CN113684222A - 一种合成母乳蛋白质及其制备方法 - Google Patents
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Abstract
本发明公开了一种合成母乳蛋白质及其制备方法,方法包括:获取编码β酪蛋白、κ酪蛋白、α乳白蛋白以及乳铁蛋白所需的CSN2、CSN3、LALBA以及LTF基因;对CSN2、CSN3、LALBA以及LTF基因进行密码子优化,针对每个基因改变20%的核苷酸,生产出DNA干粉;基因表达载体的构建,将目的基因插入基因表达载体,设计外分泌信号肽,使载体将所需的蛋白质分泌出细胞外,在所述蛋白质的N端和C端均加入his‑tag,区分所需的蛋白与其他杂质;将所述载体培养在恒温箱中,用甲醇诱导蛋白质的表达;使用SDS‑Page检查蛋白质是否生产成功;使用Ni‑NTA纯化蛋白质,提取、储存蛋白;使用缓冲液溶解蛋白质,获得合成母乳蛋白质。
Description
技术领域
本申请涉及基因工程领域,特别涉及一种合成母乳蛋白质及其制备方法。
背景技术
作为生命物质基础的蛋白质在人的一生中扮演着最为重要的角色,从生命酝酿之初的卵子或精子,到形成的人体结构组织,以及人体结构组织的修补、机体内的代谢都离不开蛋白质。除了构成生命之外,蛋白质还影响人的思维、人的情绪,甚至影响着人们的生活质量。因此科学家说,蛋白质是生命的核心。
人体内的蛋白质绝大多数是来自于食物,有些由自身代谢产物合成,其中食物是人体内蛋白质的关键来源。蛋白质是母乳中的重要组成部分。人乳的主要蛋白质是牛β-酪蛋白、α-乳白蛋白、乳铁蛋白、免疫球蛋白IgA、溶菌酶和血清白蛋白的同源酪蛋白。母乳喂养有许多好处。第一,母乳中含有的免疫物质可以增强婴儿机体抵抗力;第二,母乳本身作为一种婴儿的食物来源可以促进消化,减少过敏;第三,母乳中含有的钙质可以促进骨骼及肌肉发育;第四,研究表明母乳喂养可以有效增进婴儿的智力。也有文章表明,用强化母乳喂养的早产儿摄入的蛋白质比他们需要的要少。在Arslanoglu等人的研究里,他们测量了32名早产儿的实际营养摄入量,发现实际的蛋白质摄入量显著且持续地低于假设的蛋白质摄入量。平均摄入量的差异很大,大概每天在0.5到0.8g/κg。综合分析表明,母乳喂养可以预防儿童感染和错牙合,提高智力,并可能减少超重和糖尿病。
按照中国居民膳食营养素参考摄入量,儿童每天需摄入蛋白质40~85g,成年人每天需摄入蛋白质75g左右。而母乳或者类似于母乳的牛乳中蛋白质含量仅为1~3%,因此仅依靠母乳或牛乳不能够满足人体蛋白质地需求。通常人体需要多种来源的蛋白质以满足机体对于蛋白质的需求,营养学家建议人们每天食用不同的动物源类蛋白质搭配不同的植物源类蛋白质以弥补各自蛋白质组份的不足之处。随着我国城市人口的增加,生活节奏的加快以及工作竞争压力越来越大,很多城市居民日常饮食习惯也发生了很大的变化,不规律饮食习惯已经成为多数城市居民的生活特点之一。不规律饮食的直接后果就会导致营养摄入的不均衡,从而导致发病率的升高,所以从食物入手保证营养供应成为预防疾病、提高人体素质的关键环节。提供一种高生物价值的方便蛋白质食品,就显得十分重要。此外,当前社会中有许多母亲们在母乳喂养中遇到了许多问题和麻烦。这些问题包括婴幼儿吸吮困难,喂乳期间乳头皲裂,乳汁分泌过少,乳胀和乳腺炎、以及产妇营养不良等。这就对了母乳替代产品提出了市场需求。目前市面上主要的母乳替代类产品有奶粉、羊奶、配方母乳等。奶粉的冲泡需要使用开水,有烫伤婴儿的风险存在。且奶粉并不能完美提供婴儿所需的全部营养。未加热的生羊奶对婴儿有细菌感染的危险,而加工后的羊奶粉不能满足婴儿所有的营养需求。羊奶粉缺乏叶酸,而且存在维生素B,C和D的含量较低的可能性。其钙和磷含量不高,有可能造成低血钙症。
附图说明
图1是编码β酪蛋白的CSN2基因的示意图;
图2是编码κ酪蛋白的CSN3基因的示意图;
图3是编码α乳白蛋白的LALBA基因的示意图;
图4是编码乳铁蛋白的LTF基因的示意图。
发明内容
本发明的目的是提供一种合成母乳蛋白质及其制备方法,本发明的蛋白质食品中的成分可以提供当今市场上的乳品所不能提供的营养。在这样的前提下,人造母乳产品能解决问题,满足人们的需求。人造母乳尽量贴近天然母乳中的蛋白质、碳水化合物等成分。所生产的母乳将为常温,不存在用开水冲泡后烫伤婴儿的危险。和未加热过的羊奶相比,人造母乳将减少细菌感染的风险。在之后的研究中将在人造母乳中加入必要的维生素和钙磷等微量元素,满足婴幼儿需求。因此本发明对于提高人们的健康水平具有积极的意义。
具体实施方式
实施例1
为保证蛋白质品质,除检验过程外的操作过程均在万级以上的净化间内操作实施。
本实施例主要针对母乳中的四种主要蛋白进行表达,分别是β酪蛋白(β-casein),κ酪蛋白,α乳白蛋白,以及乳铁蛋白。编码所需蛋白质的基因分别为CSN2,CSN3,LALBA,以及LTF。
其中,如图1-4,分别为CSN2,CSN3,LALBA,以及LTF基因示意图。
在表达为生物上的选择上,考虑到应用广泛性、安全性,所以使用酵母进行表达。由于酿酒酵母无法将蛋白分泌出体外,因此本实施例选择毕赤酵母作为底盘生物,并采用了分泌载体pPIC9K。
在以上选择的基础上,使用pPIC9K载体(酵母)通过基因编辑手段插入上述四个基因。
具体步骤如下:
1.对β酪蛋白(β-casein),κ酪蛋白,α乳白蛋白,以及乳铁蛋白的四个基因进行了密码子优化,通过对氨基酸序列进行设计与改动,针对每个基因改变20%的核苷酸,并交由生物公司生产出DNA干粉。
选择毕赤酵母pPIC9K载体,针对EcoRI和NotI两个酶切位点,进行infusion引物设计,通过限制性双酶切的方法将目的基因片段插入载体。设计外分泌信号肽,使酵母将所需的蛋白质分泌出细胞外。为了方便蛋白质的纯化,在蛋白质的N端和C端都加入his-tag,将所需的蛋白与其他杂质区别出来。
具体地,在本步骤中通过将设计好的引物,目的基因片段,以及载体进行PCR,成功构建酵母的四个基因表达载体:pPIC9K-CSN2、pPIC9K-CSN3、pPIC9K-LTF和pPIC9K-LALBA。在插入目的基因的同时,将his-tag分别加入N端与C端,用以区分所需的蛋白。
接下来,通过化学转化法和电转化法,将四个目的基因载体转入到毕赤酵母中并在37摄氏度的恒温培养箱中培养了两天。测序验证转染结果后,采集真菌并在液体培养基中培养。
使用SDS-Page检查蛋白质是否生产成功,进一步提取、使用Ni-NTA纯化、储存蛋白,寻找合适的缓冲液溶解蛋白质。从而获取到合成母乳蛋白质。
实施例2
为保证蛋白质品质,除检验过程外的操作过程均在万级以上的净化间内操作实施。在本实施例中,由于母乳蛋白主要有四种蛋白被表达,即β酪蛋白,κ酪蛋白,α乳白蛋白,以及乳铁蛋白。表达这些蛋白的基因分别为:CSN2,CSN3,LALBA,和LTF。
通过将设计好的引物,目的基因片段,以及载体进行PCR,成功构建酵母的四个基因表达载体:pPIC9K-CSN2、pPIC9K-CSN3、pPIC9K-LTF和pPIC9K-LALBA。在插入目的基因的同时,本实施例将his-tag分别加入N端与C端,用以区分所需的蛋白。
接下来,通过化学转化法和电转化法,将四个目的基因片段转入到大肠杆菌中用来扩增片段,以此扩大有限数量的基因片段,并对目的菌液进行甘油管藏。至此,得到了插入pPIC9K-CSN2、pPIC9K-CSN3、pPIC9K-LTF和pPIC9K-LALBA载体的大肠杆菌,且妥善地保存了质粒。
随后,所述将所述载体培养在恒温箱中的步骤包括,将大肠杆菌培养在30摄氏度恒温箱中,用甲醇诱导蛋白质的表达。使用SDS-Page检查蛋白质是否生产成功;使用Ni-NTA纯化蛋白质,提取、储存蛋白;使用缓冲液溶解蛋白质,获得合成母乳蛋白质。
Claims (6)
1.一种合成母乳蛋白质的制备方法,其特征在于,包括以下步骤:
获取编码β酪蛋白、κ酪蛋白、α乳白蛋白以及乳铁蛋白所需的CSN2、CSN3、LALBA以及LTF基因;
对CSN2、CSN3、LALBA以及LTF基因进行了密码子优化,针对每个基因改变20%的核苷酸,生产出DNA干粉;
基因表达载体的构建,将目的基因插入基因表达载体,设计外分泌信号肽,使载体将所需的蛋白质分泌出细胞外,在蛋白质的N端和C端均加入his-tag,区分所需的蛋白质与其他杂质;
将所述载体培养在恒温箱中,用甲醇诱导蛋白质的表达;
使用SDS-Page检查蛋白质是否生产成功;
使用Ni-NTA纯化蛋白质,提取、储存蛋白;
使用缓冲液溶解蛋白质,获得合成母乳蛋白质。
2.根据权利要求1所述的合成母乳蛋白质的制备方法,其特征在于,所述基因表达载体的构建的步骤包括:
选择毕赤酵母pPIC9K载体,针对EcoRI和NotI两个酶切位点,进行infusion引物设计,通过限制性双酶切的方法将目的基因片段插入毕赤酵母pPIC9K载体。
3.根据权利要求2所述的合成母乳蛋白质的制备方法,其特征在于,所述将所述载体培养在恒温箱中的步骤包括,将酵母培养在37摄氏度酵母恒温箱中,用甲醇诱导蛋白质的表达。
4.根据权利要求1所述的合成母乳蛋白质的制备方法,其特征在于,所述基因表达载体的构建的步骤包括:
选择pET28a载体,针对EcoRI和NotI两个酶切位点,进行infusion引物设计,通过限制性双酶切的方法将目的基因片段插入pET28a载体。
5.根据权利要求4所述的合成母乳蛋白质的制备方法,其特征在于,所述将所述载体培养在恒温箱中的步骤包括,将大肠杆菌培养在30摄氏度恒温箱中,用甲醇诱导蛋白质的表达。
6.一种合成母乳蛋白质,其特征在于,它是根据权利要求1-5中任一项所述的制备方法制备的。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020044989A1 (en) * | 1988-09-22 | 2002-04-18 | Gaull Gerald E. | Infant formula compositions and nutrition |
US20030074700A1 (en) * | 2000-05-02 | 2003-04-17 | Ning Huang | Expression of human milk proteins in transgenic plants |
CN103014007A (zh) * | 2012-11-22 | 2013-04-03 | 新疆旺源驼奶实业有限公司 | 双峰驼α-乳白蛋白基因、重组蛋白及其克隆方法 |
CN104313048A (zh) * | 2014-10-21 | 2015-01-28 | 长沙中科晶博生物科技有限公司 | 一种用酿酒酵母生产乳铁蛋白的方法 |
CN107205409A (zh) * | 2014-08-21 | 2017-09-26 | 完美日公司 | 包含酪蛋白的组合物及其生产方法 |
CN108813632A (zh) * | 2018-07-13 | 2018-11-16 | 李钟� | 一种含乳铁蛋白的营养组合物及其应用 |
-
2021
- 2021-08-20 CN CN202110960609.3A patent/CN113684222A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020044989A1 (en) * | 1988-09-22 | 2002-04-18 | Gaull Gerald E. | Infant formula compositions and nutrition |
US20030074700A1 (en) * | 2000-05-02 | 2003-04-17 | Ning Huang | Expression of human milk proteins in transgenic plants |
CN103014007A (zh) * | 2012-11-22 | 2013-04-03 | 新疆旺源驼奶实业有限公司 | 双峰驼α-乳白蛋白基因、重组蛋白及其克隆方法 |
CN107205409A (zh) * | 2014-08-21 | 2017-09-26 | 完美日公司 | 包含酪蛋白的组合物及其生产方法 |
CN104313048A (zh) * | 2014-10-21 | 2015-01-28 | 长沙中科晶博生物科技有限公司 | 一种用酿酒酵母生产乳铁蛋白的方法 |
CN108813632A (zh) * | 2018-07-13 | 2018-11-16 | 李钟� | 一种含乳铁蛋白的营养组合物及其应用 |
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