CN113943724A - 一种人源溶菌酶-防御素融合蛋白及其构建方法 - Google Patents
一种人源溶菌酶-防御素融合蛋白及其构建方法 Download PDFInfo
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- CN113943724A CN113943724A CN202010689597.0A CN202010689597A CN113943724A CN 113943724 A CN113943724 A CN 113943724A CN 202010689597 A CN202010689597 A CN 202010689597A CN 113943724 A CN113943724 A CN 113943724A
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- fusion protein
- defensin
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- lysozyme
- human lysozyme
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Abstract
本发明公开了一种人源溶菌酶‑防御素融合蛋白及其构建方法，融合蛋白包括人源溶菌酶蛋白和人防御素多肽，所述人源溶菌酶蛋白和人防御素多肽由一段多肽串联构成融合蛋白，所述融合蛋白的宿主菌为毕赤酵母；本发明重组菌株合成的融合蛋白具有高效广谱的杀菌特性，不产生耐药性，且是来源于人体中的活性物质，不具有免疫原性，可作为消毒剂、抗菌剂或抗生素的安全替代品，不会对环境造成污染。
Description
技术领域
本发明具体涉及一种人源溶菌酶融合蛋白的基因工程领域，具体是一种人源溶菌酶-防御素融合蛋白及其构建方法。
背景技术
微生物无处不在，与人类活动息息相关，很多微生物对人类及自然环境有益，被人类开发与利用。但也有很多微生物严重威胁到人类的生存，例如使人得病、使食物腐坏等，某些致病菌还会在生物体之间进行交叉感染。数千年来，人们一直在用不同的方法对微生物进行抑制与杀灭，从古代的用水封坛隔绝外界微生物及抑制好氧微生物的生长，到现在各种物理、化学、生物的消毒技术层出不穷，无时不反映着人们对杀灭有害微生物的需求。近期在全球肆虐的新冠病毒，更是对人类及人类社会造成了无法估计的损失。因此，研制开发天然安全的杀菌剂变得尤为必要。
溶菌酶是一种水解酶，能水解细菌细胞壁中粘多糖的β-1,4糖苷键,对革兰氏阳性细菌具有直接的溶解作用，它可以在不破坏其他组织的前提下，选择性地分解微生物的细胞壁。同时，溶菌酶也能够对病毒产生抑制或灭活作用，其抗病毒机制是双方所带的异种电荷能够紧密结合在一起,与病毒DNA、RNA、脱辅基蛋白形成复盐,破坏病毒内部结构以达到抑制或灭活病毒的目的。这些特性使溶菌酶成为一种天然安全的杀菌剂、防腐剂，在医药、食品行业、饲料工业中有着广泛的应用。目前市面上溶菌酶产品来源常以蛋清提取为主，然而，溶菌酶同样存在于人体当中，如眼泪、唾液和乳汁等。相比于其他来源溶菌酶，人源溶菌酶具有更高的溶菌活性和热稳定性，具有天然相容性，更安全可靠，然而其缺点是无法直接从人体中大规模提取。因此，利用微生物进行重组蛋白的生物合成使得大量生产人源溶菌酶成为可能。
防御素是一类富含二硫键的阳离子型多肽，广泛分布于真菌、植物及动物中，是生物免疫系统中的重要调节分子。此外，防御素具有直接的杀菌功能，是一类重要的抗菌肽。在人体中，防御素主要存在于中性粒细胞。防御素发挥抗菌作用可分为三个阶段:
(1)由静电吸引。防御素带正电荷，可通过静电作用与带负电荷的细菌膜脂层结合，完成与靶细胞膜的结合；
(2)通道形成。带正电荷的防御素分子或其多聚体与细菌质膜上带负电荷的磷脂头部和水分子相互作用，显著增加生物膜的通透性，于膜上形成多个稳定的通道；
(3)内容物外泄。通道形成后，防御素进入细胞内的同时，其他胞外分子也伴随进入(如肽、蛋白质或无机离子)，而靶细胞的重要物质(如盐离子和大分子)渗出，致使靶细胞发生不可逆损伤而死亡。
防御素作为一种新型生物活性肽，其抗菌谱广泛，与抗生素阻断大分子生物合成的作用机制完全不同，能够快速杀灭广谱病原微生物以及某些病毒。同时，防御素作为机体本身的一种活性物质，不具有免疫原性，也不易产生耐药性。因此，防御素可以替代抗生素发挥广谱高效的抗菌作用。
现有技术中，专利名称为“一种重组溶菌酶抗菌肽融合蛋白及其制备(专利申请号为：201911406640.1)”的专利中，利用重组溶菌酶抗菌肽融合蛋白进行抗菌。但是，其融合蛋白是利用大肠杆菌进行构建，大肠杆菌容易产生内毒素，重组蛋白安全性低，而且溶菌酶对革兰氏阴性菌的杀菌能力不足。因此，针对上述问题，我们需要一种更加安全可靠，且可以提高抗菌效率的溶酶菌重组蛋白及其构建方法。
发明内容
本发明的目的在于提供一种人源溶菌酶-防御素融合蛋白及其构建方法，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
一种人源溶菌酶-防御素融合蛋白，包括人源溶菌酶蛋白和人防御素多肽，所述人源溶菌酶蛋白的氨基酸序列如SEQ ID NO.2所示，所述人防御素多肽的氨基酸序列如SEQID NO.4所示，所述人源溶菌酶蛋白和人防御素多肽由一段多肽串联构成融合蛋白，所述多肽的氨基酸序列如SEQ ID NO.6所示，所述融合蛋白的氨基酸序列如SEQ ID NO.1所示。
更进一步的方案：所述Linker多肽经密码子偏好性优化后的核苷酸序列如SEQ IDNO.7所示。
更进一步的方案：所述人源溶菌酶蛋白经密码子偏好性优化后的核苷酸序列如SEQ ID NO.3所示。
更进一步的方案：所述人防御素多肽经密码子偏好性优化后的核苷酸序列如SEQID NO.5所示。
更进一步的方案：所述融合蛋白的宿主菌为毕赤酵母。
本发明的另一目的在于提供一种人源溶菌酶-防御素融合蛋白的构建方法，具体包括以下步骤：
1、融合蛋白的核苷酸序列按照设计在生物公司进行合成，按照分子克隆常规操作，通过聚合酶链式反应(PCR)获取目的片段，片段N端有EcoRI酶切位点，C端有NotI酶切位点；
2、采用pPIC9K为质粒载体，使用EcoRI以及NotI限制性内切酶对质粒载体pPIC9K和目的片段进行双酶切，将酶切产物进行连接，转化大肠杆菌DH5α感受态，在AMP抗性平板中进行阳性克隆的筛选；
3、测序验证目的片段后，提取重组质粒pPIC9K-Lysozyme-Defensin，对重组质粒进行SalI单酶切，37℃孵育6h后回收片段；
4、制作毕赤酵母感受态，将SalI单酶切后的重组质粒导入毕赤酵母感受态中进行同源重组，在MD平板上进行阳性克隆的筛选；
5、经过测序验证后的菌株在BMMY培养基中诱导表达，每24h添加甲醇至终浓度为1％，发酵产物过滤，上清液浓缩纯化进行SDS-PAGE实验，检测到重组融合蛋白。
与现有技术相比，本发明的有益效果是：
1.本发明选择的宿主菌为毕赤酵母，这是已经被公认并广泛应用在工业中的模式菌株。相较于大肠杆菌会产生内毒素的缺点，毕赤酵母在表达重组蛋白方面更安全可靠，同时，更适合表达修饰真核来源蛋白；
2.本发明选择人源的溶菌酶及人防御素来进行融合蛋白的构建，并根据酵母表达系统进行密码子优化，表达的融合蛋白更具安全性和高效性；
3.该融合蛋白能够弥补溶菌酶对革兰氏阴性菌杀菌能力不足的缺点，配合人防御素提高抗菌的广谱性，能够高效杀灭大肠杆菌、金黄色葡萄球菌、沙门氏菌等多种致病菌以及病毒等。
附图说明
图1为重组质粒pPIC9K-Lysozyme-Defensin的质粒图谱。
图2为人源溶菌酶-防御素融合蛋白结构区的构建示意图。
图3为人源溶菌酶-防御素融合蛋白的SDS-PAGE电泳检测图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
1、融合蛋白的核苷酸序列按照设计在生物公司进行合成，以下实验均按照分子克隆常规操作：PCR获取目的片段:
引物1：CCGGAATTCAAGGTTTTCGAAAGATGTGAAT，
引物2：CTAGGCGGCCGCACAACAGAAAGCCCACAATC，
该片段N端有EcoRI酶切位点，C端有NotI酶切位点；
2、使用EcoRI以及NotI限制性内切酶对质粒载体pPIC9K和目的片段进行双酶切，将酶切产物进行连接，转化大肠杆菌DH5α感受态，在AMP抗性平板中进行阳性克隆的筛选。筛选引物F：CAGCATTGCTGCTAAAGAAG；R：GAACAGTCATGTCTAAGGCG，目的片段长度600bp；
3、测序验证目的片段后，提取重组质粒pPIC9K-Lysozyme-Defensin，对重组质粒进行SalI单酶切，37℃孵育6h后回收片段；
4、制作毕赤酵母感受态，将单酶切后的重组质粒导入毕赤酵母感受态中进行同源重组，在MD平板上进行阳性克隆的筛选；
5、经过测序验证后的菌株在BMMY培养基中诱导表达，每24h添加甲醇至终浓度为1％，发酵产物过滤，上清液浓缩纯化进行SDS-PAGE实验，检测到重组融合蛋白。
实施例2
产人源溶菌酶-防御素融合蛋白的酵母菌株经发酵、微滤、超滤浓缩，去除酵母菌体，并进行干燥，得到的融合蛋白粉末测定最小抑菌浓度(MIC)，结果如下：
由实验结果可知：本发明设计并制备的人源溶菌酶-防御素融合蛋白对大肠杆菌、金黄葡萄球菌、沙门氏菌和铜绿假单胞菌均具有良好的抗菌性。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
序列表
<110> 沐一生物科技（深圳）有限公司
<120> 一种人源溶菌酶-防御素融合蛋白及其构建方法
<130> 2020.06.25
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Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly
35 40 45
Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp
50 55 60
Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser
65 70 75 80
Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys Ala
85 90 95
Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp
100 105 110
Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys
115 120 125
Gly Val Gly Gly Gly Gly Ser Ala Cys Tyr Cys Arg Ile Pro Ala Cys
130 135 140
Ile Ala Gly Glu Arg Arg Tyr Gly Thr Cys Ile Tyr Gln Gly Arg Leu
145 150 155 160
Trp Ala Phe Cys Cys
165
<210> 2
<211> 130
<212> PRT
<213> 人源溶菌酶蛋白(Lysozyme)
<400> 2
Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly
1 5 10 15
Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala
20 25 30
Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly
35 40 45
Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp
50 55 60
Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser
65 70 75 80
Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys Ala
85 90 95
Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp
100 105 110
Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys
115 120 125
Gly Val
130
<210> 3
<211> 390
<212> DNA
<213> 人源溶菌酶蛋白(Lysozyme)
<400> 3
aaggttttcg aaagatgtga attggctaga actttgaaga gattgggtat ggacggttac 60
agaggtatct ctttggctaa ctggatgtgt ttggctaagt gggaatctgg ttacaacact 120
agagctacta actacaacgc tggtgacaga tctactgact acggtatctt ccaaatcaac 180
tctagatact ggtgtaacga cggtaagact ccaggtgctg ttaacgcttg tcacttgtct 240
tgttctgctt tgttgcaaga caacatcgct gacgctgttg cttgtgctaa gagagttgtt 300
agagacccac aaggtatcag agcttgggtt gcttggagaa acagatgtca aaacagagac 360
gttagacaat acgttcaagg ttgtggtgtt 390
<210> 4
<211> 30
<212> PRT
<213> 人防御素(Defensin -1)
<400> 4
Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr
1 5 10 15
Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys
20 25 30
<210> 5
<211> 90
<212> DNA
<213> 人防御素(Defensin -1)
<400> 5
gcttgttact gtagaatccc agcttgtatc gctggtgaaa gaagatacgg tacttgtatc 60
taccaaggta gattgtgggc tttctgttgt 90
<210> 6
<211> 5
<212> PRT
<213> 多肽(Linker)
<400> 6
Gly Gly Gly Gly Ser
1 5
<210> 7
<211> 15
<212> DNA
<213> 多肽(Linker)
<400> 7
ggtggtggtg gttct 15
Claims (6)
1.一种人源溶菌酶-防御素融合蛋白，其特征在于，包括人源溶菌酶蛋白和人防御素多肽，所述人源溶菌酶蛋白的氨基酸序列如SEQ ID NO.2所示，所述人防御素多肽的氨基酸序列如SEQ ID NO.4所示，所述人源溶菌酶蛋白和人防御素多肽由一段多肽串联构成融合蛋白，所述多肽的氨基酸序列如SEQ ID NO.6所示，所述融合蛋白的氨基酸序列如SEQ IDNO.1所示。
2.根据权利要求1所述的人源溶菌酶-防御素融合蛋白，其特征在于，所述人源溶菌酶蛋白经密码子偏好性优化后的核苷酸序列如SEQ ID NO.3所示。
3.根据权利要求1所述的人源溶菌酶-防御素融合蛋白，其特征在于，所述人防御素多肽经密码子偏好性优化后的核苷酸序列如SEQ ID NO.5所示。
4.根据权利要求1所述的人源溶菌酶-防御素融合蛋白，其特征在于，所述串联人源溶菌酶蛋白和人防御素之间多肽经密码子偏好性优化后的核苷酸序列如SEQ ID NO.7所示。
5.根据权利要求1所述的人源溶菌酶-防御素融合蛋白，其特征在于，所述融合蛋白的宿主菌为毕赤酵母。
6.根据权利要求1-5任一所述的人源溶菌酶-防御素融合蛋白的构建方法，具体包括以下步骤：
(1)融合蛋白的核苷酸序列按照设计在生物公司进行合成，按照分子克隆常规操作，通过聚合酶链式反应(PCR)获取目的片段，片段N端有EcoRI酶切位点，C端有NotI酶切位点；
(2)采用pPIC9K为质粒载体，使用EcoRI以及NotI限制性内切酶对质粒载体pPIC9K和目的片段进行双酶切，将酶切产物进行连接，转化大肠杆菌DH5α感受态，在AMP抗性平板中进行阳性克隆的筛选；
(3)测序验证目的片段后，提取重组质粒pPIC9K-Lysozyme-Defensin，对重组质粒进行SalI单酶切，37℃孵育6h后回收片段；
(4)制作毕赤酵母感受态，将SalI单酶切后的重组质粒导入毕赤酵母感受态中进行同源重组，在MD平板上进行阳性克隆的筛选；
(5)经过测序验证后的菌株在BMMY培养基中诱导表达，每24h添加甲醇至终浓度为1％，发酵产物过滤，上清液浓缩纯化进行SDS-PAGE实验，检测到重组融合蛋白。
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