CN116656516A - 基于两拷贝基因筛选高表达眼镜王蛇联合肽damp4-oh30重组菌株的方法 - Google Patents
基于两拷贝基因筛选高表达眼镜王蛇联合肽damp4-oh30重组菌株的方法 Download PDFInfo
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Abstract
本发明公开了基于两拷贝基因筛选高表达眼镜王蛇联合肽DAMP4‑OH3重组菌株的方法。本发明通过毕赤酵母密码子偏好性优化设计核苷酸序列，构建眼镜王蛇联合肽DAMP4‑OH30两拷贝载体，获得两拷贝的重组表达载体，对重组酵母菌株的诱导表达，制备眼眼镜王蛇联合肽。本发明采用基因工程技术，构建串联两拷贝的质粒，以毕赤酵母为宿主，成功表达两拷贝串联蛋白，将眼镜王蛇联合肽表达产量提高多倍；本发明提供的方法具有通用性强、表达效率较高、具有生物活性、成本低、易规模化生产等优点。
Description
技术领域
本发明涉及基因工程技术领域，尤其涉及基于两拷贝基因筛选高表达眼镜王蛇联合肽DAMP4-OH30重组菌株的方法。
背景技术
利用基因工程的方法异源抗菌肽有两大困难，首先，抗菌肽对某些宿主细胞如毕赤酵母有毒，具极强的杀伤作用；其次，抗菌肽分子量很小，且大多数都是强阳离子抗菌肽，不稳定且容易被表达菌株自身表达的蛋白酶降解。为了解决抗菌肽表达所面临的困难，大规模生产抗菌肽经常与融合标签串联表达。抗菌肽表达常用的融合标签有 MBP、谷胱甘肽S 转移酶（GST）、弹性蛋白样多肽（ELP）、SUMO、硫氧还蛋白 A（Trx A）等。这些融合标签不仅能帮助抗菌肽以可溶性的方式表达，提高抗菌肽的表达量，而且在后续的分离纯化过程中起到一定的作用。
DAMP4是一个能够耐高温高盐的融合蛋白标签，由四个α螺旋结构构成的小分子蛋白，能够通过非层析的方法实现重组蛋白的分离纯化，节省了繁琐的分离纯化步骤，从而降低了生产成本。眼镜王蛇抗菌肽OH-CATH30是由30个氨基酸组成的cathelicidins的截短肽，目前研究发现其对大肠杆菌（Escherichia coli）、铜绿假单胞菌（Pseudomona aeruginosa ）、金黄色葡萄球菌（Staphylococcus aureus）和产气肠杆菌(Enterobacter aerogenes)等多种革兰氏阴性菌和革兰氏阳性菌均具有较好的抑菌效果。因此，眼镜王蛇抗菌肽OH-CATH30是一种非常有前景的药物候选分子，可以用来治疗各种常规抗生素耐药菌引起的感染。
目前，无论是通过化学合成或者生物合成，所获得的眼镜王蛇抗菌肽OH-CATH30产量都无法满足实际使用中的需求。本发明采用眼镜王蛇抗菌肽基因与DAMP4蛋白标签构建联合肽，将其通过酶切酶连形成两拷贝基因，构建酵母表达的重组质粒，建立酵母表达系统用于获取眼镜王蛇抗菌肽联合肽。
发明内容
本发明的目的是要提供基于两拷贝基因筛选高表达眼镜王蛇联合肽DAMP4-OH30重组菌株的方法，采用基因工程技术，构建串联两拷贝的质粒，以毕赤酵母为宿主，成功表达两拷贝串联眼镜王蛇联合肽DAMP4-OH30。
为实现上述目的，本发明采用如下技术方案：
（1）眼镜王蛇联合肽DAMP4-OH30两拷贝载体的构建
①根据之前已经构建好的DAMP4-OH30-pPICZαA重组质粒，分别利用两组限制性内切酶对重组质粒进行双酶切，获得目的片段；同时对pPICZαA质粒进行双酶切，回收酶切后的线性化pPICZαA质粒。
②利用T4连接酶，将双酶切后获得的目的片段进行连接，随后将连接产物与双酶切后回收的pPICZαA质粒进行连接，获得多拷贝数的重组表达载体，对连接产物进行鉴定。
（2）重组酵母菌株的诱导表达及检测
①重组表达载体转入毕赤酵母中，筛选重组酵母表达菌。
②对同源重组成功的毕赤酵母进行诱导表达，从而得到分泌表达的两拷贝眼镜王蛇抗菌肽联合肽蛋白。
优选的，所述的pPICZαA表达载体构建包括以下内容：pPICZαA载体的酶切及酶切产物回收；眼镜王蛇抗菌肽联合肽序列和载体的连接；连接产物的鉴定。
优选的，所述的连接产物的鉴定包括制备感受态细胞、转化反应、重组质粒提取和鉴定。
其中，所述的眼镜王蛇抗菌肽基因的核苷酸序列可以是任何一种基因的核苷酸序列。核苷酸序列是通过毕赤酵母密码子偏好性优化后的核苷酸序列。
本发明选用毕赤酵母为宿主菌。毕赤酵母具有真核生物特有的蛋白高效分泌表达，翻译后修饰，因此，近年来毕赤酵母成为表达外源目的蛋白，特别是真核来源的外源蛋白的高效表达系统。
本发明对测序鉴定后的质粒酶切线性化后电转化X33毕赤酵母感受态细胞，转化后的细胞涂布于YPD平板，挑选单克隆，对克隆进行小量发酵培养，发酵液采用SDS-PAGE分离后，以获得表达眼镜王蛇联合肽的发酵液蛋白。发酵液蛋白经Ni-NTA琼脂糖树脂亲和层析后，使用相应的蛋白酶进行切割，再对所得到的眼镜王蛇联合肽纯化。
本发明的有益效果在于：
（1）本发明采用基因工程技术，构建串联两拷贝的质粒，以毕赤酵母为宿主，成功表达两拷贝串联蛋白，将眼镜王蛇联合肽表达产量提高多倍；本发明提供的方法具有通用性强、表达效率较高、具有生物活性、成本低、易规模化生产等优点。
（2）本发明提供的构建方法获得的两拷贝表达相较于眼镜王蛇联合肽的单拷贝表达，使得眼镜王蛇联合肽的产量提高2倍以上，且并未增加生产工序，生产成本基本与单拷贝表达持平。
附图说明
图1为眼镜王蛇联合肽DAMP4-OH30单拷贝基因在pPICZαA质粒上的插入位点示意图（括号内基因序列为被联合肽基因替代的pPICZαA质粒序列基因）；
图2为两拷贝载体的构建的方案设计图；
图3为抑菌活性实验结果图，眼镜王蛇联合肽DAMP4-OH30对大肠杆菌抑菌（Escherichia coli）效果的抑菌圈，编号1-6代表6组发酵罐号，发酵诱导表达培养时间为96h，左图为单拷贝联合肽抑菌结果，右图为两拷贝抑菌结果。
图4为抑菌活性实验结果图，眼镜王蛇联合肽DAMP4-OH30对金黄色葡萄球菌（S.aureus）的抑菌结果，编号1-6代表6组发酵罐号，发酵诱导表达培养时间为96h，左图为单拷贝联合肽抑菌结果，右图为两拷贝抑菌结果。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
蛋白胨Peptone、硫酸铵、甲醇、琼脂、葡萄糖、磷酸钾缓冲液、甘油、山梨醇购于国药集团化学试剂有限公司；
YNB购于北京索莱宝科技有限公司；
酵母提取物Yeast extract购于OXOID公司；
细菌菌株购自中国普通微生物菌种保藏管理中心。
除非特别指明，本发明以下实施例所用的试剂均为分析纯试剂，且可从常规渠道商购获得。
本发明中的YPD培养基的配制:酵母提取物Yeast extract 1%,蛋白胨Peptone2%, 葡萄糖 2%，固体培养基则添加琼脂粉 2%。
本发明中的BMMY培养基的配制：
酵母提取物Yeast extract 1%，蛋白胨Peptone 2%, 磷酸钾缓冲液(pH=6.0)100mmol/L, YNB 1.34%, 硫酸铵 1%，加水配置成100ml的培养基。毕赤酵母诱导表达培养基，YNB过滤除菌。摇瓶培养时每24小时之后加入0.5%的甲醇诱导，一般诱导72小时。
BMGY培养基的配制：
酵母提取物Yeast extract 1%,蛋白胨Peptone 2%, 磷酸钾缓冲液(pH6.0)100mmol/L, YNB 1.34%, 硫酸铵 1%,甘油 1%，加水配置成100ml的培养基。
山梨醇培养基的配制：
取18.217g山梨醇溶于100ml无菌水中配置成的1M山梨醇培养基，0.22μm滤膜过滤除菌。
实施例1
1、眼镜王蛇抗菌肽基因与DAMP4蛋白标签构建联合肽DAMP4-OH30，根据构建的眼镜王蛇联合肽氨基酸序列，按照毕赤酵母对遗传密码子的偏好性对眼镜王蛇联合肽基因进行密码子优化，全基因合成并插入到pPICZαA载体上。
GAACCATCTATGAAGCAATTGGCTGACTCTTTGCACCAATTGGCTAGACAAGTTTCTCGTTTGGAACACGCTGAACCATCTATGAAGCAATTGGCTGACTCTTTGCACCAATTGGCTAGACAAGTTTCTCGTTTGGAACACGCTGAACCATCTATGAAGCAATTGGCTGACTCTTTGCACCAATTGGCTAGACAAGTTTCTCGTTTGGAACACGCTGAACCATCTATGAAGCAATTGGCTGACTCTTTGCACCAATTGGCTAGACAAGTTTCTCGTTTGGAACACGCTCCAGGCGGCGGCGGCTCTGGAGGCGGCGGATCTTTGGTTCCAAGAGGCTCTAAGTTCTTCAAAAAGTTGAAGAACTCTGTAAAGAAAAAGGCTAAGAAATTCTTCAAGAAACCAAGAGTTATTGGCGTCTCTATTCCATTC
具体操作为pPICZαA载体的KEX2酶切位点之后到TGA之间的序列全部删除，用已经密码子偏爱性优化后的眼镜王蛇联合肽基因替换，末尾加TAA终止，再连接载体TGA之后的序列。眼镜王蛇联合肽单拷贝基因全合成图如图1所示。
实施例2
1）分别利用两组限制性内切酶Bgl II-Age I和Xcm I-BamH I对DAMP4-OH30-pPICZαA重组质粒进行双酶切，对酶切后的目的基因片段进行切胶回收，获得两段包含有联合肽DAMP4-OH30的基因片段；同时，利用限制性内切酶Xcm I和Age I对pPICZαA质粒进行双酶切，回收酶切后的线性化质粒；
2）利用T4连接酶，将Bgl II-Age I和Xcm I-BamH I双酶切后获得的目的片段16℃连接8h，将连接后获得的目的基因片段与Xcm I和Age I双酶切后的pPICZαA质粒16℃连接8h，将连接产物转化至E.coli DH5α感受态细胞中，涂布于含有博来霉素（100 μg/mL）的LB琼脂平板上，37℃培养12h后挑取单菌落，以上游引物5′AOX primer和下游引物3′AOXprimer进行菌液PCR，随后将重组菌株送往生工公司进行DNA核苷酸测序；
3）将含测序正确的重组质粒的菌株划线培养，挑取单克隆添加到LB培养基中，37℃ 180rpm摇培12h，然后利用试剂盒提取重组质粒。
实施例3
将80 μL酵母感受态细胞和5µg Sac I线性化后的质粒混合后转移至电转杯中，冰浴5min。电击后立刻向电转杯加入200 μL山梨醇培养基，混均后，将全部菌液涂布在含有博来霉素（100 μg/mL）的YPD琼脂平板上，在28℃ 培养3 d。挑取YPD平板上长出的单克隆提取酵母基因组进行PCR鉴定，筛选Mut+重组酵母表达菌株；
2）挑取Mut+重组酵母表达菌株单菌落接入BMGY培养基，30℃，280rpm振荡培养，培养菌体浓度至OD600=2.0-6.0，更换培养基为BMMY培养基，每隔24h，补加终浓度为0 .5%的甲醇。发酵120h，收集的上清液，通过SDS-PAGE凝胶分析。分析结果显示酵母成功表达蛋白，且不同拷贝数间蛋白量无明显差异。
实施例4
分别取不同拷贝数的眼镜王蛇联合肽DAMP4-OH30表达菌株在相同情况下进行发酵（采用BMMY培养基进行发酵，初始OD600为2.0-6.0，转速280rpm，每隔24h添加培养基总量的0.5%的甲醇，共发酵120h。
取部分上清进行SDS-PAGE检测，以此衡量其蛋白产量。发酵上清经过HPLC纯化，然后经质谱鉴定，得到纯品，用微量BCA蛋白定量分析试剂盒测其浓度。不同拷贝数眼镜王蛇联合肽DAMP4-OH30产量对比如下表：
表1不同拷贝数眼镜王蛇联合肽DAMP4-OH30产量对比
| 拷贝数 | 1 | 2 |
| 产量 | 26 mg/L | 60 mg/L |
实施例5
用25mL LB液体培养基培养大肠杆菌和金黄色葡萄球菌，37℃ 180rpm过夜培养后，再转接到25mL LB液体培养基中，同样条件培养1.5h-2h，OD600值在0.4-0.6之间，取30μL菌液到100 mL LB固体培养基中，混匀后倒平板；待其凝固后将牛津杯放在表面，分别加入50μL重组表达的眼镜王蛇抗菌肽联合肽样品，同时用pPICZαA空载体做空白对照组，37℃过夜培养后测定其透明圈的大小，一共做3组实验。得出重组表达的眼镜王蛇抗菌肽联合肽对大肠杆菌和金黄色葡萄球菌有明显抑制效果。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
Claims (1)
1.基于两拷贝基因筛选高表达眼镜王蛇联合肽DAMP4-OH3重组菌株的方法，其特征在于包括以下步骤：
（1）眼镜王蛇联合肽DAMP4-OH3两拷贝载体的构建
①根据已构建好的DAMP4-OH30-pPICZαA重组质粒，分别利用两组限制性内切酶对重组质粒进行双酶切，同时对pPICZαA质粒进行双酶切，回收酶切后的线性化质粒；
②利用T4连接酶，将双酶切后获得的目的片段进行连接，随后将连接产物与双酶切后回收的pPICZαA质粒进行连接，获得多拷贝数的重组表达载体，对连接产物进行鉴定；
（2）重组酵母菌株的诱导表达及检测
①重组表达载体转入毕赤酵母中，筛选重组酵母表达菌；
②对同源重组成功的毕赤酵母进行诱导表达，从而得到分泌表达的两拷贝眼镜王蛇抗菌肽联合肽蛋白。
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