CN115074340B - 一种新内含肽及其在合成人源原弹性蛋白上的应用 - Google Patents
一种新内含肽及其在合成人源原弹性蛋白上的应用 Download PDFInfo
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Abstract
本发明公开了一种新内含肽及其在合成人源原弹性蛋白上的应用。本发明提供了一种内含肽突变体(SEQ ID NO.1)；还提供使用内含肽突变体来制备和纯化目的分子的方法，使用内含肽变体和人源原弹性蛋白融合成高效表达的融合蛋白，并构建到表达载体pET‑28a上，重组载体核苷酸序列。利用大肠杆菌BL21(DE3)作为表达宿主，获得了高效表达原弹性蛋白的重组菌株。并通过Ni‑NTA亲和层析获得高纯度的原弹性蛋白。利用内含肽的自切割效果，得到了完整的人源原弹性蛋白。可以有效解决人源原弹性蛋白原核表达存在的问题，提高人源原弹性蛋白的产能，在食品、医疗、美容和护肤品等领域具有重要的使用价值。
Description
技术领域
本发明属于基因工程领域，涉及一种新内含肽及其在合成人源原弹性蛋白上的应用。
背景技术
弹性蛋白(ELN，elastin)是生皮组织中弹性纤维(elastic fibers)的主要成分，主要存在于韧带和脉管壁。弹性纤维与胶原纤维共同赋予组织弹性和抗张能力。弹性蛋白占真皮中总蛋白的2％，在维持皮肤弹性上起着重要作用。弹性蛋白减少是皮肤发生老化松弛、下垂和细皱纹的主要原因，随着皮肤自然老化，弹性纤维分解越来越显著。因此，弹性蛋白在化妆品、医疗、美容和日常生活具有极其重要的应用价值。人源原弹性蛋白肽链序列中含有713个以上的氨基酸。不同于胶原和角蛋白，弹性蛋白的氨基酸序列中不存在贯穿整个肽链的重复性周期结构，但是存在交替的亲水和疏水性肤段。由氧化赖氨酰形成的锁链素和开链锁链素是弹性蛋白特有的交联结构。人源原弹性蛋白(又成为弹性蛋白肽链和弹性蛋白单体)经脯氨酸和赖氨酸羟基化形成羟基化的弹性蛋白单体，并通过交联形成弹性蛋白复合物和弹性纤维。人源原弹性蛋白分子中非极性氨基酸占95％，甘氨酸含量接近总量的1/3，脯氨酸占10％，羟脯氨酸占1％；这使得人源原弹性蛋白的原核表达和纯化变得极其困难，极大地限制了弹性蛋白的生产和应用。因此亟需一种高效表达纯化人源原弹性蛋白的方法。
内含肽是一段可以催化自身从前体蛋白质中去除的氨基酸序列，插入到外显肽基因中与靶基因一起表达成为嵌合蛋白前体，再通过自催化作用从前体蛋白中切除并将两侧外显肽通过肽键连接起来成为成熟蛋白质。正因为内含肽的自剪接的性质和功能，内含肽在对蛋白的表达及修饰作用方面的应用也越来越广泛。至今，内含肽在蛋白质工程领域有着广泛的应用。这些应用包括蛋白质纯化、蛋白质连接、环肽制备以及蛋白标记等方面。其中在蛋白纯化方面的研究最为广泛和深入。但在应用内含肽时，仍存在内含肽种类少、偏好性大和效率不足的问题。
发明内容
本发明第一个方面的目的，在于提供一种内含肽突变体。
本发明第二个方面的目的，在于提供一种融合蛋白。
本发明第三个方面的目的，在于提供一种核酸分子。
本发明第四个方面的目的，在于提供一种载体。
本发明第五个方面的目的，在于提供一种重组细胞。
本发明第六个方面的目的，在于提供上述的内含肽突变体或上述的融合蛋白或上述的核酸分子或上述的载体或上述的重组细胞在蛋白表达、纯化中的应用。
本发明第七个方面的目的，在于提供制备目的分子的方法。
本发明所采取的技术方案是：
本发明的第一方面，提供一种内含肽突变体，所述内含肽突变体的氨基酸序列为：
(a)SEQ ID NO.1；或
(b)与SEQ ID NO.1所示的氨基酸序列相比具有90％以上序列一致性的氨基酸序列，且具有(a)所限定的氨基酸序列所具有的功能。
本发明的第二方面，提供一种融合蛋白，包括本发明第一方面所述的内含肽突变体。
在本发明的一些实施方式中，所述融合蛋白还包括目的分子。
在本发明的一些实施方式中，所述目的分子为寡肽、多肽或者大分子蛋白。
在本发明的一些优选实施方式中，所述大分子蛋白为人源性弹性蛋白。
在本发明的一些实施方式中，所述融合蛋白还包括纯化标签。
在本发明的一些实施方式中，所述纯化标签为亲和型标签。
在本发明的一些优选实施方式中，所述纯化标签：His-tag、Flag-tag、c-Myc-tag、HA-tag、SNAP-tag、Halo-Tag、Spy-Tag、SUMO-Tag、GST-tag等。
本发明的第三方面，提供一种核酸分子，所述核酸分子编码本申请第一方面所述的内含肽突变体或本发明第二方面所述的融合蛋白。
本发明的第四方面，提供一种载体，包含本发明第三方面所述的核酸分子。
本发明的第五方面，提供一种重组细胞，包含本发明第三方面所述的载体，所述细胞非植物细胞或动物细胞。
在本发明的一些实施方式中，所述细胞为原核生物或真核生物。
在本发明的一些实施方式中，所述原核生物为大肠杆菌。
本发明的第六方面，提供本发明第一方面所述的内含肽突变体或本发明第二方面所述的融合蛋白或本发明第三方面所述的核酸分子或本发明第四方面所述的载体或本发明第五方面所述的重组细胞在合成目的蛋白的应用。
本发明的第七方面，提供制备目的分子的方法，所述方法包括以下步骤：
S1：培养本发明第五方面所述的重组细胞诱导表达所述融合蛋白；
S2：收集步骤S1获得的重组细胞，破碎细胞后离心得上清液；
S3：将上清液纯化得融合蛋白后进一步切割得所述目的分子。
在本发明的一些实施方式中，步骤S1中所述诱导表达的条件为：当重组细胞培养至OD600值为0.4-0.8时，加入IPTG至终浓度为0.1-2mM后16-37℃100-250rpm继续培养4-6h。
在本发明的一些实施方式中，步骤S2中所述收集步骤S1获得的重组细胞的重组条件为：8000-13000rpm，3～6℃离心5-30min。
在本发明的一些实施方式中，步骤S2中所述离心条件为10000-15000rpm，3～6℃离心10-30min。
在本发明的一些实施方式中，步骤S2中离心后进一步过滤，优选为使用0.4～0.5μm滤膜过滤。
在本发明的一些实施方式中，所述破碎菌体为压力或超声破碎。
在本发明的一些实施方式中，所述纯化为亲和层析。
在本发明的一些实施方式中，所述亲和层析柱为Ni-NTA亲和层析柱。
在本发明的一些实施方式中，所述切割的条件为：调节pH至6～7使得所述目的分子从所述融合蛋白被切割。
在本发明的一些实施方式中，所述切割的时间为8～20h。
在本发明的一些实施方式中，所述细胞为细菌；优选为大肠杆菌。
本发明的有益效果是：
本发明提供了一种Chl DnaB内含肽突变体，氨基酸序列如SEQ ID NO.1所示；本发明还提供使用Chl DnaB内含肽突变体来制备和纯化目的分子的方法，使用Chl DnaB内含肽变体(SEQ ID NO.1)和人源原弹性蛋白(SEQ ID NO.3)融合成高效表达的融合蛋白(SEQ IDNO.4)，并构建到表达载体pET-28a上，重组载体核苷酸序列见SEQ ID NO.4。利用大肠杆菌BL21(DE3)作为表达宿主，获得了高效表达原弹性蛋白的重组菌株。并通过Ni-NTA亲和层析获得高纯度的原弹性蛋白。利用内含肽的自切割效果，得到了完整的人源原弹性蛋白。可以有效解决人源原弹性蛋白原核表达存在的问题，提高人源原弹性蛋白的产能；这在食品、医疗、美容和护肤品等领域具有重要的使用价值。
附图说明
图1为PJH75770.1与传统Ssp DnaB的序列比对；上面序列为PJH75770.1的氨基酸序列，下面为Ssp DnaB的氨基酸序列。
图2为Chl DnaB与传统Ssp DnaB的三级结构预测；灰色为Chl DnaB，黑色为SspDnaB的氨基酸序列。
图3为包含内含肽Chl DnaB的pET28a-Chl DnaB-ELN表达质粒图谱。
图4为不包含内含肽Chl DnaB的pET28a-ELN表达质粒图谱。
图5为SDS-PAGE检测ELN蛋白肽链融合蛋白的表达。
图6为6his抗体检测ELN蛋白肽链融合蛋白的表达水平。
图7为SDS-PAGE检测亲和层析纯化Chl DnaB-ELN肽链融合蛋白。
图8为6his抗体检测亲和层析纯化Chl DnaB-ELN肽链融合蛋白。
图9为SDS-PAGE检测Chl DnaB自切割效果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例1内含肽Chl DnaB变体的构建
申请人利用生物信息学技术分析了GenBank:PJH75770.1的氨基酸序列，并与内含肽Ssp DnaB的氨基酸序列进行序列比对。结果见图1，在PJH75770.1的氨基酸序列中发现了与Ssp DnaB的氨基酸序列具有高度的同源性。同时，申请人利用AlphaFold2对PJH75770.1中的Chl DnaB和内含肽Ssp DnaB进行肽段三级结构模拟分析，结果见图2，Chl DnaB与内含肽Ssp DnaB在三级结构上高度相似，因此申请人截取GenBank:PJH75770.1氨基酸序列410-561片段即为内含肽的序列，取得了内含肽截短体(SEQ ID NO.2)；申请人进一步将ChlDnaB的N端半胱氨酸突变成丙氨酸(SEQ ID NO.1)，因为内含肽N端的半胱氨酸C对N端切割活性是必须的，但并不适用于本申请，因此将其进一步突变成无活性的丙氨酸；从而去除内含肽N端半胱氨酸依赖性的蛋白酶活性，并应用于后续的蛋白表达和纯化应用。
SEQ ID NO.2：
CLTGDALVAVADSGRNVPIRELEGKSNFNIWALNPDTLKMESMSVSRAFCTGKKSVFKIKTRLGREIRATANHQFLTFNGWKRLDELTTSDYLALPRILPTVVQSSELTTLAESDIYWDTILSIEPDGEEQVYDLTVPGHHNFVANNIIVHN；
SEQ ID NO.1：
ALTGDALVAVADSGRNVPIRELEGKSNFNIWALNPDTLKMESMSVSRAFCTGKKSVFKIKTRLGREIRATANHQFLTFNGWKRLDELTTSDYLALPRILPTVVQSSELTTLAESDIYWDTILSIEPDGEEQVYDLTVPGHHNFVANNIIVHN。
实施例2内含肽Chl DnaB对人源弹性蛋白肽链表达的影响。
在本实施例中，比较了包含内含肽Chl DnaB变体(SEQ ID NO.1)和不包含ChlDnaB对ELN蛋白肽链表达的影响。其中使用Chl DnaB内含肽变体(SEQ ID NO.1)和人源原弹性蛋白ELN(SEQ ID NO.3)融合成高效表达的融合蛋白(SEQ ID NO.4)；并构建到表达载体pET-28a上，重组载体核苷酸序列见SEQ ID NO.5；载体图谱见图3；不包含Chl DnaB的pET28a-ELN表达载体的载体图谱见图4。将表达载体转化到大肠杆菌BL21(DE3)中，挑取单克隆进行鉴定。鉴定成功后的表达菌株培养至OD600达到0.6-0.8后，加入终浓度为1mM的IPTG进行诱导表达。表达后的菌体经10000rpm/min离心富集后，进行SDS-PAGE电泳检测蛋白表达水平。
SEQ ID NO.3：
MAGLTAAAPRPGVLLLLLSILHPSRPGGVPGAIPGGVPGGVFYPGAGLGALGGGALGPGGKPLKPVPGGLAGAGLGAGLGAFPAVTFPGALVPGGVADAAAAYKAAKAGAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGVGTPAAAAAKAAAKAAQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGVAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK。
SEQ ID NO.4：
MGSSHHHHHHALTGDALVAVADSGRNVPIRELEGKSNFNIWALNPDTLKMESMSVSRAFCTGKKSVFKIKTRLGREIRATANHQFLTFNGWKRLDELTTSDYLALPRILPTVVQSSELTTLAESDIYWDTILSIEPDGEEQVYDLTVPGHHNFVANNIIVHNMAGLTAAAPRPGVLLLLLSILHPSRPGGVPGAIPGGVPGGVFYPGAGLGALGGGALGPGGKPLKPVPGGLAGAGLGAGLGAFPAVTFPGALVPGGVADAAAAYKAAKAGAGLGGVPGVGGLGVSAGAVVPQPGAGVKPGKVPGVGLPGVYPGGVLPGARFPGVGVLPGVPTGAGVKPKAPGVGGAFAGIPGVGPFGGPQPGVPLGYPIKAPKLPGGYGLPYTTGKLPYGYGPGGVAGAAGKAGYPTGTGVGPQAAAAAAAKAAAKFGAGAAGVLPGVGGAGVPGVPGAIPGIGGIAGVGTPAAAAAAAAAAKAAKYGAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIPVVPGAGIPGAAVPGVVSPEAAAKAAAKAAKYGARPGVGVGGIPTYGVGAGGFPGFGVGVGGIPGVAGVPGVGGVPGVGGVPGVGISPEAQAAAAAKAAKYGVGTPAAAAAKAAAKAAQFGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGVAAAAKSAAKVAAKAQLRAAAGLGAGIPGLGVGVGVPGLGVGAGVPGLGVGAGVPGFGAVPGALAAAKAAKYGAAVPGVLGGLGALGGVGIPGGVVGAGPAAAAAAAKAAAKAAQFGLVGAAGLGGLGVGGLGVPGVGGLGGIPPAAAAKAAKYGAAGLGGVLGGAGQFPLGGVAARPGFGLSPIFPGGACLGKACGRKRK。
SEQ ID NO.5：
tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccattcatgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaaccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatataccatgggcagcagccatcatcatcatcatcacGCTCTAACAGGAGATGCATTAGTTGCGGTAGCTGATAGCGGTCGCAACGTGCCGATTCGC GAGTTGGAGGGCAAGTCTAATTTCAACATCTGGGCATTAAATCCGGACACCCTGAAAATGGAATCGATGAGCGTGA GCCGTGCGTTCTGCACCGGTAAGAAGTCCGTTTTTAAGATCAAAACCCGTCTGGGTCGCGAGATCCGTGCGACCGC AAACCACCAGTTTCTGACGTTTAATGGCTGGAAACGTCTGGATGAGTTGACGACCAGCGACTACCTCGCGCTGCCA CGTATTCTGCCGACCGTGGTTCAATCCAGCGAATTGACGACTCTGGCCGAATCTGACATCTACTGGGATACCATCC TGAGCATTGAACCGGACGGCGAAGAGCAGGTTTATGATCTGACCGTCCCGGGTCACCATAATTTCGTGGCGAACAA CATCATTGTTCATAACatggcgggtctgacggcggcggccccgcggcccggagtcctcctgctcctgctgtccatc ctccacccctctcggcctggaggggtccctggggccattcctggtggagttcctggaggagtcttttatccagggg ctggtctcggagcccttggaggaggagcgctggggcctggaggcaaacctcttaagccagttcccggagggcttgc gggtgctggccttggggcagggctcggcgccttccccgcagttacctttccgggggctctggtgcctggtggagtg gctgacgctgctgcagcctataaagctgctaaggctggcgctgggcttggtggtgtcccaggagttggtggcttag gagtgtctgcaggtgcggtggttcctcagcctggagccggagtgaagcctgggaaagtgccgggtgtggggctgcc aggtgtatacccaggtggcgtgctcccaggagctcggttccccggtgtgggggtgctccctggagttcccactgga gcaggagttaagcccaaggctccaggtgtaggtggagcttttgctggaatcccaggagttggaccctttgggggac cgcaacctggagtcccactggggtatcccatcaaggcccccaagctgcctggtggctatggactgccctacaccac agggaaactgccctatggctatgggcccggaggagtggctggtgcagcgggcaaggctggttacccaacagggaca ggggttggcccccaggcagcagcagcagcggcagctaaagcagcagcaaagttcggtgctggagcagccggagtcc tccctggtgttggaggggctggtgttcctggcgtgcctggggcaattcctggaattggaggcatcgcaggcgttgg gactccagctgcagctgcagctgcagcagcagccgctaaggcagccaagtatggagctgctgcaggcttagtgcct ggtgggccaggctttggcccgggagtagttggtgtcccaggagctggcgttccaggtgttggtgtcccaggagctg ggattccagttgtcccaggtgctgggatcccaggtgctgcggttccaggggttgtgtcaccagaagcagctgctaa ggcagctgcaaaggcagccaaatacggggccaggcccggagtcggagttggaggcattcctacttacggggttgga gctgggggctttcccggctttggtgtcggagtcggaggtatccctggagtcgcaggtgtccctggtgtcggaggtg ttcccggagtcggaggtgtcccgggagttggcatttcccccgaagctcaggcagcagctgccgccaaggctgccaa gtacggagtggggaccccagcagctgcagctgctaaagcagccgccaaagccgcccagtttgggttagttcctggt gtcggcgtggctcctggagttggcgtggctcctggtgtcggtgtggctcctggagttggcttggctcctggagttg gcgtggctcctggagttggtgtggctcctggcgttggcgtggctcccggcattggccctggtggagttgcagctgc agcaaaatccgctgccaaggtggctgccaaagcccagctccgagctgcagctgggcttggtgctggcatccctgga cttggagttggtgtcggcgtccctggacttggagttggtgctggtgttcctggacttggagttggtgctggtgttc ctggcttcggggcagtacctggagccctggctgccgctaaagcagccaaatatggagcagcagtgcctggggtcct tggagggctcggggctctcggtggagtaggcatcccaggcggtgtggtgggagccggacccgccgccgccgctgcc gcagccaaagctgctgccaaagccgcccagtttggcctagtgggagccgctgggctcggaggactcggagtcggag ggcttggagttccaggtgttgggggccttggaggtatacctccagctgcagccgctaaagcagctaaatacggtgc tgctggccttggaggtgtcctagggggtgccgggcagttcccacttggaggagtggcagcaagacctggcttcgga ttgtctcccattttcccaggtggggcctgcctggggaaagcttgtggccggaagagaaaaTAAgctagcatgactg gtggacagcaaatgggtcgcggatccgaattcgagctccgtcgacaagcttgcggccgcactcgagcaccaccacc accaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat(下划线表示融合蛋白对应的核苷酸序列)。
结果见图5，ELN肽链的理论分子量在62kDa，融合Chl DnaB内含肽的ELN肽链理论分子量为80kDa，不包含Chl DnaB内含肽的pET28a-ELN肽链检测不到ELN肽链的表达条带，但包含Chl DnaB的pET28a-Chl DnaB-ELN肽链融合蛋白的表达载体菌株检测出明显的表达条带，随后进行的Western blot验证，Chl DnaB-ELN肽链融合蛋白的表达产量约为ELN肽链表达产量的100倍(见图6)。这说明融合Chl DnaB能够有效提高ELN肽链的原核表达能力。
实施例3 Chl DnaB-ELN肽链融合蛋白的纯化
在本实施例中，进一步说明Chl DnaB-ELN肽链融合蛋白的纯化方法。具体实施方式如下：
1)诱导表达：将构建好的质粒转化到BL21(DE3)表达菌株中，挑取单克隆菌株至LB培养基中扩大培养中OD600值为0.8，加入1/20体积1M Tris-HCl缓冲液(pH 8.5)和终浓度为1mM的IPTG，37度200rpm继续培养4-6h。10000rpm，4度离心20min，收集菌体。菌体用PBS清洗2次。
2)菌体破碎：将菌体重悬在裂解缓冲液(20mM Tris，500mM NaCl，pH 8.0)中，使用600Mpa压力进行菌体破碎，镜检染色直至没有明显的菌体。12000rpm，4度离心20min，收集上清，0.45μm滤膜过滤。
3)亲和层析：Ni-NTA亲和层析柱用20倍柱体积裂解缓冲液充分平衡后，加入过滤后的细菌裂解缓冲液，0.5ml/min流速，再用5倍柱体积含40mM咪唑裂解缓冲液充分清洗。再用5倍柱体积含200mM咪唑裂解缓冲液洗脱目的蛋白。
4)蛋白检测：分别使用SDS-PAGE和Western blot检测纯化的目的蛋白。使用BCA法对纯化的蛋白进行定量。
纯化结果见图7和图8，Ni-NTA树脂亲和层析可以高效地纯化6his-Chl DnaB-ELN肽链融合蛋白。但可以看到的是，ELN蛋白肽链在纯化后发生轻微的降解(如图8)，这可能是由于ELN蛋白肽链在原核表达过程中无法被羟基化修饰，导致ELN蛋白容易发生降解。通过BCA定量，检测出ELN肽链融合蛋白的产能为0.2g/L。
实施例4 Chl DnaB内含肽切割和ELN肽链纯蛋白获取
在本实施例中，申请人进行了Chl DnaB的切割条件和ELN肽链纯蛋白的获取。具体实施方式如下：将实施例3得到的纯化的蛋白进行12000rpm 10min离心超滤除去咪唑，再使用盐酸将pH调至6.5，室温切割过夜后，SDS-PAGE检测切割效果。
结果如图9，Chl DnaB内含肽可以进行高效的自切割，释放出完整的ELN肽链蛋白。
综述，本发明提供了一种高效表达和切割的内含肽变体序列，且在人源原弹性蛋白肽链的原核表达及纯化上发挥重要的作用，具有很高的商业价值。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 广州市乾相生物科技有限公司
<120> 一种新内含肽及其在合成人源原弹性蛋白上的应用
<130>
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 152
<212> PRT
<213> 人工序列
<400> 1
Ala Leu Thr Gly Asp Ala Leu Val Ala Val Ala Asp Ser Gly Arg Asn
1 5 10 15
Val Pro Ile Arg Glu Leu Glu Gly Lys Ser Asn Phe Asn Ile Trp Ala
20 25 30
Leu Asn Pro Asp Thr Leu Lys Met Glu Ser Met Ser Val Ser Arg Ala
35 40 45
Phe Cys Thr Gly Lys Lys Ser Val Phe Lys Ile Lys Thr Arg Leu Gly
50 55 60
Arg Glu Ile Arg Ala Thr Ala Asn His Gln Phe Leu Thr Phe Asn Gly
65 70 75 80
Trp Lys Arg Leu Asp Glu Leu Thr Thr Ser Asp Tyr Leu Ala Leu Pro
85 90 95
Arg Ile Leu Pro Thr Val Val Gln Ser Ser Glu Leu Thr Thr Leu Ala
100 105 110
Glu Ser Asp Ile Tyr Trp Asp Thr Ile Leu Ser Ile Glu Pro Asp Gly
115 120 125
Glu Glu Gln Val Tyr Asp Leu Thr Val Pro Gly His His Asn Phe Val
130 135 140
Ala Asn Asn Ile Ile Val His Asn
145 150
<210> 2
<211> 152
<212> PRT
<213> 人工序列
<400> 2
Cys Leu Thr Gly Asp Ala Leu Val Ala Val Ala Asp Ser Gly Arg Asn
1 5 10 15
Val Pro Ile Arg Glu Leu Glu Gly Lys Ser Asn Phe Asn Ile Trp Ala
20 25 30
Leu Asn Pro Asp Thr Leu Lys Met Glu Ser Met Ser Val Ser Arg Ala
35 40 45
Phe Cys Thr Gly Lys Lys Ser Val Phe Lys Ile Lys Thr Arg Leu Gly
50 55 60
Arg Glu Ile Arg Ala Thr Ala Asn His Gln Phe Leu Thr Phe Asn Gly
65 70 75 80
Trp Lys Arg Leu Asp Glu Leu Thr Thr Ser Asp Tyr Leu Ala Leu Pro
85 90 95
Arg Ile Leu Pro Thr Val Val Gln Ser Ser Glu Leu Thr Thr Leu Ala
100 105 110
Glu Ser Asp Ile Tyr Trp Asp Thr Ile Leu Ser Ile Glu Pro Asp Gly
115 120 125
Glu Glu Gln Val Tyr Asp Leu Thr Val Pro Gly His His Asn Phe Val
130 135 140
Ala Asn Asn Ile Ile Val His Asn
145 150
<210> 3
<211> 724
<212> PRT
<213> 人工序列
<400> 3
Met Ala Gly Leu Thr Ala Ala Ala Pro Arg Pro Gly Val Leu Leu Leu
1 5 10 15
Leu Leu Ser Ile Leu His Pro Ser Arg Pro Gly Gly Val Pro Gly Ala
20 25 30
Ile Pro Gly Gly Val Pro Gly Gly Val Phe Tyr Pro Gly Ala Gly Leu
35 40 45
Gly Ala Leu Gly Gly Gly Ala Leu Gly Pro Gly Gly Lys Pro Leu Lys
50 55 60
Pro Val Pro Gly Gly Leu Ala Gly Ala Gly Leu Gly Ala Gly Leu Gly
65 70 75 80
Ala Phe Pro Ala Val Thr Phe Pro Gly Ala Leu Val Pro Gly Gly Val
85 90 95
Ala Asp Ala Ala Ala Ala Tyr Lys Ala Ala Lys Ala Gly Ala Gly Leu
100 105 110
Gly Gly Val Pro Gly Val Gly Gly Leu Gly Val Ser Ala Gly Ala Val
115 120 125
Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro Gly Val
130 135 140
Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala Arg Phe
145 150 155 160
Pro Gly Val Gly Val Leu Pro Gly Val Pro Thr Gly Ala Gly Val Lys
165 170 175
Pro Lys Ala Pro Gly Val Gly Gly Ala Phe Ala Gly Ile Pro Gly Val
180 185 190
Gly Pro Phe Gly Gly Pro Gln Pro Gly Val Pro Leu Gly Tyr Pro Ile
195 200 205
Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly Leu Pro Tyr Thr Thr Gly
210 215 220
Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly Val Ala Gly Ala Ala Gly
225 230 235 240
Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val Gly Pro Gln Ala Ala Ala
245 250 255
Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe Gly Ala Gly Ala Ala Gly
260 265 270
Val Leu Pro Gly Val Gly Gly Ala Gly Val Pro Gly Val Pro Gly Ala
275 280 285
Ile Pro Gly Ile Gly Gly Ile Ala Gly Val Gly Thr Pro Ala Ala Ala
290 295 300
Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Ala
305 310 315 320
Gly Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val Val Gly Val
325 330 335
Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ile Pro
340 345 350
Val Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro Gly Val Val
355 360 365
Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys Tyr Gly
370 375 380
Ala Arg Pro Gly Val Gly Val Gly Gly Ile Pro Thr Tyr Gly Val Gly
385 390 395 400
Ala Gly Gly Phe Pro Gly Phe Gly Val Gly Val Gly Gly Ile Pro Gly
405 410 415
Val Ala Gly Val Pro Gly Val Gly Gly Val Pro Gly Val Gly Gly Val
420 425 430
Pro Gly Val Gly Ile Ser Pro Glu Ala Gln Ala Ala Ala Ala Ala Lys
435 440 445
Ala Ala Lys Tyr Gly Val Gly Thr Pro Ala Ala Ala Ala Ala Lys Ala
450 455 460
Ala Ala Lys Ala Ala Gln Phe Gly Leu Val Pro Gly Val Gly Val Ala
465 470 475 480
Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly
485 490 495
Leu Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly
500 505 510
Val Gly Val Ala Pro Gly Ile Gly Pro Gly Gly Val Ala Ala Ala Ala
515 520 525
Lys Ser Ala Ala Lys Val Ala Ala Lys Ala Gln Leu Arg Ala Ala Ala
530 535 540
Gly Leu Gly Ala Gly Ile Pro Gly Leu Gly Val Gly Val Gly Val Pro
545 550 555 560
Gly Leu Gly Val Gly Ala Gly Val Pro Gly Leu Gly Val Gly Ala Gly
565 570 575
Val Pro Gly Phe Gly Ala Val Pro Gly Ala Leu Ala Ala Ala Lys Ala
580 585 590
Ala Lys Tyr Gly Ala Ala Val Pro Gly Val Leu Gly Gly Leu Gly Ala
595 600 605
Leu Gly Gly Val Gly Ile Pro Gly Gly Val Val Gly Ala Gly Pro Ala
610 615 620
Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Gln Phe Gly
625 630 635 640
Leu Val Gly Ala Ala Gly Leu Gly Gly Leu Gly Val Gly Gly Leu Gly
645 650 655
Val Pro Gly Val Gly Gly Leu Gly Gly Ile Pro Pro Ala Ala Ala Ala
660 665 670
Lys Ala Ala Lys Tyr Gly Ala Ala Gly Leu Gly Gly Val Leu Gly Gly
675 680 685
Ala Gly Gln Phe Pro Leu Gly Gly Val Ala Ala Arg Pro Gly Phe Gly
690 695 700
Leu Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly Lys Ala Cys Gly
705 710 715 720
Arg Lys Arg Lys
<210> 4
<211> 886
<212> PRT
<213> 人工序列
<400> 4
Met Gly Ser Ser His His His His His His Ala Leu Thr Gly Asp Ala
1 5 10 15
Leu Val Ala Val Ala Asp Ser Gly Arg Asn Val Pro Ile Arg Glu Leu
20 25 30
Glu Gly Lys Ser Asn Phe Asn Ile Trp Ala Leu Asn Pro Asp Thr Leu
35 40 45
Lys Met Glu Ser Met Ser Val Ser Arg Ala Phe Cys Thr Gly Lys Lys
50 55 60
Ser Val Phe Lys Ile Lys Thr Arg Leu Gly Arg Glu Ile Arg Ala Thr
65 70 75 80
Ala Asn His Gln Phe Leu Thr Phe Asn Gly Trp Lys Arg Leu Asp Glu
85 90 95
Leu Thr Thr Ser Asp Tyr Leu Ala Leu Pro Arg Ile Leu Pro Thr Val
100 105 110
Val Gln Ser Ser Glu Leu Thr Thr Leu Ala Glu Ser Asp Ile Tyr Trp
115 120 125
Asp Thr Ile Leu Ser Ile Glu Pro Asp Gly Glu Glu Gln Val Tyr Asp
130 135 140
Leu Thr Val Pro Gly His His Asn Phe Val Ala Asn Asn Ile Ile Val
145 150 155 160
His Asn Met Ala Gly Leu Thr Ala Ala Ala Pro Arg Pro Gly Val Leu
165 170 175
Leu Leu Leu Leu Ser Ile Leu His Pro Ser Arg Pro Gly Gly Val Pro
180 185 190
Gly Ala Ile Pro Gly Gly Val Pro Gly Gly Val Phe Tyr Pro Gly Ala
195 200 205
Gly Leu Gly Ala Leu Gly Gly Gly Ala Leu Gly Pro Gly Gly Lys Pro
210 215 220
Leu Lys Pro Val Pro Gly Gly Leu Ala Gly Ala Gly Leu Gly Ala Gly
225 230 235 240
Leu Gly Ala Phe Pro Ala Val Thr Phe Pro Gly Ala Leu Val Pro Gly
245 250 255
Gly Val Ala Asp Ala Ala Ala Ala Tyr Lys Ala Ala Lys Ala Gly Ala
260 265 270
Gly Leu Gly Gly Val Pro Gly Val Gly Gly Leu Gly Val Ser Ala Gly
275 280 285
Ala Val Val Pro Gln Pro Gly Ala Gly Val Lys Pro Gly Lys Val Pro
290 295 300
Gly Val Gly Leu Pro Gly Val Tyr Pro Gly Gly Val Leu Pro Gly Ala
305 310 315 320
Arg Phe Pro Gly Val Gly Val Leu Pro Gly Val Pro Thr Gly Ala Gly
325 330 335
Val Lys Pro Lys Ala Pro Gly Val Gly Gly Ala Phe Ala Gly Ile Pro
340 345 350
Gly Val Gly Pro Phe Gly Gly Pro Gln Pro Gly Val Pro Leu Gly Tyr
355 360 365
Pro Ile Lys Ala Pro Lys Leu Pro Gly Gly Tyr Gly Leu Pro Tyr Thr
370 375 380
Thr Gly Lys Leu Pro Tyr Gly Tyr Gly Pro Gly Gly Val Ala Gly Ala
385 390 395 400
Ala Gly Lys Ala Gly Tyr Pro Thr Gly Thr Gly Val Gly Pro Gln Ala
405 410 415
Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Phe Gly Ala Gly Ala
420 425 430
Ala Gly Val Leu Pro Gly Val Gly Gly Ala Gly Val Pro Gly Val Pro
435 440 445
Gly Ala Ile Pro Gly Ile Gly Gly Ile Ala Gly Val Gly Thr Pro Ala
450 455 460
Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Ala
465 470 475 480
Ala Ala Gly Leu Val Pro Gly Gly Pro Gly Phe Gly Pro Gly Val Val
485 490 495
Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly
500 505 510
Ile Pro Val Val Pro Gly Ala Gly Ile Pro Gly Ala Ala Val Pro Gly
515 520 525
Val Val Ser Pro Glu Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Lys
530 535 540
Tyr Gly Ala Arg Pro Gly Val Gly Val Gly Gly Ile Pro Thr Tyr Gly
545 550 555 560
Val Gly Ala Gly Gly Phe Pro Gly Phe Gly Val Gly Val Gly Gly Ile
565 570 575
Pro Gly Val Ala Gly Val Pro Gly Val Gly Gly Val Pro Gly Val Gly
580 585 590
Gly Val Pro Gly Val Gly Ile Ser Pro Glu Ala Gln Ala Ala Ala Ala
595 600 605
Ala Lys Ala Ala Lys Tyr Gly Val Gly Thr Pro Ala Ala Ala Ala Ala
610 615 620
Lys Ala Ala Ala Lys Ala Ala Gln Phe Gly Leu Val Pro Gly Val Gly
625 630 635 640
Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly
645 650 655
Val Gly Leu Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala
660 665 670
Pro Gly Val Gly Val Ala Pro Gly Ile Gly Pro Gly Gly Val Ala Ala
675 680 685
Ala Ala Lys Ser Ala Ala Lys Val Ala Ala Lys Ala Gln Leu Arg Ala
690 695 700
Ala Ala Gly Leu Gly Ala Gly Ile Pro Gly Leu Gly Val Gly Val Gly
705 710 715 720
Val Pro Gly Leu Gly Val Gly Ala Gly Val Pro Gly Leu Gly Val Gly
725 730 735
Ala Gly Val Pro Gly Phe Gly Ala Val Pro Gly Ala Leu Ala Ala Ala
740 745 750
Lys Ala Ala Lys Tyr Gly Ala Ala Val Pro Gly Val Leu Gly Gly Leu
755 760 765
Gly Ala Leu Gly Gly Val Gly Ile Pro Gly Gly Val Val Gly Ala Gly
770 775 780
Pro Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Gln
785 790 795 800
Phe Gly Leu Val Gly Ala Ala Gly Leu Gly Gly Leu Gly Val Gly Gly
805 810 815
Leu Gly Val Pro Gly Val Gly Gly Leu Gly Gly Ile Pro Pro Ala Ala
820 825 830
Ala Ala Lys Ala Ala Lys Tyr Gly Ala Ala Gly Leu Gly Gly Val Leu
835 840 845
Gly Gly Ala Gly Gln Phe Pro Leu Gly Gly Val Ala Ala Arg Pro Gly
850 855 860
Phe Gly Leu Ser Pro Ile Phe Pro Gly Gly Ala Cys Leu Gly Lys Ala
865 870 875 880
Cys Gly Arg Lys Arg Lys
885
<210> 5
<211> 7967
<212> DNA
<213> 人工序列
<400> 5
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600
tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660
actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720
gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780
aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840
agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900
cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960
aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020
tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080
tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140
taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200
ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260
tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320
tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380
cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100
gctctaacag gagatgcatt agttgcggta gctgatagcg gtcgcaacgt gccgattcgc 5160
gagttggagg gcaagtctaa tttcaacatc tgggcattaa atccggacac cctgaaaatg 5220
gaatcgatga gcgtgagccg tgcgttctgc accggtaaga agtccgtttt taagatcaaa 5280
acccgtctgg gtcgcgagat ccgtgcgacc gcaaaccacc agtttctgac gtttaatggc 5340
tggaaacgtc tggatgagtt gacgaccagc gactacctcg cgctgccacg tattctgccg 5400
accgtggttc aatccagcga attgacgact ctggccgaat ctgacatcta ctgggatacc 5460
atcctgagca ttgaaccgga cggcgaagag caggtttatg atctgaccgt cccgggtcac 5520
cataatttcg tggcgaacaa catcattgtt cataacatgg cgggtctgac ggcggcggcc 5580
ccgcggcccg gagtcctcct gctcctgctg tccatcctcc acccctctcg gcctggaggg 5640
gtccctgggg ccattcctgg tggagttcct ggaggagtct tttatccagg ggctggtctc 5700
ggagcccttg gaggaggagc gctggggcct ggaggcaaac ctcttaagcc agttcccgga 5760
gggcttgcgg gtgctggcct tggggcaggg ctcggcgcct tccccgcagt tacctttccg 5820
ggggctctgg tgcctggtgg agtggctgac gctgctgcag cctataaagc tgctaaggct 5880
ggcgctgggc ttggtggtgt cccaggagtt ggtggcttag gagtgtctgc aggtgcggtg 5940
gttcctcagc ctggagccgg agtgaagcct gggaaagtgc cgggtgtggg gctgccaggt 6000
gtatacccag gtggcgtgct cccaggagct cggttccccg gtgtgggggt gctccctgga 6060
gttcccactg gagcaggagt taagcccaag gctccaggtg taggtggagc ttttgctgga 6120
atcccaggag ttggaccctt tgggggaccg caacctggag tcccactggg gtatcccatc 6180
aaggccccca agctgcctgg tggctatgga ctgccctaca ccacagggaa actgccctat 6240
ggctatgggc ccggaggagt ggctggtgca gcgggcaagg ctggttaccc aacagggaca 6300
ggggttggcc cccaggcagc agcagcagcg gcagctaaag cagcagcaaa gttcggtgct 6360
ggagcagccg gagtcctccc tggtgttgga ggggctggtg ttcctggcgt gcctggggca 6420
attcctggaa ttggaggcat cgcaggcgtt gggactccag ctgcagctgc agctgcagca 6480
gcagccgcta aggcagccaa gtatggagct gctgcaggct tagtgcctgg tgggccaggc 6540
tttggcccgg gagtagttgg tgtcccagga gctggcgttc caggtgttgg tgtcccagga 6600
gctgggattc cagttgtccc aggtgctggg atcccaggtg ctgcggttcc aggggttgtg 6660
tcaccagaag cagctgctaa ggcagctgca aaggcagcca aatacggggc caggcccgga 6720
gtcggagttg gaggcattcc tacttacggg gttggagctg ggggctttcc cggctttggt 6780
gtcggagtcg gaggtatccc tggagtcgca ggtgtccctg gtgtcggagg tgttcccgga 6840
gtcggaggtg tcccgggagt tggcatttcc cccgaagctc aggcagcagc tgccgccaag 6900
gctgccaagt acggagtggg gaccccagca gctgcagctg ctaaagcagc cgccaaagcc 6960
gcccagtttg ggttagttcc tggtgtcggc gtggctcctg gagttggcgt ggctcctggt 7020
gtcggtgtgg ctcctggagt tggcttggct cctggagttg gcgtggctcc tggagttggt 7080
gtggctcctg gcgttggcgt ggctcccggc attggccctg gtggagttgc agctgcagca 7140
aaatccgctg ccaaggtggc tgccaaagcc cagctccgag ctgcagctgg gcttggtgct 7200
ggcatccctg gacttggagt tggtgtcggc gtccctggac ttggagttgg tgctggtgtt 7260
cctggacttg gagttggtgc tggtgttcct ggcttcgggg cagtacctgg agccctggct 7320
gccgctaaag cagccaaata tggagcagca gtgcctgggg tccttggagg gctcggggct 7380
ctcggtggag taggcatccc aggcggtgtg gtgggagccg gacccgccgc cgccgctgcc 7440
gcagccaaag ctgctgccaa agccgcccag tttggcctag tgggagccgc tgggctcgga 7500
ggactcggag tcggagggct tggagttcca ggtgttgggg gccttggagg tatacctcca 7560
gctgcagccg ctaaagcagc taaatacggt gctgctggcc ttggaggtgt cctagggggt 7620
gccgggcagt tcccacttgg aggagtggca gcaagacctg gcttcggatt gtctcccatt 7680
ttcccaggtg gggcctgcct ggggaaagct tgtggccgga agagaaaata agctagcatg 7740
actggtggac agcaaatggg tcgcggatcc gaattcgagc tccgtcgaca agcttgcggc 7800
cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga 7860
agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa 7920
acgggtcttg aggggttttt tgctgaaagg aggaactata tccggat 7967
Claims (8)
1.一种内含肽突变体，其特征在于，所述内含肽突变体的氨基酸序列为SEQ ID NO.1。
2.一种融合蛋白，其特征在于，所述融合蛋白由权利要求1所述的内含肽突变体和人源原弹性蛋白组成。
3.一种核酸分子，其特征在于，所述核酸分子编码权利要求1所述的内含肽突变体或权利要求2所述的融合蛋白。
4.一种载体，其特征在于，包含权利要求3所述的核酸分子。
5.一种重组细胞，其特征在于，包含权利要求4所述的载体。
6.权利要求1所述内含肽突变体或权利要求2所述融合蛋白或权利要求3所述核酸分子或权利要求4所述载体或权利要求5所述重组细胞在合成目的蛋白中的应用。
7.制备目的分子的方法，所述方法包括以下步骤：
S1：培养权利要求5所述的重组细胞诱导表达所述融合蛋白；
S2：收集步骤S1获得的重组细胞，破碎细胞后离心得上清液；
S3：将上清液纯化得融合蛋白，进一步切割得所述目的分子。
8.根据权利要求7所述的制备方法，其特征在于，所述切割的条件为：调节pH至6～7使得所述目的分子从所述融合蛋白被切割。
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| Spontaneous C-cleavage of a mini-intein without its conserved N-terminal motif A;Xingmei Qi等;《FEBS Letters》;20110705;第585卷(第2011期);第2513-2518页 * |
| Use of Ssp dnaB derived mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli;Ziyong Sun等;《Protein Expression and Purification》;20050608;第43卷(第2005期);第26-32页 * |
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