tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc 4320
ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg 4380
cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg 4440
cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg 4500
gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc 4560
gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc 4620
ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg 4680
cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg 4740
tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc 4800
attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca 4860
ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc 4920
gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc 4980
gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc 5040
agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt 5100
gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca 5160
atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga 5220
agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc 5280
acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg 5340
tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt 5400
tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc 5460
gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca 5520
ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga 5580
ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc 5640
gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt 5700
gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag 5760
t 5761
Claims (10)
1.一种重组PLB-hbFGF融合蛋白,其特征在于,包括由蛋白L的B结构域与人碱性成纤维细胞生长因子蛋白融合得到的融合蛋白,其中,所述蛋白L的B结构域包括如SEQ ID NO:1~5所示的PLB1~PLB5蛋白中的任意一个,所述人碱性成纤维细胞生长因子蛋白的氨基酸序列如SEQ ID NO:8所示。
2.如权利要求1所述的重组PLB-hbFGF融合蛋白,其特征在于,所述蛋白L的B结构域为如SEQ ID NO:1所示的PLB1蛋白。
3.如权利要求2所述的重组PLB-hbFGF融合蛋白,其特征在于,所述PLB1蛋白的N端添加有翻译起始优化序列,而形成N端优化的PLB1蛋白,所述N端优化的PLB1蛋白的氨基酸序列如SEQ ID NO:7所示。
4.如权利要求3所述的重组PLB-hbFGF融合蛋白,其特征在于,所述N端优化的PLB1蛋白的C端添加有柔性接头、内置组氨酸标签、蛋白酶识别区和优化的hbFGF编码区,其中,
所述柔性接头包括6~30个氨基酸;
所述内置组氨酸标签包含5~10个His;
所述蛋白酶识别区包括凝血因子Xa蛋白酶识别区、凝血酶识别区、肠激酶识别区和TEV酶识别区中的任意一种。
5.如权利要求4所述的重组PLB-hbFGF融合蛋白,其特征在于,所述柔性接头包括7~20个氨基酸;
所述组氨酸标签包含9个His;
所述蛋白酶识别区为凝血因子Xa蛋白酶识别区。
6.如权利要求5所述的重组PLB-hbFGF融合蛋白,其特征在于,所述柔性接头的序列如SEQ ID NO:14,15所示;
所述组氨酸标签的序列如SEQ ID NO:16,17所示;
所述凝血因子Xa蛋白酶识别区的序列如SEQ ID NO:18,19所示。
7.如权利要求6所述的重组PLB-hbFGF融合蛋白,其特征在于,所述重组PLB1-hbFGF融合蛋白的氨基酸序列如SEQ ID NO:10所示。
8.一种如权利要求1至7任意一项所述的重组PLB-hbFGF融合蛋白的编码基因。
9.如权利要求8所述的重组PLB-hbFGF融合蛋白的编码基因,其特征在于,所述重组PLB-hbFGF融合蛋白的编码基因的核苷酸序列如SEQ ID NO:11所示。
10.一种包含如权利要求8或9所述的重组PLB-hbFGF融合蛋白的编码基因的重组PLB-hbFGF融合蛋白表达载体或表达工程菌。
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EP1237574A2 (en) * | 1999-12-17 | 2002-09-11 | Actinova Limited | Antigen delivery |
WO2003093321A1 (en) * | 2002-05-06 | 2003-11-13 | Affibody Ab | Fusion polypeptide, use thereof and methods employing it |
CN102239183A (zh) * | 2008-12-04 | 2011-11-09 | 韩国生命工学研究院 | 大量分泌的蛋白的筛选和它们作为融合配偶体在重组蛋白制备中应用 |
CN102731657A (zh) * | 2012-04-12 | 2012-10-17 | 海狸(广州)生物科技有限公司 | 一种利用slp将功能蛋白定向固定的方法 |
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EP1237574A2 (en) * | 1999-12-17 | 2002-09-11 | Actinova Limited | Antigen delivery |
WO2003093321A1 (en) * | 2002-05-06 | 2003-11-13 | Affibody Ab | Fusion polypeptide, use thereof and methods employing it |
CN102239183A (zh) * | 2008-12-04 | 2011-11-09 | 韩国生命工学研究院 | 大量分泌的蛋白的筛选和它们作为融合配偶体在重组蛋白制备中应用 |
CN102731657A (zh) * | 2012-04-12 | 2012-10-17 | 海狸(广州)生物科技有限公司 | 一种利用slp将功能蛋白定向固定的方法 |
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