CN116836263A - 一种重组人源iii型胶原蛋白及其毕赤酵母重组表达系统 - Google Patents
一种重组人源iii型胶原蛋白及其毕赤酵母重组表达系统 Download PDFInfo
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Abstract
本发明公开了一种高活性重组人源III型三螺旋天然构型胶原蛋白及其制备方法。本发明对人全长Ⅲ型胶原蛋白的氨基酸序列进行优化,选取一段含有384个氨基酸序列区段,将人源III型胶原蛋白部分肽段中含有不含脯氨酸的Gly‑X‑Y结构肽以含有1个或者2个脯氨酸的Gly‑X‑Y结构肽替换,C末端连接GPPGPPGPPHHHHHH序列,根据优化后肽段获得编码肽段的基因序列连接到毕赤酵母分泌表达载体pPIC9K,构建人源重组胶原蛋白表达载体pPIC9K‑CO3A1,同时构建带有肽剪酶序列的人源P4H alpha亚基和beta亚基插入到pPIC9K载体中,然后转化毕赤酵母宿主菌,筛选P4H高活性以及脯氨酸高度羟基化重组人源III型三螺旋天然构型胶原蛋白的菌株。本发明通过胶原蛋白结构的和脯氨酸羟基化酶的人工设计和优化,有效的提高了胶原分子间的相互作用力,水溶性好,热稳定性和抗酶解水平,有效解决天然胶原在发酵中发生降解的问题,表达产物稳定性高、生物相容性好,可用于大规模生产。
Description
技术领域
本发明属于基因工程领域,具体涉及利用毕赤酵母表达重组人源III型胶原蛋白。
背景技术
胶原蛋白(collagen)是体内含量最多的一种蛋白质,是细胞外基质(ECM)的主要成分,也是人体中含量最多的蛋白质,占到蛋白质总量的25%至35%。它的主要功能体现在维护细胞外环境,维持组织器官的正常生理功能和修复肌体损伤等方面。胶原蛋白是天然的生物资源,具有其它高分子材料无法比拟的优异的生物组织相容性,对细胞的支撑弹性和可降解性,其对维护细胞、组织、器官的正常生理功能和损伤修复具有重要作用,已广泛应用于医药、保健品及化妆品行业中。
当前市场上销售的胶原蛋白产品都是取自猪、牛、鱼等动物组织中,很难避免病毒感染,会导致免疫排斥和过敏症状,天然胶原蛋白无法溶于水,性质不均一,难以被人体利用,往往需要经过化学手段处理后才能使用。会导致免疫排斥和过敏症状。所以,目前的胶原蛋白只能在化妆品和保健品中使用,无法发挥胶原蛋白的原本生物学功能。
随着生物技术的发展,利用基因重组技术,通过微生物发酵法获得重组胶原蛋白,解决了传统提取法存在的病毒隐患,同时与天然胶原蛋白相比,人工设计的重组胶原蛋白的亲水性和生物相容性显著提高。
但研究表明利用重组菌株,大肠杆菌表达表达的胶原蛋白无法进行蛋白翻译后修饰,对于一些具有高级构象的重组人胶原蛋白,利用大肠杆菌表达后无法糖基化。同时,由于大肠杆菌是原核生物,没有内质网(ER),因此也没有能帮助胶原蛋白质折叠成高级构象的分子伴侣蛋白及一些修饰合成蛋白如羟基化、形成二硫键的酶。因此大肠杆菌系统很难表达具有天然高级结构的三螺旋胶原蛋白。
酵母发酵产生的人源重组胶原蛋白克服了动物源以及大肠杆菌表达的胶原蛋白的缺陷。酵母表达系统具备分子伴侣以及蛋白翻译后修饰的酶(如糖基化、羟基化、乙酰基化酶等),非常适合于具有高级结构的三螺旋胶原蛋白的形成。酵母细胞的培养条件相对于哺乳动物细胞较低,非常适宜于大规模的工业化发酵生产。酵母系统取代动物源胶原和大肠杆菌原核表达系统源重组胶原在生物医学应用方面有重要研究价值。酵母表达的人源胶原蛋白通过基因工程的设计和改造能够具有形成天然胶原蛋白三螺旋构象的能力。三螺旋构象更接近与胶原蛋白天然的生物学结构,具有更优异的生物学活性(血小板凝聚、细胞、组织、器官的正常生理功能的维护、调节及损伤修复)、理化性质和可加工性,是更高级的生物材料。但是现有的毕赤酵母基因工程菌表达胶原蛋白时,在发酵过程中重组菌株表达的胶原蛋白会出现明显的降解现象,不利于后续的纯化及应用,同时也提高了胶原蛋白的生产消耗和成本。
在分子结构上,每条胶原肽链主要由Gly-X-Y结构肽构成(X、Y是Gly之外的任何氨基酸残基),这种肽链结构是形成胶原纤维高级结构所必需的,决定了胶原蛋白优良的生物相容性和低免疫原性。甘氨酸是胶原蛋白氨基酸组成中分子量最小的一个,是具有单个氢原子作为其侧链的氨基酸,由于甘氨酸的侧键非常小,它可以胶原蛋白合成中占据其它氨基酸无法占据的空间,比如作为胶原螺旋内的氨基酸,它可以在胶原蛋白α-链中氨基酸序列的第三个位置找到。
这一特性注定了甘氨酸成为胶原蛋白合成中的一个重要关键,因此甘氨酸是胶原蛋白合成中最重要的氨基酸,且是非常必需的。
在形成胶原蛋白的所有氨基酸中,脯氨酸对促进皮肤健康的影响可能是最受关注的。随着年龄增长或者但遇到某些健康问题,人体对脯氨酸等氨基酸的需求会增加。皮肤健康状况差、愈合缓慢、关节疼痛、胃肠道问题以及心脏病高风险的人都可能从获得更多这种氨基酸中获得健康益处。
脯氨酸的结构是独特的,因为它是具有仲胺的唯一蛋白原氨基酸(生物合成形成蛋白质的一种类型)。它有助于“构建”胶原蛋白。
羟脯氨酸也是胶原蛋白的主要成分,占胶原蛋白总氨基和亚氨酸含量的约14%。羟脯氨酸(Hyp)对于正确形成胶原纤维至关重要。它是通过「酶4-脯氨酰羟化酶」对脯氨酸进行翻译后修饰而产生的。而且羟脯氨酸只在年轻肌肤中独一无二的存在。
羟脯氨酸和脯氨酸对胶原蛋白的稳定性起着关键作用。羟脯氨酸上的额外OH参与与三螺旋中的其他链的氢键合,从而稳定胶原蛋白结构。另外,脯氨酸和羟脯氨酸通过允许胶原螺旋的急剧扭曲赋予胶原分子刚性。
Gly-X-Y结构中,Pro的羟化对胶原蛋白三重螺旋结构的稳定期这非常重要的作用,Pro的羟化是由P4H来催化完成的。P4H是由α2β2构成的四聚体,每一个α亚基包含一个将Pro羟化的催化位点。虽然Hyp对胶原蛋白三重螺旋结构的稳定期这非常重要的作用,但在现有的酵母发酵条件下,Hyp还是非常难以获得。为了在毕赤酵母工程菌得到稳定的三螺旋结构,并具有更高生物活性的胶原蛋白,本发明针对现有毕赤酵母工程菌表达胶原蛋白的局限性,引入人源P4H 基因,构建人源III型胶原alpha 1链、P4H alpha亚基和beta亚基的三重启动子的pPICZ 重组质粒,表达含有Hyp残基的高活性三螺旋天然结构的胶原蛋白。
三螺旋构象更接近与胶原蛋白天然的生物学结构,具有更优异的生物学活性(血小板凝聚、细胞、组织、器官的正常生理功能的维护、调节及损伤修复)、理化性质和可加工性,是更高级的生物材料。因此,研发和设计酵母重组人源三螺旋胶原蛋白作为现有胶原产品的替代和补充具有巨大的市场潜力与前景,而在国内市场,尚无获批的酵母重组三螺旋胶原蛋白生物材料。
天然胶原蛋白结构中含有约23%的羟脯氨酸与羟赖氨酸。这些羟基化位点与胶原结构形成、原纤维功能、组织发育、以及多种病理学密切相关。例如,羟赖氨酸的缺乏会造成爱唐综合征(Ehlers-Danlos syndrome type VI)、布鲁克综合征(Bruck syndrome,BS)、以及骨骼发育不良等疾病。随着年龄的增长,人类皮肤组织中赖氨酸羟化酶的含量会呈现大幅度地下降。
中国发明专利申请CN201810707153.8公开了一种通过将源于囊孢菌RH1的脯氨酸羟基化酶与人源胶原基因在大肠杆菌中进行共表达,实现了胶原蛋白的脯氨酸羟基化的方法。此类方法虽在一定程度上解决了重组胶原蛋白的部分羟基化问题,但也存在以下不足:1)重组人源胶原蛋白结构功能设计依旧不明确;2)对于羟基化完全的大肠杆菌来说,大肠杆菌属于非GRAS表达宿主(Generally Recognized as Safe,为美国FDA评价食品添加剂的安全性指标),3)下游纯化工艺复杂,去除内毒素难度大。3)大肠杆菌属于原核表达,表达的胶原蛋白无法进行蛋白翻译后修饰,对于一些具有高级构象的重组人胶原蛋白,利用大肠杆菌表达后无法糖基化和形成高活性构型。
中国发明专利CN112626074B一种在酵母中表达含羟脯氨酸修饰化的重组人iii型胶原蛋白成熟肽及其制备方法与应用,其表达重组载体是将脯氨酸羟化酶表达载体pPZAStA-P4HA2-P4HB2与编码重组人III型胶原蛋白成熟肽的基因的核苷酸序列连接得到,pPZAStA-P4HA2-P4HB2载体将P4HA2-P4HB2一起表达,脯氨酸羟化酶的α亚基和β亚基没有切开,很难保证形成活性较高的α2β2四聚体构型,得到的重组人iii型胶原蛋白成熟肽的脯氨酸的羟基化可能不充分,从而影响iii型胶原蛋白成熟肽天然构型和活性。
发明内容
本发明的目的在于提供一种高活性重组人源III型三螺旋胶原蛋白及其毕赤酵母重组表达系统。
本发明旨在至少解决现有技术中存在的技术问题之一。
为此,本发明提出一种生物活性高、水溶性好酵母重组人源III型三螺旋构象胶原蛋白及其制备方法。
本发明的另一目的在于提供一种与上述重组人III型胶原蛋白成熟肽相关的生物材料。
本发明的再一目的在于提供一种重组载体,其特征在于引入了脯氨酸羟化酶P4H基因,为了提高脯氨酸羟化酶在毕赤酵母中的羟基化活性,将编码内部核糖体进入位点(internal ribosome entry site,IRES)或自剪切多肽2A的核酸序列插入到pPIC9K-P4HA2-P4HB重组载体的P4HA2-P4HB中间,形成高活性的α2β2四聚体构型。自剪切多肽可以是P2A,T2A,E2A和F2A,优选地,自剪切多肽选择T2A,构建pPIC9K- P4HA2-T2A-P4HB载体。
本发明的第四个目的在于提供一种含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的制备方法。
本发明的第五个目的在于提供上述制备方法制备得到的含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽。
本发明的第六个目的在于提供上述含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的应用。
本发明的目的通过下述技术方案实现:
一种优化设计的编码重组人III型胶原蛋白成熟肽的基因,氨基酸序列如SEQ IDNO:1所示,编码氨基酸序列的核苷酸序列如SEQ ID NO:2 所示。
一种可表达含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的酵母,是将上述重组载体转入酵母中得到;
所述的酵母优选为毕赤酵母G115;
一种含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的制备方法,包含如下步骤:
将上述可表达含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的酵母诱导表达后,离心收集上清,上清通过硫酸铵分级沉淀和Ni2+亲和层析纯化和G25分子筛过滤,得到含脯氨酸高度羟基化的高活性重组人III型胶原蛋白成熟肽。
一种脯氨酸高度羟基化的重组人III型胶原蛋白成熟肽,通过上述制备方法制备得到;
脯氨酸高度羟基化的重组人III型胶原蛋白成熟肽可以用于应用于美白肌肤、水嫩滋润、抗皱除衰、抗敏修复,也可以应用于组织再生医学领域,合成角膜可以治疗与角膜盲相关的视力缺陷。
附图说明:
图1 示毕赤酵母菌株的发酵上清中重组Ⅲ胶原蛋白分析结果。
实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1含有III型胶原蛋白的毕赤酵母表达系统构建
以pPIC9K (购于Invitrogen公司)为骨架,在多克隆位点分别引入优化后的CO3A1基因序列,得到pPIC9K- CO3A1,最后转化至毕赤酵母GS115中,详细步骤如下:
1、依据蛋白质资源数据库UniProt(网址https://www.uniprot.org/)公布的人III型胶原蛋白成熟肽序列(P02461-1),将人源III型胶原蛋白部分肽段中含有不含脯氨酸的Gly-X-Y结构肽以含有1个或者2个脯氨酸的Gly-X-Y结构肽替换, C末端连接GPPGPPGPPHHHHHH序列,得到优化后的III型胶原蛋白氨基酸序列如SEQ ID NO:1所示:
GAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGGSGPAGPPGPQGVKGERGSPGGPGAAGFPGARGLPGPPGSNGNPGPPGPSGSPGKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPPGPPGPPHHHHHH
2、利用在线设计工具Jcat(http://www .jcat .de/)反向设计基因序列,针对宿主毕赤酵母表达所需的优选密码子,在设计过程中去除EcoR I和Not I酶切位点,有利后期基因操作,委托上海生工生物科技有限公司合成优化后Col3A1基因,优化后的基因序列为SEQ ID NO:2所示:
ATGGGTGCACCAGGTCAGAATGGTGAACCAGGTGGTAAAGGTGAAAGAGGTGCACCAGGTGAAAAAGGTGAAGGTGGTCCACCAGGTGTTGCAGGTCCACCAGGTGGTTCAGGTCCAGCAGGTCCACCAGGTCCACAGGGTGTTAAAGGTGAAAGAGGTTCACCAGGTGGTCCAGGTGCGGCGGGTTTCCCAGGAGCAAGAGGTTTACCAGGTCCACCAGGTTCAAATGGTAATCCAGGTCCACCAGGTCCATCAGGTTCACCAGGTAAAGATGGTCCACCAGGTCCAGCAGGTAATACAGGTGCCCCAGGTTCACCCGGTGTAAGCGGTCCAAAAGGTGATGCAGGTCAGCCAGGTGAAAAAGGTTCACCAGGTGCACAGGGTCCACCAGGTGCACCAGGTCCATTAGGTATAGCAGGTATAACAGGTGCAAGAGGTTTAGCAGGTCCACCAGGTATGCCAGGTCCAAGAGGTTCACCAGGTCCACAGGGTGTTAAAGGTGAATCAGGTAAACCAGGTGCAAATGGTTTATCAGGTGAAAGAGGCCCACCAGGACCACAAGGTCTCCCAGGTCTCGCGGGTACAGCGGGTGAACCAGGTAGAGATGGTAATCCAGGTTCAGATGGTTTACCAGGTAGAGATGGTTCACCAGGTGGTAAAGGTGATAGAGGTGAAAATGGTTCACCAGGTGCACCAGGTGCACCAGGTCATCCAGGTCCACCAGGTCCAGTTGGTCCAGCAGGTAAATCAGGTGATAGAGGTGAATCAGGTCCAGCAGGTCCAGCAGGTGCACCAGGTCCAGCAGGTTCAAGAGGTGCACCAGGTCCACAGGGTCCAAGAGGTGATAAAGGTGAAACAGGTGAAAGAGGTGCAGCAGGTATAAAAGGTCATAGAGGTTTTCCAGGTAATCCAGGTGCACCAGGTTCACCAGGTCCAGCAGGTCAGCAGGGTGCAATAGGTTCACCAGGTCCAGCAGGTCCAAGAGGTCCAGTTGGTCCATCAGGTCCACCAGGTAAAGATGGTACATCAGGTCATCCAGGTCCAATAGGTCCACCAGGTCCAAGAGGTAATAGAGGTGAAAGAGGTTCAGAAGGTTCACCAGGTCATCCAGGTCAGCCAGGTCCACCAGGTCCACCAGGTGCACCAGGTCCATGCGGTCCACCAGGTCCACCAGGTCCACCACATCATCATCATCATCATTGA。
实施例2 脯氨酸羟化酶表达载体pPIC9K-P4HA2-T2A-P4HB的制备
1. 依据蛋白质资源数据库UniProt(网址https://www.uniprot.org/)公布的人脯氨酸羟化酶α亚基和β亚基对应的成熟肽序列(O15460,P07237),利用在线设计工具Jcat(http://www .jcat .de/)反向设计基因序列,针对宿主毕赤酵母表达所需的优选密码子,在设计过程中去除EcoR I和Not I酶切位点,有利后期基因操作,优化后的基因序列为SEQID NO:4和SEQ ID NO:6所示,对应的氨基酸和核苷酸序列如下所示:
SEQ ID NO:3 P4HA2氨基酸序列:
MKLWVSALLMAWFGVLSCVQAEFFTSIGHMTDLIYAEKELVQSLKEYILVEEAKLSKIKSWANKMEALTSKSAADAEGYLAHPVNAYKLVKRLNTDWPALEDLVLQDSAAGFIANLSVQRQFFPTDEDEIGAAKALMRLQDTYRLDPGTISRGELPGTKYQAMLSVDDCFGMGRSAYNEGDYYHTVLWMEQVLKQLDAGEEATTTKSQVLDYLSYAVFQLGDLHRALELTRRLLSLDPSHERAGGNLRYFEQLLEEEREKTLTNQTEAELATPEGIYERPVDYLPERDVYESLCRGEGVKLTPRRQKRLFCRYHHGNRAPQLLIAPFKEEDEWDSPHIVRYYDVMSDEEIERIKEIAKPKLARATVRDPKTGVLTVASYRVSKSSWLEEDDDPVVARVNRRMQHITGLTVKTAELLQVANYGVGGQYEPHFDFSRRPFDSGLKTEGNRLATFLNYMSDVEAGGATVFPDLGAAIWPKKGTAVFWYNLLRSGEGDYRTRHAACPVLVGCKWVSNKWFHERGQEFLRPCGSTEVD
SEQ ID NO:4
ATGAAATTATGGGTTTCAGCATTATTAATGGCATGGTTTGGTGTTTTATCATGCGTTCAGGCAGAATTTTTTACATCAATAGGTCATATGACAGATTTAATATATGCAGAAAAAGAATTAGTTCAGTCATTAAAAGAATATATATTAGTTGAAGAAGCAAAATTATCAAAAATAAAATCATGGGCAAATAAAATGGAAGCATTAACATCAAAATCAGCAGCAGATGCAGAAGGTTATTTAGCACATCCAGTTAATGCATATAAATTAGTTAAAAGATTAAATACAGATTGGCCAGCATTAGAAGATTTAGTTTTACAGGATTCAGCAGCAGGTTTTATAGCAAATTTATCAGTTCAGAGACAGTTTTTTCCAACAGATGAAGATGAAATAGGTGCAGCAAAAGCATTAATGAGATTACAGGATACATATAGATTAGATCCAGGTACAATATCAAGAGGTGAATTACCAGGTACAAAATATCAGGCAATGTTATCAGTTGATGATTGCTTTGGTATGGGTAGATCAGCATATAATGAAGGTGATTATTATCATACAGTTTTATGGATGGAACAGGTTTTAAAACAGTTAGATGCAGGTGAAGAAGCAACAACAACAAAATCACAGGTTTTAGATTATTTATCATATGCAGTTTTTCAGTTAGGTGATTTACATAGAGCATTAGAATTAACAAGAAGATTATTATCATTAGATCCATCACATGAAAGAGCAGGTGGTAATTTAAGATATTTTGAACAGTTATTAGAAGAAGAAAGAGAAAAAACATTAACAAATCAGACAGAAGCAGAATTAGCAACACCAGAAGGTATATATGAAAGACCAGTTGATTATTTACCAGAAAGAGATGTTTATGAATCATTATGCAGAGGTGAAGGTGTTAAATTAACACCAAGAAGACAGAAAAGATTATTTTGCAGATATCATCATGGTAATAGAGCACCACAGTTATTAATAGCACCATTTAAAGAAGAAGATGAATGGGATTCACCACATATAGTTAGATATTATGATGTTATGTCAGATGAAGAAATAGAAAGAATAAAAGAAATAGCAAAACCAAAATTAGCAAGAGCAACAGTTAGAGATCCAAAAACAGGTGTTTTAACAGTTGCATCATATAGAGTTTCAAAATCATCATGGTTAGAAGAAGATGATGATCCAGTTGTTGCAAGAGTTAATAGAAGAATGCAGCATATAACAGGTTTAACAGTTAAAACAGCAGAATTATTACAGGTTGCAAATTATGGTGTTGGTGGTCAGTACGAGCCACATTTCGACTTTTCAAGAAGACCATTTGATTCAGGTTTAAAAACAGAAGGTAATAGATTAGCAACATTTTTAAATTATATGTCAGATGTTGAAGCAGGTGGTGCAACAGTTTTTCCAGATTTAGGTGCAGCAATATGGCCAAAAAAAGGTACAGCAGTTTTTTGGTATAATTTATTAAGATCAGGTGAAGGTGATTATAGAACAAGACATGCAGCGTGCCCAGTGCTGGTTGGTTGCAAATGGGTTTCAAATAAATGGTTTCATGAAAGAGGTCAGGAATTTTTAAGACCATGCGGTTCAACAGAAGTTGAT
SEQ ID NO:5 P4HB氨基酸序列:
MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVKDEL
SEQ ID NO:6
ATGTTAAGAAGAGCATTATTATGCTTAGCAGTTGCAGCATTAGTTAGAGCAGATGCACCAGAAGAAGAAGATCATGTTTTAGTTTTAAGAAAATCAAATTTTGCAGAAGCATTAGCAGCACATAAATATTTATTAGTTGAATTTTATGCACCATGGTGCGGTCATTGCAAAGCATTAGCACCAGAATATGCAAAAGCAGCAGGTAAATTAAAAGCAGAAGGTTCAGAAATAAGATTAGCAAAAGTTGATGCAACAGAAGAATCAGATTTAGCACAGCAGTATGGTGTTAGAGGTTATCCAACAATAAAATTTTTTAGAAATGGTGATACAGCATCACCAAAAGAATATACAGCAGGTAGAGAAGCAGATGATATAGTTAATTGGTTAAAAAAAAGAACAGGTCCAGCAGCAACAACATTACCAGATGGTGCAGCAGCAGAATCATTAGTTGAATCATCAGAAGTTGCAGTTATAGGTTTTTTTAAAGATGTTGAATCAGATTCAGCAAAACAGTTTTTACAGGCAGCAGAAGCAATAGATGATATACCATTTGGTATAACATCAAATTCAGATGTTTTTTCAAAATATCAGTTAGATAAAGATGGTGTTGTTTTATTTAAAAAATTTGATGAAGGTAGAAATAATTTTGAAGGTGAAGTTACAAAAGAAAATTTATTAGATTTTATAAAACATAATCAGTTACCATTAGTTATAGAATTTACAGAACAGACAGCACCAAAAATATTTGGTGGTGAAATAAAAACACATATATTATTATTTTTACCAAAATCAGTTTCAGATTATGATGGTAAATTATCAAATTTTAAAACAGCAGCAGAATCATTTAAAGGTAAAATATTATTTATATTTATAGATTCAGATCATACAGATAATCAGAGAATATTAGAATTTTTTGGTTTAAAAAAAGAAGAATGCCCAGCAGTTAGATTAATAACATTAGAAGAAGAAATGACAAAATATAAACCAGAATCAGAAGAATTAACAGCAGAAAGAATAACAGAATTTTGCCATAGATTTTTAGAAGGTAAAATAAAACCACATTTAATGTCACAGGAATTACCAGAAGATTGGGATAAACAGCCAGTTAAAGTTTTAGTTGGTAAAAATTTTGAAGATGTTGCATTTGATGAAAAAAAAAATGTTTTTGTTGAATTTTATGCACCATGGTGCGGTCATTGCAAACAGTTAGCACCAATATGGGATAAATTAGGTGAAACATATAAAGATCATGAAAATATAGTTATAGCAAAAATGGATTCAACAGCAAATGAAGTTGAAGCAGTTAAAGTTCATTCATTTCCAACATTAAAATTTTTTCCAGCATCAGCAGATAGAACAGTTATAGATTATAATGGTGAAAGAACATTAGATGGTTTTAAAAAATTTTTAGAATCAGGTGGTCAGGATGGTGCAGGTGATGATGATGATTTAGAAGATTTAGAAGAAGCAGAAGAACCAGATATGGAAGAAGATGATGATCAGAAAGCAGTTAAAGATGAATTA
2.脯氨酸羟化酶α亚基(P4HA2)和β亚基P4HB,并在中间插入带编码GSG的T2A肽序列(GSGEGRGSLLTCGDVEENPGP)的核酸序列,得到P4HA2-T2A-P4HB序列,如SEQ ID NO:7所示,委托上海生工生物科技有限公司合成P4HA2-T2A-P4HB序列:
SEQ ID NO:7
ATGAAATTATGGGTTTCAGCATTATTAATGGCATGGTTTGGTGTTTTATCATGCGTTCAGGCAGAATTTTTTACATCAATAGGTCATATGACAGATTTAATATATGCAGAAAAAGAATTAGTTCAGTCATTAAAAGAATATATATTAGTTGAAGAAGCAAAATTATCAAAAATAAAATCATGGGCAAATAAAATGGAAGCATTAACATCAAAATCAGCAGCAGATGCAGAAGGTTATTTAGCACATCCAGTTAATGCATATAAATTAGTTAAAAGATTAAATACAGATTGGCCAGCATTAGAAGATTTAGTTTTACAGGATTCAGCAGCAGGTTTTATAGCAAATTTATCAGTTCAGAGACAGTTTTTTCCAACAGATGAAGATGAAATAGGTGCAGCAAAAGCATTAATGAGATTACAGGATACATATAGATTAGATCCAGGTACAATATCAAGAGGTGAATTACCAGGTACAAAATATCAGGCAATGTTATCAGTTGATGATTGCTTTGGTATGGGTAGATCAGCATATAATGAAGGTGATTATTATCATACAGTTTTATGGATGGAACAGGTTTTAAAACAGTTAGATGCAGGTGAAGAAGCAACAACAACAAAATCACAGGTTTTAGATTATTTATCATATGCAGTTTTTCAGTTAGGTGATTTACATAGAGCATTAGAATTAACAAGAAGATTATTATCATTAGATCCATCACATGAAAGAGCAGGTGGTAATTTAAGATATTTTGAACAGTTATTAGAAGAAGAAAGAGAAAAAACATTAACAAATCAGACAGAAGCAGAATTAGCAACACCAGAAGGTATATATGAAAGACCAGTTGATTATTTACCAGAAAGAGATGTTTATGAATCATTATGCAGAGGTGAAGGTGTTAAATTAACACCAAGAAGACAGAAAAGATTATTTTGCAGATATCATCATGGTAATAGAGCACCACAGTTATTAATAGCACCATTTAAAGAAGAAGATGAATGGGATTCACCACATATAGTTAGATATTATGATGTTATGTCAGATGAAGAAATAGAAAGAATAAAAGAAATAGCAAAACCAAAATTAGCAAGAGCAACAGTTAGAGATCCAAAAACAGGTGTTTTAACAGTTGCATCATATAGAGTTTCAAAATCATCATGGTTAGAAGAAGATGATGATCCAGTTGTTGCAAGAGTTAATAGA
AGAATGCAGCATATAACAGGTTTAACAGTTAAAACAGCAGAATTATTACAGGTTGCAAATTATGGTGTTGGTGGTCAGTACGAGCCACATTTCGACTTTTCAAGAAGACCATTTGATTCAGGTTTAAAAACAGAAGGTAATAGATTAGCAACATTTTTAAATTATATGTCAGATGTTGAAGCAGGTGGTGCAACAGTTTTTCCAGATTTAGGTGCAGCAATATGGCCAAAAAAAGGTACAGCAGTTTTTTGGTATAATTTATTAAGATCAGGTGAAGGTGATTATAGAACAAGACATGCAGCGTGCCCAGTGCTGGTTGGTTGCAAATGGGTTTCAAATAAATGGTTTCATGAAAGAGGTCAGGAATTTTTAAGACCATGCGGTTCAACAGAAGTTGATGGTTCAGGTGAAGGTAGAGGTTCATTATTAACATGCGGTGATGTTGAAGAAAATCCAGGTCCAATGTTAAGAAGAGCATTATTATGCTTAGCAGTTGCAGCATTAGTTAGAGCAGATGCACCAGAAGAAGAAGATCATGTTTTAGTTTTAAGAAAATCAAATTTTGCAGAAGCATTAGCAGCACATAAATATTTATTAGTTGAATTTTATGCACCATGGTGCGGTCATTGCAAAGCATTAGCACCAGAATATGCAAAAGCAGCAGGTAAATTAAAAGCAGAAGGTTCAGAAATAAGATTAGCAAAAGTTGATGCAACAGAAGAATCAGATTTAGCACAGCAGTATGGTGTTAGAGGTTATCCAACAATAAAATTTTTTAGAAATGGTGATACAGCATCACCAAAAGAATATACAGCAGGTAGAGAAGCAGATGATATAGTTAATTGGTTAAAAAAAAGAACAGGTCCAGCAGCAACAACATTACCAGATGGTGCAGCAGCAGAATCATTAGTTGAATCATCAGAAGTTGCAGTTATAGGTTTTTTTAAAGATGTTGAATCAGATTCAGCAAAACAGTTTTTACAGGCAGCAGAAGCAATAGATGATATACCATTTGGTATAACATCAAATTCAGATGTTTTTTCAAAATATCAGTTAGATAAAGATGGTGTTGTTTTATTTAAAAAATTTGATGAAGGTAGAAATAATTTTGAAGGTGAAGTTACAAAAGAAAATTTATTAGATTTTATAAAACATAATCAGTTACCATTAGTTATAGAATTTACAGAACAGACAGCACCAAAAATATTTGGTGGTGAAATAAAAACACATATATTATTATTTTTACCAAAATCAGTTTCAGATTATGATGGTAAATTATCAAATTTTAAAACAGCAGCAGAATCATTTAAAGGTAAAATATTATTTATATTTATAGATTCAGATCATACAGATAATCAGAGAATATTAGAATTTTTTGGTTTAAAAAAAGAAGAATGCCCAGCAGTTAGATTAATAACATTAGAAGAAGAAATGACAAAATATAAACCAGAATCAGAAGAATTAACAGCAGAAAGAATAACAGAATTTTGCCATAGATTTTTAGAAGGTAAAATAAAACCACATTTAATGTCACAGGAATTACCAGAAGATTGGGATAAACAGCCAGTTAAAGTTTTAGTTGGTAAAAATTTTGAAGATGTTGCATTTGATGAAAAAAAAAATGTTTTTGTTGAATTTTATGCACCATGGTGCGGTCATTGCAAACAGTTAGCACCAATATGGGATAAATTAGGTGAAACATATAAAGATCATGAAAATATAGTTATAGCAAAAATGGATTCAACAGCAAATGAAGTTGAAGCAGTTAAAGTTCATTCATTTCCAACATTAAAATTTTTTCCAGCATCAGCAGATAGAACAGTTATAGATTATAATGGTGAAAGAACATTAGATGGTTTTAAAAAATTTTTAGAATCAGGTGGTCAGGATGGTGCAGGTGATGATGATGATTTAGAAGATTTAGAAGAAGCAGAAGAACCAGATATGGAAGAAGATGATGATCAGAAAGCAGTTAAAGATGAATTA
3. 将P4HA2-T2A-P4HB序列插入到pPIC9K载体中,得到pPIC9K- P4HA2-T2A-P4HB脯氨酸羟化酶表达载体。
实施例3 含有脯氨酸羟化酶基因的毕赤酵母表达系统的制备
(1) pPIC9K- P4HA2-T2A-P4HB质粒线性化:用质粒小提试剂盒(苏州易迈吉生物)抽提pPIC9K- P4HA2-T2A-P4HB质粒,加入5μL限制性内切酶Not I进行线性化;
(2)线性化pPIC9K- P4HA2-T2A-P4HB产物的纯化:用纯化试剂盒(苏州易迈吉生物)对线性化后的pPIC9K- P4HA2-T2A-P4HB产物进行纯化,具体步骤为:
加入等体积的 Buffer GRP,颠倒或涡旋混匀,把混合液转移至 DNA吸附柱子中;10,000 × g 离心 30~60 秒;弃滤液,把柱子套回收集管中;加入 500µL DNA漂洗液至柱子中;12,000 × g 离心 30~60 秒;重复此步骤一次;倒弃滤液,把柱子套回收集管中;加入500µL 80%乙醇至柱子中;12,000 × g 离心 30~60 秒;倒弃滤液,把柱子套回收集管中;12,000 x g 离心5分钟,打开柱子的盖子,风干 2分钟以彻底去除乙醇;把柱子套在1.5ml 离心管中,加入20μL超纯水至柱子膜中央溶解DNA;放置2 分钟;12,000 × g 离心1 分钟;
(3) 线性化pPIC9K- P4HA2-T2A-P4HB产物纯化产物的脱磷酸化
为了防止载体质粒DNA的自身环化,用小牛肠碱性磷酸酶(CIP)处理线性化pPIC9K- P4HA2-T2A-P4HB产物,具体操作如下:
1) 建立反应体系:线性化产物 35ul,10x CIP buffer 4ul,CIP1ul,ddH2O 补足至45ul;
2) 在PCR仪上控制反应温度(加石蜡油封闭),37℃,15 min ;50℃,15 min;56℃,30 min(灭活);
3) 加入5ul 0.1M NaOH ,56℃温育10 min,用于灭活CIP;
(4)脱磷酸化的pPIC9K- P4HA2-T2A-P4HB产物的纯化:按照步骤(2)中方法纯化脱磷酸化后的线性化pPIC9K- P4HA2-T2A-P4HB产物;
(5)毕赤酵母GS115感受态制备(GS115菌种购买于Invitrogen公司):1)挑取酵母单菌落,接种至含有5ml YPD培养基的50ml三角瓶中,30℃、250-300r/min培养过夜;2)取100-500μl的培养物接种至含有500ml新鲜培养基的2L三角摇瓶中,25~31℃、250-300r/min培养过夜,至OD600达到1.2~1.4;3) 将细胞培养物于4℃,1500g离心5min,用500ml的冰预冷的无菌水将菌体沉淀重悬;4) 按步骤3)离心,用250ml的冰预冷的无菌水将菌体沉淀重悬;5) 按步骤3) 离心,用30ml的冰预冷的1.5 mol的山梨醇溶液将菌体沉淀重悬;6) 按步骤3)离心,用1.5 ml的冰预冷的0.5 mol的山梨醇溶液将菌体沉淀重悬 7. 备注:可将其分装为40μl一份的包装冷冻起来,置于-80°C保存一个月;
(6) 将回收得到的线性化质粒加入40μL步骤(5)中制备的毕赤酵母GS115感受态中,
1)转移至预冷的0.2CM电击杯中,将电转化杯冰浴15min,1500v电击0.8S,迅速放置冰上,加入1ml冰预冷的山梨醇溶液将菌体混匀,转至1.5ml的无菌EP管中,后涂布于MD固体平板中,30℃培养2-3天知道单菌落出现,挑选转化子进行菌落PCR鉴定,根据目的基因的不同,分别设计相应的上下游验证引物,引物如下所示:
引物F-P4HA2:5’-TGAAATTATGGGTTTCAGCATT-3’;
引物R-P4HA2:5’- TCAACTTCTGTTGAACCGCATG-3’;
引物F- P4HB:5’- ATGTTAAGAAGAGCATTATTATGC-3’;
引物R-P4HB:5’- TAATTCATCTTTAACTGCTTTCTG-3’;
2)在PCR小管中加入50 μL TE,挑取不同的转化子,放入PCR小管中吹打后95℃温育10min; 2000g离心1min,取5μL上清液作为PCR反应的模板。PCR反应体系(50μL)如下:2×Taq Mix 25 μL ,上游引物(10μM) 2μL,下游引物(10μM) 2μL,模板5μL,最后用ddH2O补齐至50μL;扩增程序为:95℃,30s变性;62℃,15s退火,72℃延伸1min,整个反应32个循环;
3)将PCR产物进行核酸电泳,分析条带大小,实际值与理论值相符合的即获得目标菌株pPIC9K- P4HA2-T2A-P4HB,即为含有脯氨酸羟化酶基因的毕赤酵母表达系统。
实施例3重组人III型胶原生产过程
(1) 制备一级种子:取保藏于-80℃的菌种GS115/pPIC9K- P4HA2-T2A-P4HB,pPIC9K- CO3A1进行活化,用灭菌牙签蘸取菌液在YPD固体平板上划线;30℃培养2-3天直至有单菌落出现,挑单菌落于YPD液体培养基中,30℃,200rpm培养约24h,至OD600达到5-40范围,此即为一级种子液,种子液可根据培养体积的大小进行调整;
(2) 制备二级种子:取300-500mL一级种子液,接入50L发酵罐中(起始发酵YEPD液体培养基为30L,在28~40℃条件下,150~200r/min下振荡培养,当菌体OD600达到10.0~12.0时即得二级种子;自动流加氨水或磷酸控制pH值为5.2,调节好发酵设备的转速、空气流量、罐压(0.8个大气压);
YEPD液体培养基;
基础培养基:葡萄糖20g/L;大豆蛋白胨20g/L;酵母粉10g/L,磷酸氢二钾27mL/L,磷酸二氢钾12mg/L; 硫酸钙1g/L,硫酸钾18g/L,蔗糖20g/L,七水硫酸镁15g/L,氢氧化钠4g/L,甘油60g/L(高温灭菌);微量元素:氯化铁 2.0g/L,碘化钾0.08g/L,一水硫酸锰3.0g/L,氯化锌20.0g/L,七水合硫酸亚铁65.0g/L,浓硫酸5mL/L(过滤除菌);
(3) 由于菌体在二级种子培养阶段已经初步适应了发酵罐培养基,在发酵过程中菌体经过相对较短的适应期后进入对数生长期,此时pH值自动控制,自动流加氨水或磷酸将pH值控制在5.2,调节转速和通气量使DO维持在50y以上;当发酵罐中的甘油耗尽后(约接种21小时左右),DO值陡然上升,经检测表明甘油已经耗尽,此时以5.5 ml/min的速度流加800 mL甘油,7小时后停止补料,约2〜3小时后DO值又大幅度上升,此时菌体量达到预定要求 (本实验菌体湿重为250-300g/L);
(4)当菌体湿重达到预定值(250-300g/L)时,开始甲醇诱导,首次补加甲醇(含PTM, 12ml/L) 80ml,待甲醇耗尽后,此时以2.5 ml/min的速度再次补加250 ml甲醇,当甲醇耗尽后,以1.5 ml/min的速度流加甲醇(含PTM,12ml/L),诱导表达24小时后,再以 0.5ml/min的速度流加甲醇(含PTM,12ml/L),诱导12小时后,36小时后结束发酵;
(5)诱导结束培后,将发酵液6000xg离心10min,收集上清。将含有重组人III型胶原成熟肽发酵液上清加入8%-15%饱和硫酸铵进行沉淀,20000rpm离心20min收集上清;然后再往上清中加入2M咪唑溶液使终浓度为20mM,再过0.45μm滤膜去除颗粒,然后通过Ni2+亲和层析柱进行下一步纯化,过程如下:首先采用PH7.4平衡缓冲液(50mM磷酸缓冲液0.5MNaCl)平衡Ni2+亲和层析,然后取过滤后的上清液以10ml/min上样,用500ml平衡缓冲液洗(50mM磷酸缓冲液,0.5M NaCl,20mM咪唑)去未吸附的样品,再用100mL洗脱缓冲液(PH7.4,50mM磷酸缓冲液,0.5M NaCl,500mM咪唑)洗脱纯化后的样品,最后再过分子筛G25(GE公司),得到溶解于50 mM pH5.5磷酸缓冲液的重组人III型胶原成熟肽原液,(图1):
实施例4重组人III型胶原冻干粉的制备及粘度测试:
1)将溶于西林瓶中的实施例3制得的重组人III型胶原成熟肽(浓度为5mg/mL)过滤除菌后,装入西林瓶中;
2)将上述西林瓶放入冻干机板层,关闭机门;
3)预冻阶段:预冻温度-25℃~-30℃,预冻时间1~2小时;
4)升华干燥阶段:在-5℃升华干燥4小时,继续在10℃升华干燥5小时,压力控制为0.25±0.03mbar;
5)解吸干燥阶段:将温度升至30℃,保温2小时,将压力调至0.05~0.01mbar,保温4小时,分别得到重组人III型胶原成熟肽冻干粉;
6)准确称取一定量上述冻干粉,加入无菌的20mM pH 5.5磷酸缓冲液进行溶解,终浓度均为5 mg/mL;
7)将对照(猪皮提取胶原,购于法国罗赛洛公司)溶解于20mM pH 5.5酸缓冲液中,配置成浓度为5mg/mL溶液;
8)然后采用RS600型HAAKE旋转流变仪对溶液粘度特性进行分析,25℃下保温30min后进行测定,转速控制为32rpm;
结果显示, 5mg/mL的重组人III型胶原成熟肽粘度达到90mPa.S,而对照5mg/mL猪皮提取胶原的粘度只有45mPa.S,重组人III型胶原成熟肽粘度明显优于猪皮提取胶原。
Claims (7)
1. 一种高活性重组人源III型三螺旋天然构型胶原蛋白及其制备方法,其特征在于:选取一段含有384个氨基酸序列区段,将人源III型胶原蛋白部分肽段中含有不含脯氨酸的Gly-X-Y结构肽以含有1个或者2个脯氨酸的Gly-X-Y结构肽替换, C末端连接GPPGPPGPPHHHHHH序列。
2.如权利要求1所述,氨基酸序列如SEQ ID NO:1所示,编码氨基酸序列的核苷酸序列如SEQ ID NO:2 所示,将SEQ ID NO:2插入到pPIC9K载体中,得到pPIC9K- CO3A1,最后转化至毕赤酵母GS115中。
3.如权利要求1所述,一种含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的制备方法,其特征在于引入了脯氨酸羟化酶P4H基因。Gly-X-Y中的脯氨酸高度羟基化,形成天然三螺旋构型。
4.如权利要求3所述,其特征在将脯氨酸羟化酶序列P4HA2和-P4HB插入到表达载体pPIC9K中,表达脯氨酸羟化酶。
5.如权利要求4所述,为了提高脯氨酸羟化酶在毕赤酵母中的羟基化活性,将编码内部核糖体进入位点(internal ribosome entry site,IRES)或自剪切多肽2A的核酸序列插入到pPIC9K-P4HA2-P4HB重组载体的P4HA2-P4HB中间,形成高活性的α2β2四聚体构型。自剪切多肽可以是P2A,T2A,E2A和F2A,优选地,自剪切多肽选择T2A,构建pPIC9K- P4HA2-T2A-P4HB载体。
6.一种含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的制备方法,其特征在于包含如下步骤:将权利要求2~5任一项所述的可表达含羟脯氨酸修饰化的重组人III型胶原蛋白成熟肽的酵母诱导表达后,离心收集上清,上清通过硫酸铵分级沉淀和Ni2+亲和层析纯化和G25分子筛过滤,得到含脯氨酸高度羟基化的高活性重组人III型胶原蛋白成熟肽。
7.权利要求6所述的脯氨酸高度羟基化的重组人III型胶原蛋白成熟肽可以用于应用于美白肌肤、水嫩滋润、抗皱除衰、抗敏修复,也可以应用于组织再生医学领域,合成角膜可以治疗与角膜盲相关的视力缺陷。
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