CN114262368B - 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 - Google Patents
一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 Download PDFInfo
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Abstract
一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法,属于生物工程技术领域。为了解决现有重组类胶原蛋白自组装成水凝胶速度快的问题,本发明提供了一种重组Scl2类胶原蛋白,该蛋白通过在原有重组类胶原蛋白氨基酸序列基础上进行点突变获得。由本发明获得的重组类胶原蛋白成胶时间与氨基酸突变的个数呈正相关,同样具备规模化生产、批次间差异小及在中性条件下即可自组装成自支撑的水凝胶等特点。此外,该系列蛋白自组装成水凝胶时间存在显著差异,可组成一系列可变速的自组装水凝胶。利用本发明方法制备的重组类胶原蛋白不仅为其在医学用品等方面的商品化应用提供更多的选择,而且为研究类胶原蛋白自组装成胶开辟新的研究思路。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法。
背景技术
胶原蛋白是动物结缔组织的主要成分,也是哺乳动物体内含量最多、分布最广的功能性蛋白(CN108659117A)。它约占人体总蛋白含量的三分之一,同时也是细胞外基质(ECM)的主要成分(CN101213308A)。目前,根据序列同源性及分子结构已在脊椎动物体内确定至少28种不同类型的胶原蛋白。尽管不同种类的胶原蛋白生物功能和物理特性差异巨大,但其均由三条由重复的Gly-Xaa-Yaa序列组成的多肽链以右手螺旋的形式构成。
在过去的几十年里,胶原蛋白在食品营养、化妆品、保健品、医学用品等方面发挥着重要作用(CN108057130A)(CN105709266A)(CN108472236A)。尤其作为医学材料,其在药物递送、组织移植等方面展现出的潜在价值,越来越受到人们关注(CN1210019A)。胶原蛋白主要来源于动物组织,存在着疯牛病等疾病风险,且不同批次间提取的蛋白存在差异,这使得以胶原蛋白为原料的应用受到一定的限制。此外,在酵母和植物表达系统中生产重组胶原蛋白,又必须在表达过程中引入脯氨酰4-羟化酶实现脯氨酸的羟化和重组胶原白的稳定,这大大增加规模化生产的难度(CN106256911A)。
在申请号为202110982807X的专利申请中,公开了不需要经过复杂改性方法就能自组装成一定强度水凝胶的重组细菌性类胶原蛋白。同时,其具备生物相容性良好、无免疫原性、无细胞毒性及能够在大肠杆菌中高效表达等优点。但是该重组蛋白自组装成水凝胶的时间很迅速,这限制其在生物医疗等方面的实际应用。因此,亟需开发出可变速的细菌性类胶原蛋白自组装水凝胶,使其为后续基于细菌性重组胶原蛋白的医用产品提供更多选择。
发明内容
本发明为了解决现有重组类胶原蛋白自组装成水凝胶速度快的问题,提供了一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法,具体技术方案如下:
本发明提供了一种重组Scl2类胶原蛋白,由胶原区域和非胶原区域组成,所述重组Scl2类胶原蛋白是在scl2类胶原蛋白的基础上将非胶原区域替换为如SEQ ID No. 1或SEQ ID No. 2或SEQ ID No. 3或SEQ ID No. 4或SEQ ID No. 5或SEQ ID No. 6或SEQ IDNo. 7任一所示的氨基酸序列,并将胶原区域替换为如SEQ ID No. 8或SEQ ID No. 9所示的氨基酸序列后获得的。
本发明还提供了一种上述重组Scl2类胶原蛋白的制备方法,包括如下步骤:
(1)将化脓链球菌(Streptococcus pyogenes)Scl2.28的cDNA序列转入载体Pet28a中,获得重组载体Pet28a-Scl2,以重组载体Pet28a-Scl2为模板,根据所述重组Scl2类胶原蛋白的突变位点,设计引物,扩增DNA片段,合成重组scl2类胶原蛋白的全序列,并以合成后的全序列为模板,PCR扩增合成DNA片段,再经酶切、连接、转化大肠杆菌获得重组的Scl2类胶原蛋白重组表达载体;
(2)将步骤(1)获得的Scl2类胶原蛋白重组表达载体转化至大肠杆菌中获得阳性菌株;
(3)将步骤(2)获得的阳性菌株接种于LB培养基中培养至OD600达到0.4~0.6时,向菌液中加入IPTG,继续培养5 h~6 h,对菌液进行离心或过滤后,去除上清并收集菌体沉淀;
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶,于冰上静置1 h,获得蛋白溶液,经超声后,通过离心或过滤收集上清获得粗蛋白;
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化,经水透析后获得重组后的类胶原蛋白。
进一步地限定,步骤(1)所述扩增DNA片段是将核苷酸序列如SEQ ID No. 10所示的引物Scl2-F分别与核苷酸序列如SEQ ID No. 11所示的引物Scl2-M1-1-R1、核苷酸序列如SEQ ID No. 12所示的引物Scl2-M1-2-R1、核苷酸序列如SEQ ID No. 13所示的引物Scl2--M1-3-R1、核苷酸序列如SEQ ID No. 14所示的引物Scl2-M2-1-R1、核苷酸序列如SEQID No. 15所示的引物Scl2-M2-2-R1、核苷酸序列如SEQ ID No. 16所示的引物Scl2-M2-3-R1、核苷酸序列如SEQ ID No. 17所示的引物Scl2-M3-1-R1混合进行DNA片段扩增,并将核苷酸序列如SEQ ID No. 18所示的引物Scl2-R分别与核苷酸序列如SEQ ID No. 19所示的引物Scl2-M1-1-F1、核苷酸序列如SEQ ID No. 20所示的引物Scl2-M1-2-F1、核苷酸序列如SEQ ID No. 21所示的引物Scl2--M1-3-F1、核苷酸序列如SEQ ID No. 22所示的引物Scl2-M2-1-F1、核苷酸序列如SEQ ID No. 23所示的引物Scl2-M2-2-F1、核苷酸序列如SEQ IDNo. 24所示的引物Scl2-M2-3-F1、核苷酸序列如SEQ ID No. 25所示的引物Scl2-M3-1-F1混合进行DNA片段扩增。
进一步地限定,步骤(1)所述以合成后的全序列为模板PCR扩增合成DNA片段所用上游引物为核苷酸序列如SEQ ID No. 10所示的Scl2-F,下游引物为核苷酸序列如SEQ IDNo. 18所示的Scl2-R。
进一步地限定,步骤(1)所述酶切是将扩增后的DNA片段及Pet28a空载体经过NcoI和Xho I限制性内切酶进行双酶切。
进一步地限定,步骤(1)所述连接使用的连接酶为T4 DNA连接酶;所述转化利用的大肠杆菌为DH5α。
进一步地限定,步骤(5)所述利用已装镍的重力柱进行纯化的方法是将步骤(4)获得的粗蛋白溶液加入已装镍的重力柱中,待镍柱无剩余粗蛋白溶液时,加入Buffer A进行杂蛋白的清洗,再加入Buffer B将目的蛋白洗脱下来。
进一步地限定,所述Buffer A的组成为50 mM Tris,250 mM NaCl和30 mM咪唑;所述Buffer B的组成为50 mM Tris,250 mM NaCl和400 mM咪唑。
进一步地限定,步骤(5)所述透析共进行2次,每次6 h。
本发明还提供了一种上述重组Scl2类胶原蛋白自组装可变速水凝胶的制备方法,是将所述重组类胶原蛋白与0.1~0.2 M的盐酸或醋酸混合,制成25~50 mg/mL的酸溶液,然后向酸溶液中加入2 M的NaOH溶液将pH调节至6~8,室温静置即可成胶。
本发明的有益效果:
本发明在申请号为202110982807X的专利申请公开的重组类胶原蛋白的基础上,通过点突变的方式改变原有重组类胶原蛋白的氨基酸序列,进而获得了一系列自组装成水凝胶速度不同的重组类胶原蛋白。新开发的重组类胶原蛋白成胶时间与氨基酸突变的个数呈正相关。该系列蛋白同样具备规模化生产、批次间差异小及在中性条件下即可自组装成自支撑的水凝胶等特点。此外,利用本发明方法制备的重组类胶原蛋白能够采用稀醋酸溶解,自组装成水凝胶的反应条件更加温和。更加难得的是,该系列蛋白自组装成水凝胶时间存在显著差异,组成一系列可变速的自组装水凝胶。总而言之,利用本发明方法制备的重组类胶原蛋白不仅为其在医学用品等方面的商品化应用提供更多的选择,而且为研究类胶原蛋白自组装成胶开辟新的研究思路。
附图说明
图1为Scl-M1-2水凝胶扫描电镜图;
图2为Scl-M2-2水凝胶扫描电镜图;
图3为Scl-M3-1水凝胶扫描电镜图;
图4为Scl-M1-2水凝胶图;
图5为Scl-M2-2水凝胶图;
图6为Scl-M3-1水凝胶图。
具体实施方式
以下结合具体实施例和附图,对本发明作进一步的详细说明,本发明所用试剂及设备等均可通过商业化途径购买获得,涉及的PCR扩增,酶切连接、转化等分子生物学实验操作如无特殊说明,均为本领域常规实验操作或依照相应试剂的产品说明书进行。
实施例1:重组Scl2类胶原蛋白的制备方法
本发明中的重组Scl2类胶原蛋白,均由胶原区域和非胶原区域组成,非胶原区域位于胶原区域的羧基端。非胶原区域-1,非胶原区域-2,非胶原区域-3,非胶原区域-4,非胶原区域-5,非胶原区域-6,非胶原区域-7与胶原区域-1组成的蛋白分别命名为Scl2-M1-1,Scl2-M1-2,Scl2-M1-3,Scl2-M2-1,Scl2-M2-2,Scl2-M2-3和Scl2-M3-1;非胶原区域-1,非胶原区域-2,非胶原区域-3,非胶原区域-4,非胶原区域-5,非胶原区域-6,非胶原区域-7与胶原区域-2组成的蛋白分别命名为Scl2-2X-M1-1,Scl2-2X-M1-2,Scl2-2X-M1-3,Scl2-2X-M2-1,Scl2-2X-M2-2,Scl2-2X-M2-3和Scl2-2X-M3-1。
非胶原区域-1的氨基酸序列如SEQ ID No. 1所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’;
非胶原区域-2的氨基酸序列如SEQ ID No. 2所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQGLAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-3的氨基酸序列如SEQ ID No. 3所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQELAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-4的氨基酸序列如SEQ ID No. 4所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQGLAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-5的氨基酸序列如SEQ ID No. 5所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQELAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-6的氨基酸序列如SEQ ID No. 6所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQGLAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-7的氨基酸序列如SEQ ID No. 7所示:5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQGLAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
胶原区域-1的氨基酸序列如SEQ ID No. 8所示:5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGAGAAGAGSAAALEHHHHHH-3’
胶原区域-2的氨基酸序列如SEQ ID No. 9所示:5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
重组Scl2类胶原蛋白的制备方法具体如下:
1)将化脓链球菌(Streptococcus pyogenes)Scl2.28的cDNA序列转入载体Pet28a中,获得重组载体Pet28a-Scl2,以重组载体Pet28a-Scl2为模板,将引物Scl2-F分别和引物Scl2-M1-1-R、引物Scl2-M1-2-R1、引物Scl2-M1-3-R1、引物Scl2-M2-1-R1、引物Scl2-M2-2-R1、引物Scl2-M2-3-R1、引物Scl2-M3-1-R1混合,扩增出Scl2-M1-1-1、Scl2-M1-2-1、Scl2-M1-3-1、Scl2-M2-1-1、Scl2-M2-2-1、Scl2-M2-3-1、Scl2-M3-1-1;
2)以重组载体Pet28a-Scl2为模板,分别使用引物Scl2-M1-1-F1、引物Scl2-M1-2-F1、引物Scl2-M1-3-F1、引物Scl2-M2-1-F1、引物Scl2-M2-2-F1、引物Scl2-M2-3-F1、引物Scl2-M3-1-F1和引物Scl2-R混合,扩增出Scl2-M1-1-2、Scl2-M1-2-2、Scl2-M1-3-2、Scl2-M2-1-2、Scl2-M2-2-2、Scl2-M2-3-2、Scl2-M3-1-2片段;
3)最后,再分别以片段Scl2-M1-1-1和Scl2-M1-1-2、Scl2-M1-2-1和Scl2-M1-2-2、Scl2-M1-3-1和Scl2-M1-3-2、Scl2-M2-1-1和Scl2-M2-1-2、Scl2-M2-2-1和Scl2-M2-2-2、Scl2-M2-3-1和Scl2-M2-3-2、Scl2-M3-1-1和Scl2-M3-1-2为模板,均用引物Scl2-F和Scl2-R扩增合成Scl2-M1-1、Scl2-M1-2、Scl2-M1-3、Scl2-M2-1、Scl2-M2-2、Scl2-M2-3、Scl2-M3-1全长DNA片段;
上述步骤中涉及的引物及其序列如表1所示。
表1 引物及其序列
名称 | 序列号 | 序列(5'-3') |
Scl2-F | SEQ ID No.10 | TATACCATGGGCAGCAGCCATCATC |
Scl2-M1-1-R1 | SEQ ID No.11 | ACCGGTGCGCACTTTCGCTTTTTCTTCC |
Scl2-M1-2-R1 | SEQ ID No.12 | ACCCTGAATCAGTTCGGTGCGCACT |
Scl2-M1-3-R1 | SEQ ID No.13 | ACCAATGCCGCCCAGACCTTGC |
Scl2-M2-1-R1 | SEQ ID No.14 | GGTGCGCACTTTCGCTTTTTCTTCC |
Scl2-M2-2-R1 | SEQ ID No.15 | ACCTTGCGCCAGTTCCTGAATCAGACCGGTGCGCACTTTCGCTTTTTCT |
Scl2-M2-3-R1 | SEQ ID No.16 | GCCCAGACCTTGCGCCAGACCCTGAATCAGTTCGGTGCGCACTTT |
Scl2-M3-1-R1 | SEQ ID No.17 | ACCTTGCGCCAGACCCTGAATCAGACCGGTGCGCACTTTCGCTTTTTCT |
Scl2-R | SEQ ID No.18 | GTGCTCGAGTGCGGCCGCGCTA |
Scl2-M1-1-F1 | SEQ ID No.19 | GGAAGAAAAAGCGAAAGTGCGCACCGGTCTGATTCAGGAACTGGCGCAAGG |
Scl2-M1-2-F1 | SEQ ID No.20 | GCGCACCGAACT GATTCAGGGTCTGGCGCAAGGTCTGGGCG |
Scl2-M1-3-F1 | SEQ ID No.21 | GCAAGGTCTGGGCGGCATTGGTAAGAAAAACTTTCCGACCCTGGGCG |
Scl2-M2-1-F1 | SEQ ID No.22 | GGAAGAAAAAGCGAAAGTGCGCACCGGTCTGATTCAGGGTCTGGCGCAAGGTCTGGGC |
Scl2-M2-2-F1 | SEQ ID No.23 | ATTCAGGAACTGGCGCAAGGTCTGGGCGGCATTGGTAAGAAAAACTTTCCGACCCTGGG |
Scl2-M2-3-F1 | SEQ ID No.24 | GGTCTGGCGCAAGGTCTGGGCGGCATTGGTAAGAAAAACTTTCCGACC CTGGGCG |
Scl2-M3-1-F1 | SEQ ID No.25 | ATTCAGGGTCTGGCGCAAGGTCTGGGCGGCATTGGTAAGAAAAACTTTCCGACCCTGGG |
4)步骤3)扩增后的DNA片段及Pet28a空载体经过Nco I和Xho I限制性内切酶进行双酶切;
5)从步骤4)得到的酶切产物,用1%的琼脂糖凝胶电泳分离,切取目的片段;
6)在步骤5)得到的目的片段,用凝胶DNA回收试剂盒(TIANGEN)纯化;
7)将步骤6)纯化后的片段和载体在T4 DNA连接酶的作用下进行16℃过夜连接;
8)将步骤7)得到的连接产物,利用热激转化法转入大肠杆菌DH5α中,涂布至含有卡纳抗生素的固体LB培养基平板中;
9)在步骤8)得到的平板中挑取单克隆,转移至含有预先加入卡纳抗生素培养基的试管中,37℃震荡过夜培养;
10)将步骤9)得到的菌液收集,用质粒小提试剂盒(TIANGEN)制备质粒,并经DNA测序确认其序列;
11)将步骤10)测序正确的Scl2蛋白重组表达载体转化至大肠杆菌BL21(DE3)菌株中,涂布至含有卡纳抗生素的固体LB培养基平板中;
12)从步骤11)得到的平板中挑取单克隆,转移至含有预先加入卡纳抗生素培养基的试管中,37℃震荡过夜培养;
13)将步骤12)的菌液转移至预先加入卡纳抗生素的LB培养基中,37℃震荡培养至OD600达到0.4~0.6;
14)在步骤13)的菌液中加入IPTG,37℃培养5 h~6 h;
15)将步骤14)得到的菌液收集离心/过滤,除去上清并收集菌体沉淀;
16)向步骤15)的菌体沉淀中加入蛋白提取液和溶菌酶,冰上静止1 h;
17)将步骤16)获得的蛋白溶液超声破碎,离心/过滤收集上清;
18)将步骤17)的粗蛋白溶液加入到已装镍的重力柱中;
19)待步骤18)的镍柱无剩余粗蛋白溶液时,加入Buffer A(50 mM Tris,250 mMNaCl,30 mM咪唑)进行杂蛋白的清洗;
20)在步骤19)的镍柱中加入Buffer B(50 mM Tris,250 mM NaCl,400 mM咪唑)将目的蛋白洗脱下来;
21)将步骤20)洗脱下来的目的蛋白,直接用蒸馏水进行透析,透析时间为每次6h,共2次;
22)将步骤21)透析后的溶液进行冷冻风干即可获得重组后的细菌性类胶原蛋白。
Scl2-2X-M1-1蛋白,Scl2-2X-M1-2蛋白,Scl2-2X-M1-3蛋白,Scl2-2X-M2-1蛋白,Scl2-2X-M2-2蛋白,Scl2-2X-M2-3蛋白和Scl2-2X-M3-1蛋白的制备方法中涉及的引物与Scl2-M1-1蛋白,Scl2-M1-2蛋白,Scl2-M1-3蛋白,Scl2-M2-1蛋白,Scl2-M2-2蛋白,Scl2-M2-3蛋白和Scl2-M3-1蛋白的制备方法中涉及的引物相同,其他制备步骤也相同。
实施例2:可变速类胶原蛋白自组装水凝胶的制备方法
第一种:称取实施例1获得的重组Scl2类胶原蛋白,将其与0.1~0.2 M HCl充分混合,配制成浓度为25~50 mg/mL的酸溶液;之后缓慢加入2 M NaOH调节溶液pH,使最终的PH在6~8;最后,室温静置即可成胶。Scl2-M1-1,Scl2-M1-2,Scl2-M1-3成胶时间为1~5 min;Scl2-M2-1,Scl2-M2-2,Scl2-M2-3成胶时间为5~20 min;Scl2-M3-1成胶时间为40~90 min。
第二种:称取实施例1获得的重组的Scl2类胶原蛋白,将其与0.1~0.2 M CH3COOH充分混合,配制成浓度为25~50 mg/mL的酸溶液;之后缓慢加入2 M NaOH调节溶液pH,使最终的PH在6~8;最后,室温静置即可成胶。Scl2-M1-1,Scl2-M1-2,Scl2-M1-3成胶时间为1~5min;Scl2-M2-1,Scl2-M2-2,Scl2-M2-3成胶时间为5~10 min;Scl2-M3-1成胶时间为30~70min。
制备得到的Scl-M1-2水凝胶、Scl-M2-2水凝胶、Scl-M3-1水凝胶的扫描电镜图分别如图1、图2和图3所示。
由Scl-M1-2蛋白、Scl-M2-2蛋白和Scl-M3-1蛋白分别制备的水凝胶图分别如图4、图5和图6所示。由本发明制备的另外7种重组类胶原蛋白制成的水凝胶同样表现出自组装成水凝胶具有时间差异的特点。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法
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<213> 人工合成
<400> 5
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Gly Leu Ile Gln Glu
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 6
<211> 89
<212> PRT
<213> 人工合成
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile Gln Gly
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 7
<211> 89
<212> PRT
<213> 人工合成
<400> 7
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Gly Leu Ile Gln Gly
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 8
<211> 281
<212> PRT
<213> 人工合成
<400> 8
Gly Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Phe Pro Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Leu Ala
85 90 95
Gly Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly
100 105 110
Glu Lys Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro
115 120 125
Ala Gly Glu Arg Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys
130 135 140
Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Asp Lys Gly
145 150 155 160
Glu Thr Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu
165 170 175
Arg Gly Pro Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp
180 185 190
Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly
195 200 205
Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro
225 230 235 240
Gly Lys Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
245 250 255
Gln Pro Gly Lys Pro Gly Ala Gly Ala Ala Gly Ala Gly Ser Ala Ala
260 265 270
Ala Leu Glu His His His His His His
275 280
<210> 9
<211> 524
<212> PRT
<213> 人工合成
<400> 9
Gly Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Phe Pro Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Leu Ala
85 90 95
Gly Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly
100 105 110
Glu Lys Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro
115 120 125
Ala Gly Glu Arg Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys
130 135 140
Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Asp Lys Gly
145 150 155 160
Glu Thr Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu
165 170 175
Arg Gly Pro Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp
180 185 190
Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly
195 200 205
Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro
225 230 235 240
Gly Lys Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
245 250 255
Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro
260 265 270
Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu Gln
275 280 285
Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly
290 295 300
Phe Pro Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly Lys
305 310 315 320
Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro Gln
325 330 335
Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly
340 345 350
Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Glu
355 360 365
Lys Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala
370 375 380
Gly Glu Arg Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly
385 390 395 400
Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Asp Lys Gly Glu
405 410 415
Thr Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu Arg
420 425 430
Gly Pro Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp Gly
435 440 445
Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu
450 455 460
Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro
465 470 475 480
Gly Lys Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro Gly
485 490 495
Lys Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln
500 505 510
Pro Gly Lys Pro Leu Glu His His His His His His
515 520
<210> 10
<211> 25
<212> DNA
<213> 人工合成
<400> 10
tataccatgg gcagcagcca tcatc 25
<210> 11
<211> 28
<212> DNA
<213> 人工合成
<400> 11
accggtgcgc actttcgctt tttcttcc 28
<210> 12
<211> 25
<212> DNA
<213> 人工合成
<400> 12
accctgaatc agttcggtgc gcact 25
<210> 13
<211> 22
<212> DNA
<213> 人工合成
<400> 13
accaatgccg cccagacctt gc 22
<210> 14
<211> 25
<212> DNA
<213> 人工合成
<400> 14
ggtgcgcact ttcgcttttt cttcc 25
<210> 15
<211> 49
<212> DNA
<213> 人工合成
<400> 15
accttgcgcc agttcctgaa tcagaccggt gcgcactttc gctttttct 49
<210> 16
<211> 45
<212> DNA
<213> 人工合成
<400> 16
gcccagacct tgcgccagac cctgaatcag ttcggtgcgc acttt 45
<210> 17
<211> 49
<212> DNA
<213> 人工合成
<400> 17
accttgcgcc agaccctgaa tcagaccggt gcgcactttc gctttttct 49
<210> 18
<211> 22
<212> DNA
<213> 人工合成
<400> 18
gtgctcgagt gcggccgcgc ta 22
<210> 19
<211> 51
<212> DNA
<213> 人工合成
<400> 19
ggaagaaaaa gcgaaagtgc gcaccggtct gattcaggaa ctggcgcaag g 51
<210> 20
<211> 41
<212> DNA
<213> 人工合成
<400> 20
gcgcaccgaa ctgattcagg gtctggcgca aggtctgggc g 41
<210> 21
<211> 47
<212> DNA
<213> 人工合成
<400> 21
gcaaggtctg ggcggcattg gtaagaaaaa ctttccgacc ctgggcg 47
<210> 22
<211> 58
<212> DNA
<213> 人工合成
<400> 22
ggaagaaaaa gcgaaagtgc gcaccggtct gattcagggt ctggcgcaag gtctgggc 58
<210> 23
<211> 59
<212> DNA
<213> 人工合成
<400> 23
attcaggaac tggcgcaagg tctgggcggc attggtaaga aaaactttcc gaccctggg 59
<210> 24
<211> 55
<212> DNA
<213> 人工合成
<400> 24
ggtctggcgc aaggtctggg cggcattggt aagaaaaact ttccgaccct gggcg 55
<210> 25
<211> 59
<212> DNA
<213> 人工合成
<400> 25
attcagggtc tggcgcaagg tctgggcggc attggtaaga aaaactttcc gaccctggg 59
Claims (8)
1.一种重组Scl2类胶原蛋白,其特征在于,由胶原区域和非胶原区域组成,非胶原区域位于胶原区域的羧基端,非胶原区域的序列为SEQ ID No. 1或SEQ ID No. 2或SEQ ID No.3或SEQ ID No. 4或SEQ ID No. 5或SEQ ID No. 6或SEQ ID No. 7任一所示的氨基酸序列,胶原区域的序列为SEQ ID No. 8或SEQ ID No. 9所示的氨基酸序列。
2.一种权利要求1所述重组Scl2类胶原蛋白的制备方法,其特征在于,包括如下步骤:
(1)将化脓链球菌(Streptococcus pyogenes)Scl2.28的cDNA序列转入载体Pet28a中,获得重组载体Pet28a-Scl2,以重组载体Pet28a-Scl2为模板,根据所述重组Scl2类胶原蛋白的突变位点,设计引物,扩增DNA片段,合成重组scl2类胶原蛋白的全序列,并以合成后的全序列为模板,PCR扩增合成DNA片段,再经酶切、连接、转化大肠杆菌获得重组的Scl2类胶原蛋白重组表达载体;
所述扩增DNA片段是将核苷酸序列如SEQ ID No. 10所示的引物Scl2-F分别与核苷酸序列如SEQ ID No. 11所示的引物Scl2-M1-1-R1、核苷酸序列如SEQ ID No. 12所示的引物Scl2-M1-2-R1、核苷酸序列如SEQ ID No. 13所示的引物Scl2-M1-3-R1、核苷酸序列如SEQID No. 14所示的引物Scl2-M2-1-R1、核苷酸序列如SEQ ID No. 15所示的引物Scl2-M2-2-R1、核苷酸序列如SEQ ID No. 16所示的引物Scl2-M2-3-R1、核苷酸序列如SEQ ID No. 17所示的引物Scl2-M3-1-R1混合进行DNA片段扩增,并将核苷酸序列如SEQ ID No. 18所示的引物Scl2-R分别与核苷酸序列如SEQ ID No. 19所示的引物Scl2-M1-1-F1、核苷酸序列如SEQ ID No. 20所示的引物Scl2-M1-2-F1、核苷酸序列如SEQ ID No. 21所示的引物Scl2-M1-3-F1、核苷酸序列如SEQ ID No. 22所示的引物Scl2-M2-1-F1、核苷酸序列如SEQ IDNo. 23所示的引物Scl2-M2-2-F1、核苷酸序列如SEQ ID No. 24所示的引物Scl2-M2-3-F1、核苷酸序列如SEQ ID No. 25所示的引物Scl2-M3-1-F1混合进行DNA片段扩增;
所述以合成后的全序列为模板PCR扩增合成DNA片段所用上游引物为核苷酸序列如SEQID No. 10所示的Scl2-F,下游引物为核苷酸序列如SEQ ID No. 18所示的Scl2-R;
(2)将步骤(1)获得的Scl2类胶原蛋白重组表达载体转化至大肠杆菌中获得阳性菌株;
(3)将步骤(2)获得的阳性菌株接种于LB培养基中培养至OD600达到0.4~0.6时,向菌液中加入IPTG,继续培养5 h~6 h,对菌液进行离心或过滤后,去除上清并收集菌体沉淀;
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶,于冰上静置1 h,获得蛋白溶液,经超声后,通过离心或过滤收集上清获得粗蛋白;
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化,经水透析后获得重组后的类胶原蛋白。
3.根据权利要求2所述的制备方法,其特征在于,步骤(1)所述酶切是将扩增后的DNA片段及Pet28a空载体经过Nco I和Xho I限制性内切酶进行双酶切。
4.根据权利要求2所述的制备方法,其特征在于,步骤(1)所述连接使用的连接酶为T4DNA连接酶;所述转化利用的大肠杆菌为DH5α。
5.根据权利要求2所述的制备方法,其特征在于,步骤(5)所述利用已装镍的重力柱进行纯化的方法是将步骤(4)获得的粗蛋白溶液加入已装镍的重力柱中,待镍柱无剩余粗蛋白溶液时,加入Buffer A进行杂蛋白的清洗,再加入Buffer B将目的蛋白洗脱下来。
6.根据权利要求5所述的制备方法,其特征在于,所述Buffer A的组成为50 mM Tris,250 mM NaCl和30 mM咪唑;所述Buffer B的组成为50 mM Tris,250 mM NaCl和400 mM咪唑。
7.根据权利要求2所述的制备方法,其特征在于,步骤(5)所述透析共进行2次,每次6h。
8.一种用权利要求1所述的重组Scl2类胶原蛋白制备自组装可变速水凝胶的方法,其特征在于,将所述重组Scl2类胶原蛋白与0.1~0.2 M的盐酸或醋酸混合,制成25~50 mg/mL的酸溶液,然后向酸溶液中加入2 M的NaOH溶液将pH调节至6~8,室温静置即可成胶。
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