CN110028575B - 一种小分子胶原蛋白的制备方法 - Google Patents
一种小分子胶原蛋白的制备方法 Download PDFInfo
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- CN110028575B CN110028575B CN201910316626.6A CN201910316626A CN110028575B CN 110028575 B CN110028575 B CN 110028575B CN 201910316626 A CN201910316626 A CN 201910316626A CN 110028575 B CN110028575 B CN 110028575B
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Abstract
本发明提供了一种小分子胶原蛋白的制备方法,其特征在于,包括如下步骤:将未经色谱纯化过的胶原蛋白于25~37℃下放置3~14天。本发明能够有效制备分子量在10~50KD大小的胶原蛋白,无需借助γ射线发生装置、蛋白酶等装置或试剂,成本低廉。本发明还能通过调节制备时间来得到特定主蛋白分子量的胶原蛋白,操作易行。本发明得到的胶原蛋白可有效促进细胞增殖,发明人经实验证明,本发明的小分子胶原蛋白相比大分子胶原蛋白对IEC‑6细胞的促增殖生物活性显著更高。根据常识,组织修复与细胞增殖息息相关,因此,将本发明应用于组织修复相关的产品(例如药物、保健品等)的生产上,具有十分良好的前景。
Description
技术领域
本发明涉及胶原蛋白制备领域,尤其涉及一种小分子胶原蛋白的制备方法。
背景技术
胶原蛋白是一种广泛存在于有机体中的结构蛋白,是皮肤、软骨、动脉血管壁及结缔组织的主要成分。胶原蛋白适度降解后分子量降低,成为小分子胶原蛋白。后者除了具备易吸收(吸收率可达95-100%)、低抗原性、低过敏性、载体运输能力强等肽的典型特征外,还具有抗氧化、免疫调节、增强骨强度和促进软骨再生等多种生理活性,在医药、食品、化妆品领域有着广阔的应用前景。
目前,小分子胶原蛋白的制备主要通过蛋白酶降解、色谱分离或γ射线照射等方法处理胶原蛋白,其成本较高。
发明内容
为了解决上述问题,本发明提供了一种小分子胶原蛋白的制备方法,包括如下步骤:
将未经色谱纯化过的胶原蛋白于一定温度下放置一定时间;
所述小分子胶原蛋白的分子量为10~50KD;
所述一定温度为25~37℃;
所述一定时间为3~14天。
如前述的制备方法,所述胶原蛋白的氨基酸序列如SEQ ID NO.1所示。
进一步地,所述胶原蛋白由特定DNA表达而来,所述特定DNA含有如SEQ ID NO.2所示的序列。
如前述的制备方法,所述一定温度为25℃。
进一步地,所述一定时间为4天。
如前述的制备方法,所述一定温度为37℃。
进一步地,所述一定时间为3天。
本发明还提供了前述方法制备得到的小分子胶原蛋白产品。
本发明还提供了前述小分子胶原蛋白产品在药物或保健品制备中的用途。
如前述的用途,所述药物或保健品是促进细胞增殖或组织修复的药物或保健品。
本发明能够有效制备分子量在10~50KD大小的胶原蛋白,无需借助γ射线发生装置、蛋白酶等装置或试剂,成本低廉。
本发明还能通过调节制备时间来得到特定主蛋白分子量的胶原蛋白,操作易行。
本发明得到的胶原蛋白可有效促进细胞增殖,发明人经实验证明,本发明的小分子胶原蛋白相比大分子胶原蛋白对IEC-6细胞的促增殖生物活性显著更高。根据常识,组织修复与细胞增殖息息相关,因此,将本发明应用于组织修复相关的产品(例如药物、保健品等)的生产上,具有十分良好的前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过具体实施方式对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1:实施例中胶原蛋白4℃放置的蛋白电泳图。
图2:实施例中胶原蛋白25℃放置的蛋白电泳图。
图3:实施例中胶原蛋白37℃放置的蛋白电泳图。
图4:胶原蛋白20170504流穿样品4℃放置的蛋白电泳图。
图5:胶原蛋白20170509流穿样品4℃放置的蛋白电泳图。
图6:胶原蛋白20170510流穿样品4℃放置的蛋白电泳图。
图7:胶原蛋白20170504高盐洗脱样品4℃放置的蛋白电泳图。
图8:胶原蛋白20170509高盐洗脱样品4℃放置的蛋白电泳图。
图9:胶原蛋白20170510高盐洗脱样品4℃放置的蛋白电泳图。
图10:胶原蛋白20170504流穿样品25℃放置的蛋白电泳图。
图11:胶原蛋白20170509流穿样品25℃放置的蛋白电泳图。
图12:胶原蛋白20170510流穿样品25℃放置的蛋白电泳图。
图13:胶原蛋白20170504高盐洗脱样品25℃放置的蛋白电泳图。
图14:胶原蛋白20170509高盐洗脱样品25℃放置的蛋白电泳图。
图15:胶原蛋白20170510高盐洗脱样品25℃放置的蛋白电泳图。
图16:胶原蛋白20170504流穿样品37℃放置的蛋白电泳图。
图17:胶原蛋白20170509流穿样品37℃放置的蛋白电泳图。
图18:胶原蛋白20170510流穿样品37℃放置的蛋白电泳图。
图19:胶原蛋白20170504高盐洗脱样品37℃放置的蛋白电泳图。
图20:胶原蛋白20170509高盐洗脱样品37℃放置的蛋白电泳图。
图21:胶原蛋白20170510高盐洗脱样品37℃放置的蛋白电泳图。
图22:小分子胶原蛋白样品与大分子胶原蛋白样品在不同浓度条件下对IEC-6细胞增殖数量的比较。
具体实施方式
实施例小分子胶原蛋白的制备
1.胶原蛋白材料
重组胶原蛋白发酵上清,该蛋白的氨基酸序列如SEQ ID NO.1所示,表达该蛋白的基因具有SEQ ID NO.2所示序列;该蛋白的理论氨基酸数目是1067,理论分子量是95549。
SEQ ID NO.1:
SEQ ID NO.2:
2.方法
蛋白样品经无菌过滤后,分装到EP管中,0.5-1ml/支,分别于4℃、25℃及37℃放置,为期14天,每天取样并电泳观察降解情况。每次电泳上样量为10μg蛋白。
3.结果
(1)如图1所示,于4℃放置的胶原蛋白,降解缓慢,在第14天仍可观察到100KD~150KD处的蛋白条带,主带降解至50KD附近(37~50KD)。
(2)如图2所示,于25℃放置的胶原蛋白发酵液上清,降解迅速,在第3天100KD~150KD处蛋白基本降解完毕,在第4天,观察到50KD处附近蛋白明显增加,主带降至50KD附近(37~50KD),且25KD以下蛋白较少。于15KD处蛋白呈现递增趋势,在第14天,主带降至15KD处附近(10~20KD)。
(3)如图3所示,于37℃放置,发酵液上清降解迅速且趋势明显,第2天100KD~150KD处的蛋白带基本降解完全,在第3天50KD附近(37~50KD)蛋白明显增加,且25KD以下分子量蛋白较少。随实验进行,主蛋白带逐渐降至15KD附近(10~20KD)。
4.结论
制备小分子胶原蛋白(10~50KD的胶原蛋白)可在25℃条件下放置4天,或是于37℃放置3天,皆可使主蛋白带降至50KD附近(37~50KD),且此条件下,高分子量(75KD及以上)蛋白基本降解完全,低分子量(25KD及以下)蛋白只部分降解。
对比例 色谱纯化后的胶原蛋白自然降解实验
1.胶原蛋白材料
经过色谱纯化后的胶原蛋白
色谱纯化的大致步骤为:平衡、上样、再平衡、洗脱、再生、CIP(在位清洗)。色谱参数如下:
层析柱:50mm×200mm,层析填料:GE Sephrose SP HP,柱体积:400ml
平衡液:20mM PB+130mM NaCl,pH 6.2
洗脱液:20mM PB+280mM NaCl,pH 6.2
再生液:2MNaCl
CIP液:0.5MNaOH
上样流速:10mL/min,平衡及洗脱流速:20mL/min
其样品信息如表1所示。
表1流穿及高盐洗脱样品信息
2.方法
同实施例。
3.结果
3.1于4℃放置结果
(1)流穿样品电泳
由图4(纯化日期/批号20170504)、5(纯化日期/批号20170509)和6(纯化日期/批号20170510)可知,于4℃放置的样品在14天的实验中无明显的降解趋势,较为稳定。虽然20170510样品在第13天的电泳结果呈现蛋白降解,条带变浅的现象,但由于其第14天的结果与前12天保持一致,无明显降解,故考虑第13天取样的样品可能染菌,则第13天的电泳结果不作为评判的依据。
(2)洗脱样品电泳
图7~9依次为20170504、20170509及20170510的高盐洗脱样品的电泳。由图可知,在4℃条件下放置14天,同流穿相同,皆无明显降解趋势。20170510样品在第14天的电泳,可能电泳上样时,由于操作原因上样部分沉淀,致条带颜色略深,但不影响降解情况的判断。
3.2于25℃放置的结果
(1)流穿样品电泳
图10~12依次为20170504、20170509及20170510的流穿样品。在25℃放置条件下,观察到有降解趋势,20170509降解缓慢,20170504及20170510流穿降解明显,且在第12天两者100KD~150KD处蛋白基本降解完全。第14天仍有高分子量蛋白未完全降解,含75KD及以上蛋白。
(2)高盐洗脱样品电泳
于25℃放置的高盐洗脱样品,20170504(图13)及20170509(图14)样品降解缓慢,但20170510样品(图15)降解明显,可观察到第7天开始100KD~150KD处蛋白基本降解完全,第11天50~100KD蛋白基本降解,在第12天,条带基本降至25KD及15KD,且50KD以上无明显蛋白。
3.3于37℃放置的结果
(1)流穿样品电泳
于37℃放置的流穿样品,同样观察到有降解趋势。20170509(图17)降解缓慢,20170504(图16)及20170510(图18)流穿降解明显;在第8、9天,100KD~150KD处蛋白降解完全。20170510样品的50~100KD蛋白在第11天基本降解,同样在第12天,条带基本降至30KD及15KD附近,且50KD以上无明显蛋白(图18)。
(2)高盐洗脱样品电泳
于37℃放置的高盐洗脱样品,20170504(图19)及20170509(图20)降解稍缓,20170510(图21)样品在第4天开始100KD~150KD处蛋白基本降解完全,第6天50~100KD蛋白基本降解,第8、9天已无明显高分子量蛋白条带,主带已降解至50KD以下。20170510在第3天的电泳不符合整体降解趋势,猜测样品染菌加速了降解,但不影响样品降解趋势的判断。
4.结论
选取的三次色谱纯化的样品之间,不存在降解一致性。部分样品可进行不同分子量降解情况的确认,其余样品虽然有降解趋势,但降解缓慢,不能进行蛋白降解分子量区间。
所以,色谱纯化的胶原蛋白样品不适合用于本发明的方法,
本发明的方法不宜以色谱纯化的胶原蛋白样品为原料,制备小分子胶原蛋白。
实验例 小分子胶原蛋白的促细胞增殖能力检测
1.目的
比较小分子胶原蛋白(通过实施例的方法制备得到的10~50KD的胶原蛋白,下同)与大分子胶原蛋白对IEC-6细胞的促增殖生物活性差异。
2.实验材料
(1)试剂
完全培养液:DMEM+10%FBS;
维持培养液:DMEM+0.5%FBS;
0.25%胰蛋白酶;
PBS;
CCK-8:日本同仁。
(2)设备
生物安全柜、二氧化碳培养箱、离心机、倒置生物显微镜、酶标仪、漩涡混匀器
(3)器具
T75方瓶、移液枪、移液管、50ml离心管、1.5ml EP管、96孔板、1ml枪头、200ul枪头。
3.操作方法
(1)细胞培养及传代
复苏IEC-6细胞,用完全培养液培养至细胞状态良好,汇合度约80%,按1∶2传代,传代后24-36h后用于生物活性检测。
(2)细胞接种
弃培养瓶中的培养液,加入胰酶消化,离心1000rpm、4min收集细胞。加入维持培养液重悬,调整细胞密度为4×104cells/ml,接种96孔板,100ul/孔,37℃、8%CO2培养。
(3)样品稀释
用维持培养液稀释小分子胶原蛋白样品与大分子胶原蛋白样品,待测样品起始终浓度为500μg/ml,2倍稀释6个稀释度,分别为500、250、125、50、25、12.5μg/ml,每个浓度做3个孔。
(4)加样
加入稀释的胶原蛋白样品100ul/孔,再于培养箱37℃、8%CO2培养48h。
(5)检测
弃96孔板内培养液,加入稀释后的CCK-8试剂(CCK-8∶维持培养液=1∶10)100ul/孔,37℃、8%CO2孵育30-60min。取出96孔板,以620nm为参比波长,450nm为试验波长测定吸光度,记录测定结果。
小分子胶原蛋白检测结果见表2,大分子胶原蛋白检测结果见表3。比较两种样品对细胞增殖数量的效果如图22。
表2小分子胶原蛋白检测结果
表3大分子胶原蛋白检测结果
4.结论
小分子胶原蛋白较大分子胶原蛋白对细胞IEC-6的增殖效果更好。
SEQUENCE LISTING
<110> 成都中医药大学
<120> 一种小分子胶原蛋白的制备方法
<130> GY041-18P1749
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1067
<212> PRT
<213> 人工序列
<400> 1
Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser Val
1 5 10 15
Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro
20 25 30
Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly
35 40 45
Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly Pro
50 55 60
Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro
65 70 75 80
Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly
85 90 95
Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly Leu
100 105 110
Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro
115 120 125
Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg Gly
130 135 140
Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala
145 150 155 160
Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr
165 170 175
Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys Gly
180 185 190
Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly Val
195 200 205
Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala
210 215 220
Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly
225 230 235 240
Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro
245 250 255
Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn Ser
260 265 270
Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly
275 280 285
Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Glu
290 295 300
Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu Pro
305 310 315 320
Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro Gly
325 330 335
Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly Ser
340 345 350
Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro
355 360 365
Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly
370 375 380
Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln
385 390 395 400
Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala
405 410 415
Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly
420 425 430
Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly Pro
435 440 445
Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala
450 455 460
Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly
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Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala Gly
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Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala
580 585 590
Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro Ile
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Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser Gly
610 615 620
Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp
625 630 635 640
Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro
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Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly
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Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro
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Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala Arg
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Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro
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Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala
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Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val Val
785 790 795 800
Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly
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Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu
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Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro
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Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala Glu Gly Ser Pro Gly
850 855 860
Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro
865 870 875 880
Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Val
885 890 895
Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly
900 905 910
Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly Pro
915 920 925
Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg
930 935 940
Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro Gly
945 950 955 960
Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly Pro
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Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly
995 1000 1005
Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly
1010 1015 1020
Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser
1025 1030 1035
Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg
1040 1045 1050
Tyr Tyr Arg Ala Gly Pro Pro Gly Pro Cys Cys Gly Gly Gly
1055 1060 1065
<210> 2
<211> 3201
<212> DNA
<213> 人工序列
<400> 2
caattgtctt acggttacga cgaaaagtct actggtggta tctctgttcc aggtccaatg 60
ggtccatctg gtccaagagg tttgccaggt ccaccaggtg ctccaggtcc acaaggtttc 120
caaggtccac caggtgaacc aggtgaacca ggtgcttctg gtccaatggg tccaagaggt 180
ccaccaggtc caccaggtaa gaacggtgac gacggtgaag ctggtaagcc aggtagacca 240
ggtgaaagag gtccaccagg tccacaaggt gctagaggtt tgccaggtac tgctggtttg 300
ccaggtatga agggtcacag aggtttctct ggtttggacg gtgctaaggg tgacgctggt 360
ccagctggtc caaagggtga accaggttct ccaggtgaaa acggtgctcc aggtcaaatg 420
ggtccaagag gtttgccagg tgaaagaggt agaccaggtg ctccaggtcc agctggtgct 480
agaggtaacg acggtgctac tggtgctgct ggtccaccag gtccaactgg tccagctggt 540
ccaccaggtt tcccaggtgc tgttggtgct aagggtgaag ctggtccaca aggtccaaga 600
ggttctgaag gtccacaagg tgttagaggt gaaccaggtc caccaggtcc agctggtgct 660
gctggtccag ctggtaaccc aggtgctgac ggtcaaccag gtgctaaggg tgctaacggt 720
gctccaggta tcgctggtgc tccaggtttc ccaggtgcta gaggtccatc tggtccacaa 780
ggtccaggtg gtccaccagg tccaaagggt aactctggtg aaccaggtgc tccaggttct 840
aagggtgaca ctggtgctaa gggtgaacca ggtccagttg gtgttcaagg tccaccaggt 900
ccagctggtg aagaaggtaa gagaggtgct agaggtgaac caggtccaac tggtttgcca 960
ggtccaccag gtgaaagagg tggtccaggt tctagaggtt tcccaggtgc tgacggtgtt 1020
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ggtgctccag gtttgcaagg tatgccaggt gaaagaggtg ctgctggttt gccaggtcca 1740
aagggtgaca gaggtgacgc tggtccaaag ggtgctgacg gttctccagg taaggacggt 1800
gttagaggtt tgactggtcc aatcggtcca ccaggtccag ctggtgctcc aggtgacaag 1860
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aagggtgcta gaggttctgc tggtccacca ggtgctactg gtttcccagg tgctgctggt 2160
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ggtaaggaag gtggtaaggg tccaagaggt gaaactggtc cagctggtag accaggtgaa 2280
gttggtccac caggtccacc aggtccagct ggtgaaaagg gttctccagg tgctgacggt 2340
ccagctggtg ctccaggtac tccaggtcca caaggtatcg ctggtcaaag aggtgttgtt 2400
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ccaccaggtt tggctggtcc accaggtgaa tctggtagag aaggtgctcc aggtgctgaa 2580
ggttctccag gtagagacgg ttctccaggt gctaagggtg acagaggtga aactggtcca 2640
gctggtccac caggtgctcc aggtgctcca ggtgctccag gtccagttgg tccagctggt 2700
aagtctggtg acagaggtga aactggtcca gctggtccag ctggtccagt tggtccagtt 2760
ggtgctagag gtccagctgg tccacaaggt ccaagaggtg acaagggtga aactggtgaa 2820
caaggtgaca gaggtatcaa gggtcacaga ggtttctctg gtttgcaagg tccaccaggt 2880
ccaccaggtt ctccaggtga acaaggtcca tctggtgctt ctggtccagc tggtccaaga 2940
ggtccaccag gttctgctgg tgctccaggt aaggacggtt tgaacggttt gccaggtcca 3000
atcggtccac caggtccaag aggtagaact ggtgacgctg gtccagttgg tccaccaggt 3060
ccaccaggtc caccaggtcc accaggtcca ccatctgctg gtttcgactt ctctttcttg 3120
ccacaaccac cacaagaaaa ggctcacgac ggtggtagat actacagagc tggtccacca 3180
ggtccatgtt gtggtggtgg t 3201
Claims (4)
1.一种小分子胶原蛋白的制备方法,其特征在于,包括如下步骤:
将未经色谱纯化过的胶原蛋白于一定温度下放置一定时间;所述胶原蛋白的氨基酸序列如SEQ ID NO.1所示;所述胶原蛋白由特定DNA表达而来,所述特定DNA含有如SEQ IDNO.2所示的序列;
所述小分子胶原蛋白的分子量为10~50KD;
所述一定温度为25℃,所述一定时间为4天;或者
所述一定温度为37℃,所述一定时间为3天。
2.权利要求1所述方法制备得到的小分子胶原蛋白产品。
3.权利要求2所述小分子胶原蛋白产品在药物或保健品制备中的用途。
4.如权利要求3所述的用途,所述药物是促进细胞增殖或组织修复的药物。
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