CN113185612B - 一种酿酒酵母表达长效重组iii型胶原蛋白及其在化妆品中的应用 - Google Patents
一种酿酒酵母表达长效重组iii型胶原蛋白及其在化妆品中的应用 Download PDFInfo
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- CN113185612B CN113185612B CN202110358113.9A CN202110358113A CN113185612B CN 113185612 B CN113185612 B CN 113185612B CN 202110358113 A CN202110358113 A CN 202110358113A CN 113185612 B CN113185612 B CN 113185612B
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- type iii
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- iii collagen
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/50—Fusion polypeptide containing protease site
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Abstract
本发明公开了一种酿酒酵母表达长效重组III型胶原蛋白及其在化妆品中的应用。该长效重组III型胶原蛋白的氨基酸序列包括基本重复单元及末端氨基酸序列。本发明中的长效重组III型胶原蛋白具有良好的生物相容性、良好的细胞黏附性、能促进新细胞形成、具有止血功能,而且其促进上皮细胞生长功能更强。且本发明中的长效重组III型胶原蛋白与从动物组织和异体中提取的胶原蛋白相比的优势在于其可溶性、无病毒隐患和排异反应低。
Description
技术领域
本发明涉及新兴生物医学领域,尤其涉及一种酿酒酵母表达长效重组III型胶原蛋白,一种长效重组III型胶原蛋白的活性检测方法,以及一种酿酒酵母表达长效重组III型胶原蛋白在化妆品中的应用。
背景技术
胶原(collagen),早期的定义是“生成胶的产物”。胶原的形态一般是白色、透明、无分支的原纤维。胶原的氨基酸组成有以下特点:甘氨酸(Gly,G)占总氨基酸残基的33%,即每隔两个其他氨基酸(X,Y)就有一个甘氨酸,所以其肽链可以用(G-X-Y)-来表示;X与Y可以是任意氨基酸,但X通常是脯氨酸(Pro,P),Y通常是羟脯氨酸(HyP,P*)与羟赖氨酸(Hyl,K*)。目前,己经可以分离得到20余种天然胶原,根据发现次序依次被称为I型、II型胶原等,分布于动物的皮肤、骨骼、韧带、内脏、血管、虹膜、角膜等结缔组织中,具有支撑器官、保护机体的功能。胶原具有很强的生物活性及生物功能,使结缔组织具有机械强度,还能促进细胞生长,具有止血、生物相容性和生物降解性。基于这些特性,胶原在烧伤、创伤、美容、矫形、硬组织修复、创面止血、药物传递、缓释技术等医药卫生领域有着广泛的应用。
III型胶原在血管中的含量比较高,主要存在于肺泡的间质中,并且分布比较杂乱,会构成比较复杂的网状结构。正是由于这种网状结构使得肺组织,尤其是肺泡间质,可以保持良好的柔韧性和一定的弹性,同时为细胞提供所需的养分,并且直接与血管的母细胞结合,可以促进心血管的形成。这与皮肤的饱满程度、润焕程度和光泽度都具有紧密的联系。
由于天然胶原的不溶性、大分子性和特殊的翻译后修饰(羟基化、糖基化、相互交联,且受多种生物酶的复杂调控),对其原始序列直接进行原核或酵母真核异源表达,却期望获得三重螺旋构象的胶原的可行性为零。
发明内容
针对现有的技术问题:由于天然胶原的不溶性、大分子性和特殊的翻译后修饰(羟基化、糖基化、相互交联,且受多种生物酶的复杂调控),对其原始序列直接进行原核或酵母真核异源表达,却期望获得三重螺旋构象的胶原的可行性为零,本发明提供一种酿酒酵母表达长效重组III型胶原蛋白、一种长效重组III型胶原蛋白的活性检测方法、以及一种酿酒酵母表达长效重组III型胶原蛋白在化妆品中的应用。
本发明采用以下技术方案实现:一种酿酒酵母表达长效重组III型胶原蛋白,长效重组III型胶原蛋白的氨基酸序列包括基本重复单元及末端氨基酸序列;
所述长效重组III型胶原蛋白的核苷酸序列如Seq ID NO.1所示,所述长效重组III型胶原蛋白的核苷酸序列所对应的氨基酸序列如Seq ID NO.2所示。
作为上一步技术方案的改进,长效重组III型胶原蛋白的核苷酸序列包括起始密码子、终止密码子及6×His标签序列。
作为上一步技术方案的进一步改进,长效重组III型胶原蛋白的核苷酸序列还包括Not I酶切位点、Xba I酶切位点。
作为上一步技术方案的进一步改进,长效重组III型胶原蛋白的核苷酸序列是通过酵母密码子优化人胶原的核苷酸序列获得的,优化包括减少重复序列的重复频率对人胶原的核苷酸序列进行人工设计。
本发明还提供一种长效重组III型胶原蛋白的活性检测方法,该活性检测方法采用如上任意一项所述的长效重组III型胶原蛋白;活性检测方法包括试剂配制、测定操作及测定方法。
作为上一步技术方案的改进,试剂包括完全培养液、维持培养液、消化液、噻唑蓝溶液、PBS缓冲液。
作为上一步技术方案的改进,测定操作包括重组人胶原蛋白供试品制备。
作为上一步技术方案的改进,测定方法包括以下步骤:
步骤一、细胞株的培养,细胞株为BALB/C 3T3细胞株;
步骤二、制备细胞悬液,将细胞悬液接种于96孔板;
步骤三、用维持培养液维持细胞生存;
步骤四、细胞的加样处理;
步骤五、根据MTT比色法测定吸光度,记录测定结果,并完成四参数拟合计算;
步骤六、根据测定结果判断长效重组III型胶原蛋白的活性。
本发明还提供一种酿酒酵母表达长效重组III型胶原蛋白在化妆品中的应用,其特征在于,其采用如上任意一项所述的长效重组III型胶原蛋白。
本发明具有以下有益效果:
1、本发明通过减少重复序列的重复频率(获得小分子量蛋白)等手段对人胶原的序列进行人工设计,异源表达后获得长效重组III型胶原蛋白。
2、本发明提供的长效重组III型胶原蛋白具有良好的生物相容性、良好的细胞黏附性、能促进新细胞形成、具有止血功能,而且其促进上皮细胞生长功能更强。
3、本发明提供的长效重组III型胶原蛋白与从动物组织和异体中提取的胶原蛋白相比的优势在于其可溶性、无病毒隐患和排异反应低。
序列表说明(序列表内容单独提供)
Seq ID NO.1所示为本发明实施例中长效重组III型胶原蛋白的核苷酸序列。
Seq ID NO.2所示为本发明实施例中长效重组III型胶原蛋白的氨基酸序列。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明,并不用于限定本发明。
实施例1
本实施例提供了一种酿酒酵母表达长效重组III型胶原蛋白,该胶原蛋白的氨基酸序列包括基本重复单元及末端氨基酸序列。
长效重组III型胶原蛋白的核苷酸序列包括Not I酶切位点、Xba I酶切位点、起始密码子、终止密码子及6×His标签序列。Not I酶切位点的核苷酸序列为GCGGCCGC;Xba I酶切位点的核苷酸序列为TCTAGA;起始密码子的核苷酸序列为ATG;终止密码子的核苷酸序列为TAA。
所述长效重组III型胶原蛋白的核苷酸序列是通过酵母密码子优化人胶原的核苷酸序列获得的,所述优化包括减少重复序列的重复频率对所述人胶原的核苷酸序列进行人工设计。经酵母密码子优化后的核苷酸序列为:
GCGGCCGCAATGGGTGAAAGAGGTGCTCCAGGTTTTAGAGGACCAGCCGGACCAAATGGAATTCCTGGCGAGAAAGGTCCTGCTGGAGAAAGGGGAGCACCTGGTGAAAGAGGAGCTCCAGGTTTCAGAGGGCCTGCTGGTCCGAATGGAATCCCGGGTGAGAAGGGTCCTGCTGGTGAGCGTGGTGCACCTGGAGAGCGTGGAGCACCTGGTTTTAGAGGCCCGGCTGGTCCTAACGGTATACCAGGGGAAAAGGGTCCGGCAGGAGAAAGAGGTGCTCCGGGAGAAAGGGGTGCTCCAGGTTTCCGTGGCCCTGCTGGTCCAAACGGTATCCCGGGTGAAAAAGGCCCTGCCGGAGAACGTGGTGCTCCTAGAAGCGGTGAAAGGGGTGCACCTGGTTTCCGTGGTCCAGCTGGCCCTAATGGTATACCAGGCGAAAAAGGACCAGCAGGAGAACGTGGTGCACCAGGTGAACGTGGAGCACCGGGATTTAGGGGTCCTGCTGGTCCTAATGGTATACCAGGTGAAAAGGGTCCAGCAGGAGAAAGAGGTGCCCCAGGAGAAAGAGGGGCACCTGGTTTCAGAGGTCCTGCAGGACCCAATGGTATCCCTGGCGAGAAGGGACCAGCTGGAGAAAGAGGTGCTCCTGGTGAAAGAGGAGCACCGGGATTTAGAGGCCCAGCCGGTCCTAACGGTATTCCAGGAGAAAAGGGTCCAGCTGGTGAAAGAGGAGCCCCAGGACCATGTTGTGGTGGTGGTCATCACCATCACCATCACTAATCTAGA。
注:GCGGCCGC为Not I酶切位点,TCTAGA为Xba I酶切位点;ATG为起始密码子,TAA为终止密码子;CATCACCATCACCATCAC为6×His标签序列。
经优化的长效重组III型胶原蛋白的核苷酸序列所对应的氨基酸序列为:
MGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPRSGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPCCGGGHHHHHH。
在本实施例中,长效重组III型胶原蛋白的核苷酸序列如Seq ID NO.1所示,长效重组III型胶原蛋白的核苷酸序列所对应的氨基酸序列如Seq ID NO.2所示。
本实施例提供的长效重组III型胶原蛋白具有良好的生物相容性、良好的细胞黏附性、能促进新细胞形成、具有止血功能,而且其促进上皮细胞生长功能更强。且本实施例提供的长效重组III型胶原蛋白与从动物组织和异体中提取的胶原蛋白相比的优势在于其可溶性、无病毒隐患和排异反应低。
实施例2
本实施例提供了一种长效重组III型胶原蛋白的活性检测方法,用于验证实施例1所述的长效重组III型胶原蛋白的促细胞增殖活性。该检测方法的细胞株选用BALB/C 3T3细胞。
活性检测方法包括试剂配制、测定操作及测定方法。试剂包括完全培养液、维持培养液、消化液、噻唑蓝溶液、PBS缓冲液。测定操作包括重组人胶原蛋白供试品制备。
测定方法包括以下步骤:
步骤一、细胞株的培养,细胞株为BALB/C 3T3细胞株;
步骤二、制备细胞悬液,将细胞悬液接种于96孔板;
步骤三、用维持培养液维持细胞生存;
步骤四、细胞的加样处理;
步骤五、根据MTT比色法测定吸光度,记录测定结果,并完成四参数拟合计算;
步骤六、根据测定结果判断长效重组III型胶原蛋白的活性。
在本实施例中,试剂配制包括:
(1)完全培养液的配制:量取小牛血清100ml,加培养液定容至1000ml;
(2)维持培养液的配制:量取小牛血清4ml,加培养液定容至1000ml;
(3)消化液的配制:称取乙二胺四乙酸二钠0.2g、氯化钠8g、氯化钾0.2g、磷酸氢二钠1.152g、磷酸二氢钾0.2g、胰蛋白酶2.5g,加纯化水溶解并定容至1000ml,过滤除菌。
(4)噻唑蓝(MTT)溶液的配制:称取MTT粉末0.10g,加PBS 20ml使溶解,经0.22μm滤膜过滤除菌。4℃避光保存。
(5)PBS缓冲液的配制:称取氯化钠8.0g、氯化钾0.20g、磷酸氢二钠1.44g、磷酸二氢钾0.24g,加纯化水溶解并定容至1000ml,经121℃15分钟灭菌。细胞株:BALB/C 3T3细胞。
测定操作包括重组人胶原蛋白供试品制备,其具体包括以下操作:
取100μl供试品于900μl完全培养液中进行10倍稀释。取10倍稀释液在96孔细胞培养板中,从1︰4开始依次做4倍递增梯度稀释,共做10个稀释度(1~10孔),每个稀释度同时设置一个复孔。
本实施例中4倍递增梯度稀释的具体操作步骤为:首先于96孔细胞培养板中加入150μl完全培养液,取50μl 1000倍稀释的供试品溶液加入96孔板中第1列,吹打稀释。充分稀释后于第1列孔中抽取50μl于第2列孔,吹打稀释。充分稀释后于第2列孔中抽取50μl于第3列孔,吹打稀释。重复上述操作,直至第10列。
测定方法:
步骤一、BALB/C 3T3细胞株用完全培养液于37℃、5%二氧化碳培养,控制细胞浓度为每1ml含1.0×105-5.0×105个细胞,传代后24~36小时用于生物学活性测定。
步骤二、弃去培养瓶中的培养液,消化和收集细胞用完全培养液配成每1ml含5.0×104~8.0×104个细胞的细胞悬液,接种于96孔板中,接种过程中不停摇匀,保持每孔接种数相同,每孔100μl,于37℃、5%二氧化碳条件下培养。
步骤三、24小时后换成维持培养液。置37℃、5%二氧化碳培养24小时。
步骤四、制备的细胞培养板弃去维持液,加入标准品溶液和供试品溶液,每孔100μl。置37℃、5%二氧化碳培养64~72小时。
步骤五、MTT比色法:每孔加入MTT溶液20μl,于37℃、5%二氧化碳培养5小时。以上操作在无菌条件下进行。弃去培养板中的液体后,向每孔中加入DMSO 100μl,混匀后在酶标仪上,以630nm为参比波长,570nm为试验波长测定吸光度,记录测定结果,并完成四参数拟合计算。
步骤六、根据步骤五中的测定结果,判断长效重组III型胶原蛋白是否具有促BALB/c 3T3细胞增殖活性。
根据本实施例提供的长效重组III型胶原蛋白的活性检测方法,测的长效重组III型胶原蛋白具有促BALB/c 3T3细胞增殖活性,且工作效价约为6000U/ml。
实施例3
本实施例提供一种酿酒酵母表达长效重组III型胶原蛋白在化妆品中的应用,其采用如实施例1中所述的长效重组III型胶原蛋白。该长效重组III型胶原蛋白具有良好的生物相容性、良好的细胞黏附性、能促进新细胞形成、具有止血功能。且如实施例2中所述,实施例1中的长效重组III型胶原蛋白促进上皮细胞生长功能更强。由此可知,实施例1中所述的长效重组III型胶原蛋白在化妆品中具有广泛的应用前景。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 芜湖英特菲尔生物制品产业研究院有限公司
<120> 一种酿酒酵母表达长效重组III 型胶原蛋白及其在化妆品中的应用
<141> 2021-04-01
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 771
<212> DNA
<213> Saccharomyces cerevisiae
<400> 1
atgggtgaaa gaggtgctcc aggttttaga ggaccagccg gaccaaatgg aattcctggc 60
gagaaaggtc ctgctggaga aaggggagca cctggtgaaa gaggagctcc aggtttcaga 120
gggcctgctg gtccgaatgg aatcccgggt gagaagggtc ctgctggtga gcgtggtgca 180
cctggagagc gtggagcacc tggttttaga ggcccggctg gtcctaacgg tataccaggg 240
gaaaagggtc cggcaggaga aagaggtgct ccgggagaaa ggggtgctcc aggtttccgt 300
ggccctgctg gtccaaacgg tatcccgggt gaaaaaggcc ctgccggaga acgtggtgct 360
cctagaagcg gtgaaagggg tgcacctggt ttccgtggtc cagctggccc taatggtata 420
ccaggcgaaa aaggaccagc aggagaacgt ggtgcaccag gtgaacgtgg agcaccggga 480
tttaggggtc ctgctggtcc taatggtata ccaggtgaaa agggtccagc aggagaaaga 540
ggtgccccag gagaaagagg ggcacctggt ttcagaggtc ctgcaggacc caatggtatc 600
cctggcgaga agggaccagc tggagaaaga ggtgctcctg gtgaaagagg agcaccggga 660
tttagaggcc cagccggtcc taacggtatt ccaggagaaa agggtccagc tggtgaaaga 720
ggagccccag gaccatgttg tggtggtggt catcaccatc accatcacta a 771
<210> 2
<211> 256
<212> PRT
<213> Saccharomyces cerevisiae
<400> 2
Met Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn
1 5 10 15
Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly
20 25 30
Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile
35 40 45
Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Glu Arg
50 55 60
Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly
65 70 75 80
Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Glu Arg Gly Ala
85 90 95
Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys
100 105 110
Gly Pro Ala Gly Glu Arg Gly Ala Pro Arg Ser Gly Glu Arg Gly Ala
115 120 125
Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys
130 135 140
Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Glu Arg Gly Ala Pro Gly
145 150 155 160
Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro
165 170 175
Ala Gly Glu Arg Gly Ala Pro Gly Glu Arg Gly Ala Pro Gly Phe Arg
180 185 190
Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly
195 200 205
Glu Arg Gly Ala Pro Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro
210 215 220
Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg
225 230 235 240
Gly Ala Pro Gly Pro Cys Cys Gly Gly Gly His His His His His His
245 250 255
Claims (9)
1.一种酿酒酵母表达的长效重组III型胶原蛋白,其特征在于,所述长效重组III型胶原蛋白的氨基酸序列包括基本重复单元及末端氨基酸序列;
所述长效重组III型胶原蛋白的核苷酸序列如SEQ ID NO.1所示,所述长效重组III型胶原蛋白的核苷酸序列所对应的氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的酿酒酵母表达的长效重组III型胶原蛋白,其特征在于,所述长效重组III型胶原蛋白的核苷酸序列包括起始密码子、终止密码子及6×His标签序列。
3.如权利要求2所述的酿酒酵母表达的长效重组III型胶原蛋白,其特征在于,所述长效重组III型胶原蛋白的核苷酸序列还包括Not I酶切位点、Xba I酶切位点。
4.如权利要求2所述的酿酒酵母表达的长效重组III型胶原蛋白,其特征在于,所述长效重组III型胶原蛋白的核苷酸序列是通过酵母密码子优化人胶原的核苷酸序列获得的,所述优化包括减少重复序列的重复频率对所述人胶原的核苷酸序列进行人工设计。
5.一种长效重组III型胶原蛋白的活性检测方法,其特征在于,所述活性检测方法采用如权利要求1至4中任意一项所述的长效重组III型胶原蛋白;所述活性检测方法包括试剂配制、测定操作及测定方法。
6.如权利要求5所述的长效重组III型胶原蛋白的活性检测方法,其特征在于,所述试剂包括完全培养液、维持培养液、消化液、噻唑蓝溶液、PBS缓冲液。
7.如权利要求5所述的长效重组III型胶原蛋白的活性检测方法,其特征在于,所述测定操作包括重组人胶原蛋白供试品制备。
8.如权利要求5所述的长效重组III型胶原蛋白的活性检测方法,其特征在于,所述测定方法包括以下步骤:
步骤一、细胞株的培养,所述细胞株为BALB/C 3T3细胞株;
步骤二、制备细胞悬液,将所述细胞悬液接种于96孔板;
步骤三、用维持培养液维持细胞生存;
步骤四、所述细胞的加样处理;
步骤五、根据MTT比色法测定吸光度,记录测定结果,并完成四参数拟合计算;
步骤六、根据测定结果判断所述长效重组III型胶原蛋白的活性。
9.一种酿酒酵母表达的长效重组III型胶原蛋白在化妆品中的应用,其特征在于,其采用如权利要求1至4中任意一项所述的长效重组III型胶原蛋白。
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