CN101220092A - 人角质细胞生长因子-1结构类似物,其生产方法及应用 - Google Patents
人角质细胞生长因子-1结构类似物,其生产方法及应用 Download PDFInfo
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Abstract
本发明提供了一种人角质细胞生长因子－1结构类似物KGF－1Δ23KGF(40S)，其氨基酸序列的N末端缺失23个氨基酸，并且其40位半胱氨酸点突变成非极性氨基酸。本发明还涉及该结构类似物的生产方法，其是利用小分子泛素相关修饰因子成熟肽与之进行融合表达，且该融合蛋白与泛素相关修饰因子蛋白酶1在原核生物体内共表达。在发酵表达过程中，泛素相关修饰因子蛋白酶1可以水解融合蛋白，以生产可溶性KGF－1Δ23KGF(40S)，这种新型人角质细胞生长因子结构类似物能促进角质细胞的增殖，毛囊细胞的增生和抑制成纤维细胞生长，具有抗疤痕，抗纤维化和促进表皮愈合，及修复角膜损伤等功能。
Description
技术领域
本发明属于基因工程技术领域，更具体地，本发明涉及一种人角质细胞生长因子-1(Keratinocyte Growth Factor，KGF)结构类似物蛋白、编码该蛋白的基因、含有该基因的重组表达载体。本发明还涉及一种以可溶性形式表达和生产人角质细胞生长因子-1结构类似物蛋白的方法。
背景技术
角化细胞生长因子(KGF)是一类具有广泛生物学活性的肽类生长因子，属于FGF家庭成员(FGF-7)，KGF的作用主要表现抗肝肺组织纤维化、促进创伤性组织损伤、慢性组织损伤(糖尿病患者的持续性损伤)以及静脉溃疡(包括肠胃的慢性溃疡)的修复和愈合等。研究证明，KGF全身给药是安全的，正常人体对其产生很好的耐受。如美国Amgen公司的静脉注射液KepivanceTM(palifermin，译名帕利非明)，用于治疗口腔黏膜炎，且已通过美国FDA认证；美国人类基因组公司(HGSI)开发的RepiTermin(rhKGF-2)已进入II期临床试验，主要用于治疗静脉溃疡、因癌症治疗诱发的粘膜炎和溃疡性大肠炎。本研究中的人KGF由163个氨基酸组成(SEQ No.6)，即FGF-7，含5个二硫键，属于碱性蛋白，KGF-2由213个氨基酸组成，其生物活性与KGF-1类似，但不具有KGF-1具有的促进毛囊增生的作用。
成纤维细胞生长因子(FGF)成员是一类相对不稳定的蛋白家族，已有充分证据表明，酸性成纤维细胞生长因子(aFGF)与KGF的很不稳定(Tsai et al，1993；Volkin et al，1993；Chen and Arakawa，1996)，人角质细胞生长因子KGF的5个半胱氨酸分别位于1、15、40、102和106位，其中1位与15位以及102位与106位之间分别形成二对二硫键，而40位的半胱氨酸是以自由的形式存在的(Culajayet al，2000)，全长的KGF-1由于二硫键之间的错配而引发蛋白的不稳定与聚集等。研究发现，缺失第1位与15位的半胱氨酸，KGF的稳定性不但提高，活性也比原来高了5-10倍(Eric Hsu，Timothy Osslund，2006)。
利用哺乳动物细胞或者酵母生产KGF方法已有许多报道，然而利用哺乳动物细胞生产获得的可溶性KGF，产量极低且提纯步骤繁复。同时，也有大量的文献报导利用蛋白质工程技术生产人KGF的方法，且使用大肠杆菌作为以重组技术工业生产蛋白质的优选宿主细胞。尽管大肠杆菌表达系统具有表达量高、易于培养和操作以及生产成本低等优点，但是，使用该表达系统难以获得大量可溶性的KGF，其原因在于含有5个半胱氨酸，极容易形成“包涵体”。此外，即使获得大量的包涵体，为了得到有生物学活性的蛋白，还必须对包涵体进行变性-复性处理，这个过程往往损失大量的蛋白。为了解决这一难题，研究人员采用融合技术，将KGF基因与具有协助蛋白质正确折叠的融合蛋白如谷胱甘肽硫转移酶(GST)、Intein等进行融合表达。尽管蛋白的可溶性及收率有所提高，但是，耗时、昂贵的蛋白酶费用以及存在的蛋白酶水解靶蛋白的风险，常常不能获得令人满意的KGF产量。
小分子泛素样修饰蛋白(Small Ubiquitin-related Modifier，SUMO)和泛素(ubiquitin，Ub)在一级结构上只有18％的同源性，然而两者的三级结构及其生物学功能却十分相似。SUMO广泛存在于各种真核细胞中，参与调节细胞凋亡、信号转导、RNA转录、蛋白的核质运输以及细胞周期等多种生理进程(参见Johnson，E.S.2004，Annu.Rev.Biochem.，73，355-382.)。在脊椎动物中，有SUMO-1、-2、-3三个成员，而酵母和无脊椎动物仅有一个相关基因编码的SUMO。简言之，SUMO前体在泛素相似蛋白酶Ulps(ubiquitin-like proteases)的作用下失去C端的部分氨基酸残基，暴露出Gly-Gly序列，成为SUMO成熟肽；在ATP酶作用下，该成熟肽的C末端Gly与活化酶E1(SAE1/SAE2)的一个Cys残基通过硫酯键相连接；然后，SUMO经E1传递到结合酶E2(Ubc9)，并借助硫酯键与Ubc9活性位点上的Cys93残基相连接；在连接酶E3的作用下，SUMO的Gly通过异肽键与靶蛋白Lys残基的ε-氨基相结合并参与相关的生理活动；最后，通过Ulps的水解作用下，SUMO与靶蛋白分离。整个过程是一个动态迅速的反应。与泛素修饰不同，SUMO不会导致靶蛋白降解，而是通过改变靶蛋白的稳定性和亚细胞定位等调节靶蛋白的功能和生物活性。
除了上述的生物学活性外，近年来，SUMO被发现可以作为重组蛋白表达的融合标签和分子伴侣，具有抗蛋白酶水解、显著增加重组蛋白表达量以及促进靶蛋白正确折叠，提高可溶性等功能。此外，Ulp1可以根据SUMO的三级结构迅速水解融合蛋白，而不象凝血酶、Xa因子及肠激酶等只能根据蛋白一级序列切割，因此能准确无误地切除SUMO，从而获得具天然N末端的靶蛋白且不会水解目的蛋白(Butt et al.，2005，Protein Expression and Purification，43，1-9；US 60/482817；PCT/US03/00436)。在本发明中，首先利用SUMO作为分子伴侣显著增加人KGF的表达量以及溶解度，促进其正确折叠，然后在宿主内同时表达的Ulp1根据SUMO的三级结构准确水解融合蛋白，进而获得大量可溶性人KGF，这个方法有效解决上述耗时、昂贵的蛋白酶费用、存在的蛋白酶水解靶蛋白的风险以及不能获得令人满意的KGF产量的难题。
发明内容
本发明的第一个目的在于提供KGF-1结构类似物蛋白，其稳定性和活性显著优于天然蛋白。
本发明第二个目的在于提供一种制备KGF-1结构类似物蛋白的方法，其能显著提高KGF-1结构类似物蛋白的产量。
本发明的KGF-1结构类似物蛋白是通过对人KGF的40位半胱氨酸突变为丝氨酸，从而避免重组KGF形成分子间二硫键。同时通过缺失N端23个氨基酸来去掉1位与15位的半胱氨酸，形成了一种只保留一对分子内二硫键的突变体，现将其命名为KGF-1Δ23KGF(40S)，其氨基酸序列如序列表SEQ ID No.2所示。
针对KGF表达形式与表达量的研究瓶颈，本发明提供了生产一种人角质细胞生长因子-1(Keratinocyte Growth Factor，KGF)结构类似物蛋白的方法，本发明利用小分子泛素相关修饰因子成熟肽(Small Ubiquitin-related Modifier，SUMO)和KGF-1Δ23KGF(40S)进行融合表达，并将该融合蛋白与泛素相关修饰因子蛋白酶1(Ubiquitin-like protease 1)在原核生物体内共表达，在发酵表达过程中，泛素相关修饰因子蛋白酶1可以水解由分子泛素相关修饰因子成熟肽和KGF-1Δ23KGF(40S)的基因构成的融合蛋白，以生产可溶性KGF-1Δ23KGF(40S)蛋白。
可以使用任何一种适于在其中以高效率共表达所需蛋白质(由小分子泛素相关修饰因子成熟肽和人角质细胞生长因子-1结构类似物蛋白组成的融合蛋白以及泛素相关修饰因子蛋白酶1)的大肠杆菌株作为宿主，这样的菌株包括大肠杆菌JM109，DH5，BL21，Origami(DE3)，Rosetta等。本发明优选的宿主是大肠杆菌菌株BL21。
可按照本领域已知的技术进行基因的合成、克隆和表达载体的构建、DNA序列分析及鉴定、宿主细胞的转化和培养以及表达产物的分离纯化等操作(参见Sambrook et al.，Molecular Cloning：A Laboratory Manual，Cold Spring HarborLaboratory Press，Cold Spring Harbor，NY，1989)。
下面以本发明一个优选的表达载体pET-3c为例来说明重组表达载体的构建。可以首先利用PCR技术人工合成编码小分子泛素相关修饰因子成熟肽和人角质细胞生长因子-1结构类似物蛋白融合蛋白的核苷酸序列(SEQ ID No.3)，将上述基因序列插入到表达载体中的T7启动子下游，得到重组质粒pET-SUMO-KGF-1Δ23KGF(40S)，其中的重组质粒上的T7启动子与编码融合蛋白的基因构成第一转录单位。然后，再利用PCR技术人工合成编码泛素相关修饰因子蛋白酶1的核苷酸序列并直接连接到本发明选用的pET-3c表达载体中的T7启动子下游，得到重组质粒pET-Ulp1。使用基于由重组质粒pET-Ulp1上的T7启动子与泛素相关修饰因子蛋白酶1基因组成的第二转录单位的核苷酸序列合成寡核苷酸引物，并以上述方法制得的重组质粒pET-Ulp1为模板，经PCR扩增得到两端都带有BamH I位点的合成的双股T7启动子与泛素相关修饰因子蛋白酶1基因序列。将所得到的编码T7启动子与泛素相关修饰因子蛋白酶1基因序列进行BamH I单酶切并连接到用BamH I预先处理按上述方法制得的重组质粒pET-SUMO-KGF-1Δ23KGF(40S)上，进而得到以串联方式连接的，含有处于T7启动子驱动下的编码小分子泛素相关修饰因子成熟肽和人角质细胞生长因子-1结构类似物融合蛋白cDNA序列以及处于T7启动子驱动下的泛素相关修饰因子蛋白酶1基因的重组载体(pET-SUMO-KGF-1Δ23KGF(40S)-Ulp1)。
可以按常规方法(如电转化或者化学方法)将携带上述第一和第二转录单位的重组质粒转化到适当的宿主中，例如适当的大肠杆菌菌株中，并按本领域中技术人员熟知的方法，例如使用LB培养基培养用适当抗生素筛选的转化株(参见Sambrook et al.，Molecular Cloning：A Laboratory Manual，Cold Spring HarborLaboratory Press，Cold Spring Harbor，NY，1989)。
发酵过程中，首先将筛选出的单克隆重组转化子接种到含氨苄青霉素的新鲜LB培养基中(含1％葡萄糖和100μg/ml氨苄青霉素)中，30℃、250rpm振荡过夜培养。然后，取100μl细菌培养液接种于50ml新鲜LB培养基中(含100μg/ml氨苄青霉素，但不含1％葡萄糖)，至OD600到0.6-1.0时，加入终浓度为0.4mmol/l的IPTG并置于30℃，250rpm培养6小时。
发酵完成后，可以按本领域中技术人员熟知的方法收集菌体以及分离培养物上清，并用已知的三步层析方法(离子交换层析-亲合层析-分子筛)纯化可溶性KGF-1Δ23KGF(40S)，将纯化后蛋白再进行反向高效液相层析(HPLC)分离，将KGF-1Δ23KGF(40S)纯化到纯度高于98％、银染色的SDS-聚丙烯酰胺凝胶电泳呈单一条带状态。可用飞行质谱(MOLDI-TOF)、氨基酸组成分析方法以及氨基酸末端测序方法鉴定人KGF-1Δ23KGF(40S)的结构真实性，并用MTT法测定角质细胞的增殖促进作用。测定的结果与标准品相比，活性大大提高。
本发明的人角质细胞生长因子-1结构类似物具有角膜损伤修复，促进毛囊增生，抗疤痕，抗纤维化等作用。其治疗作用依赖于KGF-1结构类似物加入添加剂制成的药物配方。这些添加剂包括有赋形剂，缓冲剂，稳定剂，填充剂，载体等，然后用已知的制剂方法制成可用于外用和内用(注射和口服)等各种形式。其常用的有注射液，滴眼剂，外用膏剂，乳剂，水剂，口服液，胶囊，缓释或控释等。
本发明中的KGF突变体可进一步进行化学修饰，以改变其可能的不期望的副作用，延长体内的生物半衰期等。最常见的修饰是聚乙二醇通过KGF蛋白质上的游离的半胱氨酸反应基因连接到KGF突变体。
本发明通过将SUMO与hKGF融合表达，再经分离纯化得到hKGF，该方法有助于目的蛋白hKGF真核基因在原核中的正确翻译，并以可溶性形式存在，从而不仅提高了hKGF的表达量，增加了KGF的活性，而且简化了分离纯化工艺，提高了生产效率，降低了成本，从而适合大规模工业化生产。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。应当理解，这些实施例仅用于说明本发明，而不能限制本发明的保护范围。
实施例1
一、基因的设计与合成
1 Δ23KGF(40S)的基因合成：
根据NCBI网上的人KGF1的核苷酸序列(SEQ ID No.5)，其氨基酸序列由163个氨基酸组成如(SEQ ID No.6)所示使用DNAStar软件设计合成13条引物，应用递归PCR方法人工合成KGF全基因序列，并在KGF的两端再设计一对引物，合成Δ23KGF，再在40位设计一对引物，同时把半胱氨酸突变为丝氨酸，得到Δ23KGF(40S)。并将其内部含有的NdeI和BamH I的位点去掉。在人工合成时选择了大肠杆菌偏爱密码子，以便提高重组表达水平。
| F1(SEQ ID No.9)：GACATACCCGTAGCTATGATTATATGGAAGGTGGTGATATTCGTGTGCGTCGTCTGTTT(79-137) |
| F2(SEQ ID No.10)：AACAGATGGCGACGAATGTGAATTGCAGCAGCCCAGAAAGACATACCCGTAGCTATGAT(40-98) |
| F3(SEQ ID No.11)：ACAGAGAACAGATTGGTGGTTGTAATGATATGACCCCGGAACAGATGGCGACGAATGTG(1-59) |
| R1(SEQ ID No.12)：ACCACGTTTATCAATACGCAGATACCACTGGGTACGGCAAAACAGACGACGCACACGAA |
| R2(SEQ ID No.13)：ATTATAATTATTTTTCATTTCCTGGGTGCCTTTCACTTTACCACGTTTATCAATACGCA |
| R3(SEQ ID No.14)：CGCCACGATGCCCACTGCCACGGTACGAATTTCCATGATATTATAATTATTTTTCATTT |
| F1*(SEQ ID No.15)：CGAAGAAGGAGTGTAACGAGGATTGCAATTTCAAGGAACTGATTCTGGAGAACCATTAT |
| F2*(SEQ ID No.16)：AATTTTACCTGGCAATGAATAAGAAGGCAAACTGTATGCGAAGAAGGAGTGTAACGAG |
| F3*(SEQIDNo.17)：TGGCAGTGGGCATCGTGGCGATTAAAGGCGTGGAAAGCGAATTTTACCTGGCAATGAAT |
| R1*(SEQ ID No.18)：GCCTCCGTTATGGGTCCACTTCGCGCTCGCATAGGTATATAATGGTTCTCCAGAATCA |
| R2*(SEQ ID No.19)：AACTGGGATACCTTTCTGATTCAGTGCCACAAACATTTCGCCTCCGTTATGGGTCCACT |
| R3*(SEQ ID No.20)：GTGCGCGGTTTTCTGTTCTTTTTTGGTTTTTTTGCCACGAACTGGGATACCTTTCTGAT |
| R4*(SEQ ID No.21)：CGGGGATCCTTATCACGTAATGGCCATTGGCAGGAAGTGCGCGGTTTTCTGTTCTT |
| F：(SEQ ID No.22)：ACAGAGAACAGATTGGTGGTAGCTATGATTATATGGAAGG |
| R：(SEQ ID No.23)：CGGGGATCCTTATCACGTAATGGCC |
两个反应体系同时进行：
Pyrobest 0.5μl
10×Pyrobest Buffer 10μl
2.5mM dNTP Mixture 8μl
Primer F1 2μl
Primer R1 2μl
ddH2O 77.5μl
Total 100μl
Pyrobest 0.5μl
10×Pyrobest Buffer 10μl
2.5mM dNTP Mixture 8μl
Primer F1* 2μl
Primer R1* 2μl
ddH2O 77.5μl
Total 100μl
反应条件：
经94℃变性3min进入循环，循环温度及时间为94℃，30s；56℃，30s；72℃，30s，共30个循环，然后72℃延伸3min。
回收产物(用TaKaRa的Agarose Gel DNA Purification Kit，参照说明书回收)，命名为KI与KI*按照上述方法，以前次扩增产物为模板，依次采用引物对F2/R2、F3/R3与F2*/R2*、F3*/R3*F1*/R4*进行扩增，得到产物KIII与KIV*。反应体系：
Pyrobest 0.5μl
10×Pyrobest Buffer 10μl
2.5mM dNTP Mixture 8μl
Primer F1 2μl
Primer R4* 2μl
KIII 1μl
KIV* 1μl
ddH2O 77.5μl
Total 100μl
经94℃变性3min进入循环，循环温度及时间为94℃，50s；56℃，50s；72℃，50s，共30个循环，然后72℃延伸7min。(条件1)回收产物用TaKaRa的AgaroseGel DNA Purification Kit，(参照说明书回收)，得到全长的野生产物，命名为KGF-1。再在其两端设计F：(SEQ ID No.22)与R：(SEQ ID No.23)上下游引物，PCR反应条件同条件1，得到Δ23KGF，再在40位设计一对引物，同时把半胱氨酸突变为丝氨酸，PCR反应条件同上，得到Δ23KGF(40S)，此片段前端含有一段SUMO的后段序列。
2、SUMO的合成：
以pET28a-SUMO-EGF为模版，
P1：GGAATTCCATATGCATCATCATCATCATCACG
P2：GGCTCACAGAGAACAGATT GGT GGT作为引物合成SUMO基因序列，反应体系如下：
上游引物P1(10μmol/L) 1.5μL
下游引物P2(10μmol/L) 1.5μL
10×TaKaRa Pyrobest Buffer 5μL
2.5mM dNTP Mixture 4μL
Pyrobest DNA Polymerase(5u/μL) 0.5μL
pET28a-SUMO-EGF 1μL
ddH2O 36.5μL
Total 50μL
PCR反应条件：经94℃热冲击预变性3min进入循环，循环条件：94℃，45s；60℃，45s；72℃，20s，共28个循环，然后72℃延伸5min；最后冷却至4℃。用1％琼脂糖凝胶电泳检测，利用TaKaRa的Agarose Gel DNA Purification Kit进行回收目的SUMO片段。
3、SUMO-KGFΔ23(40S)片段合成
以KGFΔ23(40S)和SUMO为模版，P1、R4作为引物合成SUMO-KGF-1Δ23(40S)基因序列，反应体系如下：
上游引物P1(10μmol/L) 1.5μL
下游引物R4(10μmol/L) 1.5μL
10×TaKaRa Pyrobest Buffer 5μL
2.5M dNTP Mixture 4μL
Pyrobest DNA Polymerase(5u/μL)0.5μL
KGF-1Δ23(40S) 1μL
SUMO 1μL
ddH2O 35.5μL
Total 50μL
PCR反应条件：经94℃热冲击预变性3min进入循环，循环条件：94℃，45s；59℃，45s；72℃，51s，共28个循环，然后72℃延伸10min，最后冷却至4℃。用1％琼脂糖凝胶电泳检测，利用TaKaRa的Agarose Gel DNA Purification kit进行回收目的DNA片段，合成SUMO-KGF-1Δ23(40-S)由753个碱基组成，其中5‘端含有Nde I酶切位点，3’端含有终止子和BamH I酶切位点。此SUMO-KGF-1Δ23(40-S)片段插入测序质粒PET-3C后，由上海申工完成，测序正确。实施例2 pET-ULP1的重组质粒的构建
SEQ ID NO：26：Ulp-F1(5’-3’，共59个碱基)
SEQ ID NO：27：Ulp-F2(5’-3’，共59个碱基)
SEQ ID NO：28：Ulp-F3(5’-3’，共59个碱基)
SEQ ID NO：29：Ulp-F4(5’-3’，共59个碱基)
SEQ ID NO：30：Ulp-F5(5’-3’，共46个碱基)
SEQ ID NO：31：Ulp-F6(5’-3’，共33个碱基)
SEQ ID NO：32：Ulp-F7(5’-3’，共59个碱基)
SEQ ID NO：33：Ulp-F8(5’-3’，共59个碱基)
SEQ ID NO：34：Ulp-F9(5’-3’，共59个碱基)
SEQ ID NO：35：Ulp-R1(5’-3’，共59个碱基)
SEQ ID NO：36：Ulp-R2(5’-3’，共59个碱基)
SEQ ID NO：37：Ulp-R3(5’-3’，共59个碱基)
SEQ ID NO：38：Ulp-R4(5’-3’，共59个碱基)
SEQ ID NO：39：Ulp-R5(5’-3’，共59个碱基)
SEQ ID NO：40：Ulp-R6(5’-3’，共59个碱基)
SEQ ID NO：41：Ulp-R7(5’-3’，共59个碱基)
SEQ ID NO：42：Ulp-R8(5’-3’，共59个碱基)
SEQ ID NO：43：Ulp-R9(5’-3’，共59个碱基)
SEQ ID NO：44：UI(5’-3’，共215个碱基)
SEQ ID NO：45：UII(5’-3’，共254个碱基)
SEQ ID NO：46：UIII(5’-3’，共255个碱基)
SEQ ID NO：47：UIV(5’-3’，共469个碱基)
SEQ ID NO：48：Ulp1-T7-F(5’-3’，共53个碱基)
首先合成18条寡聚核苷酸链(由上海生工生物工程技术服务有限公司合成)，分别为：Ulp-F1(SEQ ID No：26)，Ulp-F2(SEQ ID No：27)，Ulp-F3(SEQ ID No：28)，Ulp-F4(SEQ ID No：29)，Ulp-F5(SEQ ID No：30)，Ulp-F6(SEQ ID No：31)，Ulp-F7(SEQ ID No：32)，Ulp-F8(SEQ ID No：33)，Ulp-F9(SEQ ID No：34)，Ulp-R1(SEQ ID No：35)，U1p-R2(SEQ ID No：36)，Ulp-R3(SEQ ID No：37)，Ulp-R4(SEQ ID No：38)，Ulp-R5(SEQ ID No：39)，Ulp-R6(SEQ ID No：40)，Ulp-R7(SEQ ID No：41)，Ulp-R8(SEQ ID No：42)和Ulp-R9(SEQ ID No：43)。利用PCR技术人工合成三个大片段，分别命名为UI(SEQ ID No：44)，UII(SEQ ID No：45)和UIII(SEQ ID No：46)。然后，以UI和UII为共同模板，加入Ulp-F6和Ulp-R3为上下游引物，按照实施例1中3的PCR反应中所述的参数及方法扩增出长度约为469的大片段UIV(SEQ ID No：47)。最后，以UIV和UIII为共同模板，以Ulp-F6和Ulp-R9为上下游引物，按照实施例1中的第二步PCR反应中所述的参数及方法扩增出长度约为702bp，两端分别含有Nde I(CATATG)和BamH I(GGATCC)酶切位点的Ulp1的PCR产物。将该PCR产物用Nde I和BamH I双酶切，然后连接到用这两种酶相同处理过的表达载体pET-3c中，构成重组表达质粒pET-Ulp1。将该质粒转化至感受态细胞DH5中。培养增殖后从转化株中抽提重组质粒，按已知的方法并使用通用引物(T7 promoterprimer和T7 terminator primer)测定正向和反向连接片段的DNA序列，结果显示所得片段为编码219个氨基酸的Ulp1蛋白的核苷酸序列。
实施例3 SUMO-KGF-1Δ23(40-S)与Ulp1的共表达重组质粒的构建
本发明用于可溶性表达人KGF-1结构类似物。蛋白的重组表达载体包括一个表达由小分子泛素相关修饰因子成熟肽和人KGF-1结构类似物组成的融合蛋白的第一转录单位和用于表达泛素相关修饰因子蛋白酶的第二转录单位，其中所述第一转录单位和第二转录单位彼此串联连接于同一重组载体内。
就两转录单位彼此间的串联连接方式而言，本发明的重组表达载体首先包括一个可操作地连接到第一启动子上的编码小分子泛素相关修饰因子成熟肽和人KGF-1结构类似物组成的融合蛋白的核苷酸序列，和一个可操作地第二转录单位的插入位点。借助这个插入位点，可以容易地在所述表达载体中插入由编码泛素相关修饰因子蛋白酶的核苷酸序列和其上游的第二启动子组成的第二转录单位。
为了构建包括以串联方式连接有处于T7启动子控制下的含有处于T7启动子控制下的编码小分子泛素相关修饰因子成熟肽和人KGF-1结构类似物融合蛋白cDNA序列以及处于T7启动子控制下的泛素相关修饰因子蛋白酶1基因的重组表达载体，首先，基于T7启动子合成一条寡聚核苷酸引物ULP1-T7-F：5’CT GGATCC GAA TTC GAG CTC AAG CTT CTC GAG TAA TAC GAC TCA CTA TAGGGA G3’(由上海生工生物工程技术服务有限公司合成)。该引物的5’端含有一个多克隆位点。按照常规的PCR方法，以重组表达载体pET-ULP1为模板，ULP1-T7-F和ULP-R9为上下游引物，94℃变性4min后进入循环，循环参数为94℃变性30秒，55℃退火30秒，72℃延伸30秒，共30个循环，扩增得到5’端含有一个多克隆位点的T7启动子与泛素相关修饰因子蛋白酶1基因组成的DNA片段，且两端均带有BamH I酶切位点。T7启动子与泛素相关修饰因子蛋白酶1基因组成的DNA片段也就是第二转录单位的DNA片段(SEQ ID NO：24)。
将该片段用BamH I进行单酶切，然后连接到用BamH I相同处理过的载体pET-SKGFΔ23(40-S)中，构建成重组表达载体pET-SKGF-1Δ23(40-S)-Ulp1。将该质粒转化至感受态细胞DH5中。培养增殖后从转化株中抽提重组质粒，按已知的方法并使用通用引物(T7 promoter primer和T7 terminator primer)正向和反向测序确定片段插入的方向以及编码的氨基酸都是正确。可以利用两个转录单位间的多克隆位点插入长度不一的核苷酸序列以调整两单位间的距离，以获得最佳的转录效率。将该重组质粒利用电转或者化学方法如CaCl2转化到大肠杆菌Origami(DE3)中，利用氨苄青霉素抗性LB固体平板(10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠，20g/L琼脂粉，100μg/ml氨苄青霉素)筛选出重组子。
实施例4发酵培养
将种子菌液先在LB培养基中(含1％葡萄糖和100μg/ml氨苄青霉素)摇瓶培养8～10h，按5％的接种量接种到1L三角瓶(装液量为200ml)中，30℃、220rpmin摇瓶培养到OD600为0.8时添加0.4mM诱导剂IPTG，继续培养6h，发酵结束。
实施例5 KGF-1Δ23(40-S)蛋白的分离纯化
发酵结束后，12000rpm离心收集菌体，加入0.02mol/L的磷酸盐缓冲液(pH7.0)重悬菌体。利用超声波破碎仪破碎细胞。18000rpm离心30分钟，收集上清。将该上清首先通过已用0.05mol/L的PB(pH7.0)使用阳离子交换层析(CM-Sephadex)将该峰中的蛋白样品进行分离。用含0.1mol/l的NaCl的PB(pH7.0)溶液洗柱后(共3-5柱床体积)，加入含0.6mol/l NaCl的PB(pH7.0)洗脱目的蛋白。收集洗脱峰，并利用12％的聚丙烯酰胺凝胶电泳(SDS-PAGE)检测目的蛋白存在的洗脱峰。将含有目的蛋白的洗脱峰再次通过用0.6mol/l NaCl的PB(pH7.0)平衡的肝素凝胶进行精制，用1.2mol/LNaCl的PB(pH7.0)洗脱目的蛋白，再过葡聚糖凝胶进行脱盐，收集的洗脱峰并利用聚丙烯酰胺凝胶电泳(SDS-PAGE)检测目的蛋白存在位置。将含有目的蛋白的溶液利用HPLC进行最后分离，用0.05％三氟乙酸+乙腈(30-60％)+水(60-30％)的梯度进行洗脱，时间为60分钟，收集的KGF-1Δ23(40-S)蛋白纯度高于98％。纯化后的KGF-1Δ23(40-S)蛋白经12％的聚丙烯酰胺凝胶电泳分离后，硝酸银染色显示分子量约16Kda的单一条带。通过飞行质谱的测定、氨基酸N端测序以及氨基酸组成分析证实按本发明制备的KGF-1Δ23(40-S)蛋白的真实性。按照本发明的方法，在摇瓶发酵水平上，每升发酵液可以得到40 mg的重组人KGF-1Δ23(40-S)蛋白。
实施例6 MTT法测定KGF-1Δ23(40-S)蛋白的活性
收集对数生长期的BalB/c3T3细胞用0.25％胰酶消化液消化后，用10％完全1640培养液吹打成细胞悬液调整细胞浓度为8.0×104/ml左右。将细胞悬液接种于96孔板中，每孔100μl，37℃5％CO2孵箱培养24小时。吸去96孔板中培养液，加入0.4％完全培养液，每孔100μl，37℃5％CO2孵箱培养24小时。将饥饿培养24小时后的细胞板吸去板内培养液待用。用0.4％完全1640培养液将标准品稀释至100IU/ml，三复孔加入1-3列A排孔中，用0.4％完全1640培养液将样品按蛋白含量稀释至50ng/ml起始，将稀释蛋白的样品加入4-12列A排孔中，每三复孔为一个样品(100μl/孔样品)。将与A排孔相同的稀释样品继续4倍稀释从B-C排，从C-D排逐依类推至G排(样品100ul/孔)；H排各孔只填加0.4％完全1640培养液作为空白孔。此板为稀释板。
将稀释板放入37℃5％CO2孵箱培养72小时，将培养72小时的稀释板吸去培养液，加入MTT(20μl/孔)，37℃5％CO2孵箱培养5小时，然后加入终止液DMSO(150μl/孔)，室温放置30分钟后，在酶标仪下比色(波长570nm)。
5计算方法：
结果显示按发明制得的KGF-1Δ23(40-S)蛋白能促进角质细胞的增殖。得到的活性值为标准品的7倍。
对序列表的说明：SEQ ID No.1&2是本发明的KGFΔ23(40-S)核苷酸及氨基酸序列；SEQ IDNo.3&4是SUMO-KGFΔ23(40-S)的核苷酸序列及氨基酸序列；SEQ ID No.5&6是hKGF-1的核苷酸序列及氨基酸序列(未改造)；SEQ ID No.7&8是SUMO的核苷酸序列及氨基酸序列；SEQID No.9～23是合成SUMO-KGFΔ23(40S)的相关引物核苷酸序列；SEQ ID No.24&25是Ulp1的核苷酸序列及氨基酸序列；SEQ ID No.26～43是合成Ulp1的相关引物核苷酸序列；SEQID No.44～47是合成过程中的中间产物核苷酸序列序列；SEQ ID No.48是用来合成T7启动子与泛素相关修饰因子蛋白酶1基因组成的DNA片段的引物。
序列表
<110>吉林农大生物反应器工程有限公司
<120>人角质细胞生长因子-1结构类似物，其生产方法及应用
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atg gtg gta cac tca 831
Met Val Val His Ser
275
<210>4
<211>277
<212>PRT
<213>人工序列
<400>4
Met His His His His His His Gly Met Ser Asp Ser Glu Val Asn Gln
1 5 10 15
Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile
20 25 30
Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys
35 40 45
Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln
50 55 60
Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile
65 70 75 80
Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile
85 90 95
Ile Glu Ala His Arg Glu GlnIle Gly Gly Gln Ala Leu Gly Gln Asp
100 105 110
Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser
115 120 125
Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn His Leu Gln Gly
130 135 140
Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys
145 150 155 160
Ile Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro
165 170 175
Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val Val Ala Val
180 185 190
Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys
195 200 205
Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg
210 215 220
Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His
225 230 235 240
Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg
245 250 255
Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro
260 265 270
Met Val Val His Ser
275
<210>5
<211>489
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(489)
<400>5
tgc aat gat atg acc ccg gaa cag atg gcg acc aat gtg aat tgc agc 48
Cys Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser
1 5 10 15
agc ccg gaa cgt cat acc cgt agc tat gat tat atg gaa ggc ggc gat 96
Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp
20 25 30
att cgt gtg cgt cgt ctg ttt tgc cgt acc cag tgg tat ctg cgt att 144
Ile Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile
35 40 45
gat aaa cgt ggc aaa gtg aaa ggc acc cag gaa atg aaa aat aat tat 192
Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr
50 55 60
aat att atg gaa att cgt acc gtg gcg gtg ggc att gtg gcg att aaa 240
Asn Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys
65 70 75 80
ggc gtg gaa agc gaa ttt tat ctg gcg atg aat aaa gaa ggc aaa ctg 288
Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu
85 90 95
tat gcg aaa aaa gaa tgc aat gaa gat tgc aat ttt aaa gaa ctg att 336
Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile
100 105 110
ctg gaa aat cat tat aat acc tat gcg agc gcg aaa tgg acc cat aat 384
Leu Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn
115 120 125
ggc ggc gaa atg ttt gtg gcg ctg aat cag aaa ggc att ccg gtg cgt 432
Gly Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg
130 135 140
ggc aaa aaa acc aaa aaa gaa cag aaa acc gcg cat ttt ctg ccg atg 480
Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met
145 150 155 160
gcg att acc 489
Ala Ile Thr
<210>6
<211>163
<212>PRT
<213>人工序列
<400>6
Cys Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser
1 5 10 15
Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp
20 25 30
Ile Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile
35 40 45
Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr
50 55 60
Asn Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys
65 70 75 80
Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu
85 90 95
Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile
100 105 110
Leu Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn
115 120 125
Gly Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg
130 135 140
Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met
145 150 155 160
Ala Ile Thr
<210>7
<211>297
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(297)
<400>7
ggc atg tcg gac tca gaa gtc aat caa gaa gct aag cca gag gtc aag 48
Gly Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys
1 5 10 15
cca gaa gtc aag cct gag act cac atc aat tta aag gtg tcc gat gga 96
Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly
20 25 30
tct tca gag atc ttc ttc aag atc aaa aag acc act cct tta aga agg 144
Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg
35 40 45
ctg atg gaa gcg ttc gct aaa aga cag ggt aag gaa atg gac tcc tta 192
Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu
50 55 60
aga ttc ttg tac gac ggt att aga att caa gct gat cag acc cct gaa 240
Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu
65 70 75 80
gat ttg gac atg gag gat aac gat atc att gag gct cac aga gaa cag 288
Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln
85 90 95
att ggt ggt 297
Ile Gly Gly
<210>8
<211>99
<212>PRT
<213>人工序列
<400>8
Gly Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys
1 5 10 15
Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly
20 25 30
Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg
35 40 45
Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu
50 55 60
Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu
65 70 75 80
Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln
85 90 95
Ile Gly Gly
<210>9
<211>59
<212>DNA
<213>人工序列
<400>9
gacatacccg tagctatgat tatatggaag gtggtgatat tcgtgtgcgt cgtctgttt 59
<210>10
<211>59
<212>DNA
<213>人工序列
<400>10
aacagatggc gacgaatgtg aattgcagca gcccagaaag acatacccgt agctatgat 59
<210>11
<211>59
<212>DNA
<213>人工序列
<400>11
acagagaaca gattggtggt tgtaatgata tgaccccgga acagatggcg acgaatgtg 59
<210>12
<211>59
<212>DNA
<213>人工序列
<400>12
accacgttta tcaatacgca gataccactg ggtacggcaa aacagacgac gcacacgaa 59
<210>13
<211>59
<212>DNA
<213>人工序列
<400>13
attataatta tttttcattt cctgggtgcc tttcacttta ccacgtttat caatacgca 59
<210>14
<211>59
<212>DNA
<213>人工序列
<400>14
cgccacgatg cccactgcca cggtacgaat ttccatgata ttataattat ttttcattt 59
<210>15
<211>59
<212>DNA
<213>人工序列
<400>15
cgaagaagga gtgtaacgag gattgcaatt tcaaggaact gattctggag aaccattat 59
<210>16
<211>59
<212>DNA
<213>人工序列
<400>16
aattttacct ggcaatgaat aaagaaggca aactgtatgc gaagaaggag tgtaacgag 59
<210>17
<211>59
<212>DNA
<213>人工序列
<400>17
tggcagtggg catcgtggcg attaaaggcg tggaaagcga attttacctg gcaatgaat 59
<210>18
<211>59
<212>DNA
<213>人工序列
<400>18
gcctccgtta tgggtccact tcgcgctcgc ataggtatta taatggttct ccagaatca 59
<210>19
<211>59
<212>DNA
<213>人工序列
<400>19
aactgggata cctttctgat tcagtgccac aaacatttcg cctccgttat gggtccact 59
<210>20
<211>59
<212>DNA
<213>人工序列
<400>20
gtgcgcggtt ttctgttctt ttttggtttt tttgccacga actgggatac ctttctgat 59
<210>21
<211>56
<212>DNA
<213>人工序列
<400>21
cggggatcct tatcacgtaa tggccattgg caggaagtgc gcggttttct gttctt 56
<210>22
<211>40
<212>DNA
<213>人工序列
<400>22
acagagaaca gattggtggt agctatgatt atatggaagg 40
<210>23
<211>25
<212>DNA
<213>人工序列
<400>23
cggggatcct tatcacgtaa tggcc 25
<210>24
<211>666
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)..(666)
<400>24
atg ggc ctg gtt ccg gaa ctg aac gaa aaa gac gac gat caa gtg cag 48
Met Gly Leu Val Pro Glu Leu Asn Glu Lys Asp Asp Asp Gln Val Gln
1 5 10 15
aaa gct ctg gca agc cgc gaa aac acc cag ctg atg aat cgt gat aac 96
Lys Ala Leu Ala Ser Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn
20 25 30
att gag att act gtc cgt gac ttc aaa acc ctc gct ccc cgc cgt tgg 144
Ile Glu Ile Thr Val Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp
35 40 45
tta aac gat acc atc att gag ttc ttc atg aaa tac att gag aaa tct 192
Leu Asn Asp Thr Ile Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser
50 55 60
acg ccc aat acg gtc gca ttc aat agc ttc ttc tac acc aac ctg tct 240
Thr Pro Asn Thr Val Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser
65 70 75 80
gaa cgt ggt tat cag ggt gtc cgc cgc tgg atg aaa cgc aag aaa act 288
Glu Arg Gly Tyr Gln Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr
85 90 95
cag att gac aag tta gac aaa atc ttt act ccg att aac tta aat cag 336
Gln Ile Asp Lys Leu Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln
100 105 110
agc cac tgg gca ctg ggc atc atc gat ctg aaa aag aaa acc atc ggt 384
Ser His Trp Ala Leu Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly
115 120 125
tat gtt gac tct ctg tcc aac ggt cca aac gca atg tcc ttc gca atc 432
Tyr Val Asp Ser Leu Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile
130 135 140
ctg act gat ctg caa aaa tac gtc atg gaa gaa tcc aaa cac acc atc 480
Leu Thr Asp Leu Gln Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile
145 150 155 160
ggt gag gac ttc gac ttg att cac tta gac tgc cca cag caa cca aac 528
Gly Glu Asp Phe Asp Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn
165 170 175
ggc tat gac tgc ggt atc tat gtc tgt atg aac act ctc tac ggt agt 576
Gly Tyr Asp Cys Gly Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser
180 185 190
gct gat gct ccc ttg gat ttc gat tac aaa gat gcc att cgt atg cgt 624
Ala Asp Ala Pro Leu Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg
195 200 205
cgc ttt att gcc cat tta att ctc aca gat gct ctg aaa tga 666
Arg Phe Ile Ala His Leu Ile Leu Thr Asp Ala Leu Lys
210 215 220
<210>25
<211>221
<212>PRT
<213>人工序列
<400>25
Met Gly Leu Val Pro Glu Leu Asn Glu Lys Asp Asp Asp Gln Val Gln
1 5 10 15
Lys Ala Leu Ala Ser Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn
20 25 30
Ile Glu Ile Thr Val Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp
35 40 45
Leu Asn Asp Thr Ile Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser
50 55 60
Thr Pro Asn Thr Val Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser
65 70 75 80
Glu Arg Gly Tyr Gln Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr
85 90 95
Gln Ile Asp Lys Leu Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln
100 105 110
Ser His Trp Ala Leu Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly
115 120 125
Tyr Val Asp Ser Leu Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile
130 135 140
Leu Thr Asp Leu Gln Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile
145 150 155 160
Gly Glu Asp Phe Asp Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn
165 170 175
Gly Tyr Asp Cys Gly Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser
180 185 190
Ala Asp Ala Pro Leu Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg
195 200 205
Arg Phe Ile Ala His Leu Ile Leu Thr Asp Ala Leu Lys
210 215 220
<210>26
<211>59
<212>DNA
<213>人工序列
<400>26
ccgctggatg aaacgcaaga aaactcagat tgacaagtta gacaaaatct ttactccga 59
<210>27
<211>59
<212>DNA
<213>人工序列
<400>27
ctacaccaac ctgtctgaac gtggttatca gggtgtccgc cgctggatga aacgcaaga 59
<210>28
<211>59
<212>DNA
<213>人工序列
<400>28
gaaatctacg cccaatacgg tcgcattcaa tagcttcttc tacaccaacc tgtctgaac 59
<210>29
<211>59
<212>DNA
<213>人工序列
<400>29
agacgacgat caagtgcaga aagctctggc aagccgcgaa aacacccagc tgatgaatc 59
<210>30
<211>46
<212>DNA
<213>人工序列
<400>30
ggcctggttc cggaactgaa cgaaaaagac gacgatcaag tgcaga 46
<210>31
<211>33
<212>DNA
<213>人工序列
<400>31
gcaattccat atgggcctgg ttccggaact gaa 33
<210>32
<211>59
<212>DNA
<213>人工序列
<400>32
gattcactta gactgcccac agcaaccaaa cggctatgac tgcggtatct atgtctgta 59
<210> 33
<211> 59
<212>DNA
<213>人工序列
<400> 33
ggaagaatcc aaacacacca tcggtgagga cttcgacttg attcacttag actgcccac 59
<210>34
<211>59
<212>DNA
<213>人工序列
<400>34
gtccttcgca atcctgactg atctgcaaaa atacgtcatg gaagaatcca aacacacca 59
<210>35
<211>59
<212>DNA
<213>人工序列
<400>35
cgatgatgcc cagtgcccag tggctctgat ttaagttaat cggagtaaag attttgtct 59
<210>36
<211>59
<212>DNA
<213>人工序列
<400>36
acagagagtc aacataaccg atggttttct ttttcagatc gatgatgccc agtgcccag 59
<210>37
<211>59
<212>DNA
<213>人工序列
<400>37
cagtcaggat tgcgaaggac attgcgtttg gaccgttgga cagagagtca acataaccg 59
<210>38
<211>59
<212>DNA
<213>人工序列
<400>38
gggttttgaa gtcacggaca gtaatctcaa tgttatcacg attcatcagc tgggtgttt 59
<210>39
<211>59
<212>DNA
<213>人工序列
<400>39
actcaatgat ggtatcgttt aaccaacggc ggggagcgag ggttttgaag tcacggaca 59
<210>40
<211>59
<212>DNA
<213>人工序列
<400>40
ccgtattggg cgtagatttc tcaatgtatt tcatgaagaa ctcaatgatg gtatcgttt 59
<210>41
<211>59
<212>DNA
<213>人工序列
<400>41
aatccaaggg agcatcagca ctaccgtaga gagtgttcat acagacatag ataccgcag 59
<210>42
<211>59
<212>DNA
<213>人工序列
<400>42
caataaagcg acgcatacga atggcatctt tgtaatcgaa atccaaggga gcatcagca 59
<210>43
<211>59
<212>DNA
<213>人工序列
<400>43
gaggatcctc atttcagagc atctgtgaga attaaatggg caataaagcg acgcatacg 59
<210>44
<211>215
<212>DNA
<213>人工序列
<400>44
gcaattccat atgggcctgg ttccggaact gaacgaaaaa gacgacgatc aagtgcagaa 60
agctctggca agccgcgaaa acacccagct gatgaatcgt gataacattg agattactgt 120
ccgtgacttc aaaaccctcg ctccccgccg ttggttaaac gataccatca ttgagttctt 180
catgaaatac attgagaaat ctacgcccaa tacgg 215
<210>45
<211>250
<212>DNA
<213>人工序列
<400>45
gaaatctacg cccaatacgg tcgcattcaa tagcttcttc tacaccaacc tgtctgaacg 60
tggttatcag ggtgtccgcc gctggatgaa acgcaagaaa actcagattg acaagttaga 120
caaaatcttt actccgatta acttaaatca gagccactgg gcactgggca tcatcgatct 180
gaaaaagaaa accatcggtt atgttgactc tctgtccaac ggtccaaacg caatgtcctt 240
cgcaatcctg 250
<210>46
<211>255
<212>DNA
<213>人工序列
<400>46
gtccttcgca atcctgactg atctgcaaaa atacgtcatg gaagaatcca aacacaccat 60
cggtgaggac ttcgacttga ttcacttaga ctgcccacag caaccaaacg gctatgactg 120
cggtatctat gtctgtatga acactctcta cggtagtgct gatgctccct tggatttcga 180
ttacaaagat gccattcgta tgcgtcgctt tattgcccat ttaattctca cagatgctct 240
gaaatgagga tcctc 255
<210>47
<211>469
<212>DNA
<213>人工序列
<400>47
gcaattccat atgggcctgg ttccggaact gaacgaaaaa gacgacgatc aagtgcagaa 60
agctctggca agccgcgaaa acacccagct gatgaatcgt gataacattg agattactgt 120
ccgtgacttc aaaaccctcg ctccccgccg ttggttaaac gataccatca ttgagttott 180
catgaaatac attgagaaat ctacgcccaa tacgggaaat ctacgcccaa tacggtcgca 240
ttcaatagct tcttctacac caacctgtct gaacgtggtt atcagggtgt ccgccgctgg 300
atgaaacgca agaaaactca gattgacaag ttagacaaaa tctttactcc gattaactta 360
aatcagagcc actgggcact gggcatcatc gatctgaaaa agaaaaccat cggttatgtt 420
gactctctgt ccaacggtcc aaacgcaatg tccttcgcaa tcctgactg 469
<210>48
<211>53
<212>DNA
<213>人工序列
<400>48
ctggatccga attcgagctc aagcttctcg agaatacgac tcactatagg gag 53
Claims (14)
1.人角质细胞生长因子-1结构类似物，其特征在于其氨基酸序列的N末端缺失23个氨基酸，并且其40位半胱氨酸点突变成非极性氨基酸KGF-1Δ23KGF(40S)。
2.如权利要求1所述的人角质细胞生长因子-1结构类似物，其特征在于所述的非极性氨基酸为丝氨酸、丙氨酸、缬氨酸、亮氨酸或异亮氨酸，优选为丝氨酸。
3.如权利要求1所述的人角质细胞生长因子-1结构类似物，其特征在于该结构类似物的氨基酸序列如序列表SEQ ID No.2所示。
4.编码权利要求1～3任一项所述人角质细胞生长因子-1结构类似物的基因。
5.权利要求1～3任一项所述人角质细胞生长因子-1结构类似物与SUMO的融合蛋白。
6.编码权利要求5所述融合蛋白的基因。
7.含有权利要求6所述基因的重组表达载体。
8.如权利要求7所述的重组表达载体，其特征在于，该表达载体具有两个启动子，分别与融合蛋白基因和编码泛素相关修饰因子蛋白酶1基因可操作地连接。
9.如权利要去7或8所述的重组表达载体为pET-3c。
10.含有权利要求7～9所述重组表达载体的转化宿主。
11.如权利要求10所述的转化宿主，其衍生于大肠杆菌JM109、DH5、BL21或Origami(DE3)，优选为BL21。
12.一种制备权利要求1～3任一项所述人角质细胞生长因子-1结构类似物的方法，包括步骤：培养权利要求10或11所述的转化宿主，诱导表达人角质细胞生长因子-1结构类似物，回收诱导产物。
13.如权利要求12所述的方法，其特征在于诱导表达的条件是30℃，0.5mmol/L IPTG诱导6小时。
14.权利要求1～3任一项所述人角质细胞生长因子-1结构类似物在制备角膜损伤修复、促进毛囊增生、抗疤痕和/或抗纤维化的药物中的应用。
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| CN2007101221745A CN101220092B (zh) | 2007-09-21 | 2007-09-21 | 人角质细胞生长因子-1结构类似物,其生产方法及应用 |
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| CN2007101221745A CN101220092B (zh) | 2007-09-21 | 2007-09-21 | 人角质细胞生长因子-1结构类似物,其生产方法及应用 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103656622A (zh) * | 2013-12-04 | 2014-03-26 | 广东暨大基因药物工程研究中心有限公司 | 重组人角质细胞因子kgf-2环境敏感型眼部传递系统及其应用 |
| CN106479998A (zh) * | 2016-08-24 | 2017-03-08 | 上海交通大学 | 一种灰略红链霉菌β‑1,4‑葡萄糖苷酶及其编码基因和应用 |
| CN107041948A (zh) * | 2009-01-21 | 2017-08-15 | 北京三有利科技发展有限公司 | 细胞生长因子治疗溃疡性疾病和肺纤维化疾病的应用 |
| CN112625140A (zh) * | 2020-12-22 | 2021-04-09 | 北京致力生科科技有限公司 | 一种pep-1-g4s-kgf2融合蛋白及其编码基因和应用 |
| CN113354745A (zh) * | 2021-07-09 | 2021-09-07 | 温州医科大学 | 一种组合物及规模化生产成纤维细胞生长因子的方法 |
| CN114292323A (zh) * | 2022-01-12 | 2022-04-08 | 中国医学科学院整形外科医院 | 一种角质细胞生长因子活性多肽及其应用 |
| WO2024065900A1 (zh) * | 2022-09-26 | 2024-04-04 | 领星生物科技（上海）有限公司 | 用于抑制肿瘤生长的sirna及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7655413B2 (en) * | 2003-06-26 | 2010-02-02 | Lifesensors, Inc. | Methods and compositions for enhanced protein expression and purification |
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2007
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107041948A (zh) * | 2009-01-21 | 2017-08-15 | 北京三有利科技发展有限公司 | 细胞生长因子治疗溃疡性疾病和肺纤维化疾病的应用 |
| CN103656622A (zh) * | 2013-12-04 | 2014-03-26 | 广东暨大基因药物工程研究中心有限公司 | 重组人角质细胞因子kgf-2环境敏感型眼部传递系统及其应用 |
| CN103656622B (zh) * | 2013-12-04 | 2015-09-02 | 广东暨大基因药物工程研究中心有限公司 | 重组人角质细胞因子kgf-2环境敏感型眼部传递系统及其应用 |
| CN106479998A (zh) * | 2016-08-24 | 2017-03-08 | 上海交通大学 | 一种灰略红链霉菌β‑1,4‑葡萄糖苷酶及其编码基因和应用 |
| CN112625140A (zh) * | 2020-12-22 | 2021-04-09 | 北京致力生科科技有限公司 | 一种pep-1-g4s-kgf2融合蛋白及其编码基因和应用 |
| CN113354745A (zh) * | 2021-07-09 | 2021-09-07 | 温州医科大学 | 一种组合物及规模化生产成纤维细胞生长因子的方法 |
| CN114292323A (zh) * | 2022-01-12 | 2022-04-08 | 中国医学科学院整形外科医院 | 一种角质细胞生长因子活性多肽及其应用 |
| WO2024065900A1 (zh) * | 2022-09-26 | 2024-04-04 | 领星生物科技（上海）有限公司 | 用于抑制肿瘤生长的sirna及其应用 |
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