CN110358770B - 一种酵母生物合成芋螺毒素的方法 - Google Patents
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Abstract
本发明公开了一种酵母表达芋螺毒素的生物学制备方法。将芋螺毒素成熟肽基因按照毕赤酵母对遗传密码子偏爱性优化，人工合成的成熟肽基因，将其克隆至带有sfGFP的表达载体，转入酵母后获得表达芋螺毒素的酵母工程菌，制备芋螺毒素。本发明采用的酵母表达系统表达具有生物活性的芋螺毒素,安全性极高,表达产物可用于神经性等疾病潜在药物,具有生产成本较低，可实现规模化生产等优点。
Description
技术领域
本发明涉及芋螺毒素生物合成的方法,尤其涉及芋螺毒素毕赤酵母酵母表达制备领域，属于芋螺毒素的基因工程制备领域。
背景技术
芋螺毒素（Conotoxins）又称芋螺肽（Conopeptides）是芋螺通过长鼻内的齿舌射出用于捕食毒液中活性肽。芋螺毒素多数由 8-86 个氨基酸残基组成，富含二硫键，包括了至今发现的最小神经肽毒素。每种芋螺的毒液中含有100-200 种不同的毒素，能选择性的作用于细胞膜上的钠、钾、钙等多种离子通道和神经递质的受体，干扰神经或其他细胞中的信号传递。
芋螺毒素不仅可作为临床药物或新药导向化合物，还可为药物分子设计提供有价值的新药效模型和结构构架，更能为发现药物新作用靶位发挥特殊作用。芋螺毒素在探讨毒理药理机制、疾病病因和建立新药物靶位方面均可发挥不可替代的特殊作用。但是目前难以获得一定量的纯化芋螺毒素严重制约其相关药理研究及应用。
目前，化学合成是芋螺毒素获得主要途径，但人工合成芋螺毒素的成本很高，还不能完全满足作为药物商业化生产的要求和相关药理活性的研究。采用芋螺毒素成熟肽基因，连接真核生物的信号肽，构建酵母表达的重组质粒，建立酵母表达系统用于获取芋螺毒素。
发明内容
本发明的目的在于提供一种采用酵母生物合成获得芋螺毒素的方法。
基于上述目的，本发明提供以下技术方案：
1）GenBank 中检索获得芋螺毒素小肽序列或成熟肽基因,按照毕赤酵母对遗传密码子的偏爱性对反翻译芋螺毒素成熟肽基因进行密码子优化；
2）对pPink-HC质粒进行改造：首先加入分泌信号a-mating facter，将质粒改造成分泌表达质粒pPink-HC-MF；其次加入sfGFP，作为筛选信号，成功构建pPink-HC-MF-GFP；最后将芋螺毒素成熟肽基因克隆至pPink-HC-MF-GFP；
3）重组表达载体转入毕赤酵母,筛选重组酵母表达菌；
4）对阳性克隆子进行诱导表达，荧光检测外源蛋白表达，获得表达芋螺毒的毕赤酵母工程菌；
其中,所述的芋螺毒素基因的核苷酸序列可以是任何一种芋螺毒素基因的核苷酸序列。核苷酸序列是通过酵母密码子偏爱优化后的核苷酸序列。
所述的带有sfGFP标签的表达载体为pPink-HC，超折叠绿色荧光蛋白（superfolder green fluorescent protein，sfGFP），sfGFP 是无毒的，它可在不同的有机体中，高水平地予以表达，而对有机体的生理学影响很小的。此外，GFP与目的蛋白融合表达时可保持其荧光的发射能力，对目的蛋白活性影响小。酵母表达系统是目前基因工程中成熟的表达方法。本发明采用了载体作为蛋白表达载体,该载体的特点是带有sfGFP 标签,会产生sfGFP和目的蛋白的融合蛋白。此外，sfGFP上游或下游带有TEV酶切位点Glu-Asn-Leu-Tyr-Phe-Gln-Gly。
该融合蛋白的序列中，芋螺毒素既可以融合GFP蛋白后，也可以在GFP蛋白前；GFP和连接部分的氨基酸序列允许有多种突变。
所述的酵母重组表达载体构建包括以下内容：pPinK-HC载体的酶切及酶切产物回收；芋螺毒素序列和载体的连接；连接产物的鉴定。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。
针对于大部分芋螺毒素二硫键较多，存在翻译后修饰的特点,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质粒,酶切线性化后电转化prb1和pep4双蛋白酶缺陷型毕赤酵母感受态细胞,转化后的细胞涂布于Pichia Adenine Dropout Agar （PAD）平板,挑选单克隆,对克隆进行小量发酵培养,对酵母菌检测荧光,发酵液采用15% SDS-PAGE分离后，胶上直接观察荧光，以获得表达芋螺毒素的毕赤酵母工程菌。对甲醇诱导目的蛋白的表达条件进行了优化,结果发现,诱导时间对于蛋白表达量有着较为显著的影响, 当96h时,蛋白表达量最高。
发酵液蛋白经Ni-NTA琼脂糖树脂亲和层析后，采用CCK8测试蛋白活性。结果显示抑制昆虫细胞系sf9生长，具有细胞毒性。
本发明的有益效果在于：
（1）芋螺毒素保守估计有5万种，而仅有两三个芋螺毒素在酵母中可直接表达成功，表明芋螺毒素在酵母中直接表达成功率极低。这可能原因是芋螺肽分子量小，具有毒性，表达量极低或易被宿主降解。本发明采用酵母表达芋螺毒素时，加入sfGFP作为荧光筛选信号，同时其可作为保护蛋白与芋螺毒素融合表达，成功制备芋螺毒素。该技术具有通用性，作为一个芋螺毒素表达平台，可将不同的芋螺毒素在酵母中成功表达。
（2）本发明采用基因工程技术，构建sfGFP融合蛋白的质粒，以毕赤酵母为宿主，成功表达芋螺毒素与GFP的融合蛋白，且融合蛋白具有活性。也可通过进一步酶切GFP后，获得芋螺毒素小肽，进行药物研发。本发明制备芋螺毒素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。
附图说明
图1 荧光观察诱导后的CalTx-GFP重组酵母菌。
图2 PAGE胶上直接荧光观察酵母不同诱导时间蛋白表达量。
图3 Western-blot检测酵母不同诱导时间蛋白表达量。
图4 MALDI-TOF/TOF测定CalTx–GFP蛋白分子量。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限此。
实施例1
CalTx基因的全合成
根据CalTx蛋白序列（NCPAGCRSQGCCM），优化为酵母菌偏爱的密码子基因片段。基因序列两端引入限制性酶切位点Sph I、Stu I，合成引物见表1。取相应的上游和下游引物（100 µmol/L）各5uL，加40uL无菌水，混匀。98℃变性1 min，立即终止反应，在PCR仪上自然冷却降温，然后放置冰上10min。即分别完成CalTx基因的全合成。 -20冰箱中保存。
表1
实施例2
（1）对pPink-HC质粒进行改造：首先将分泌信号a-mating facter，插入到经EcoRI和Sph I双酶切的质粒pPink-HC，将质粒改造成分泌表达质粒pPink-HC-MF；其次加入sfGFP，作为筛选信号。具体操作是将PCR扩增的sfGFP插入到经Stu I和Fse I双酶切的pPink-HC-MF中，成功构建pPink-HC-MF-GFP；其中a-mating facter，sfGFP序列分别如下：
a-mating facter：
atgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttacttagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctttggataaaaga；
sfGFP：
TCCAAAGGAGAAGAGCTGTTCACTGGGGTTGTACCCATTTTGGTAGAACTGGACGGAGATGTAAACGGACATAAATTCTCTGTTAGAGGTGAGGGCGAAGGCGATGCCACCAATGGTAAATTGACTCTGAAGTTTATATGCACTACGGGTAAATTACCTGTTCCTTGGCCAACCCTAGTAACAACTTTGACATATGGTGTTCAATGTTTCTCAAGATACCCAGACCATATGAAAAGGCATGATTTCTTTAAAAGTGCTATGCCAGAAGGCTACGTGCAAGAGAGAACTATCTCCTTTAAGGATGACGGTACGTATAAAACACGAGCAGAAGTGAAATTCGAAGGGGATACACTAGTTAATCGCATCGAATTAAAGGGTATAGACTTTAAGGAAGATGGTAATATTCTCGGCCATAAACTTGAGTATAATTTCAACTCGCATAATGTGTACATTACAGCTGACAAACAAAAGAACGGAATTAAAGCGAATTTTAAAATCAGGCACAACGTCGAAGATGGGTCTGTTCAACTTGCCGATCATTATCAGCAAAACACCCCTATTGGTGATGGTCCAGTCTTGTTACCCGATAATCACTACTTAAGCACACAGTCTAGATTGTCAAAAGATCCGAATGAAAAGCGTGATCACATGGTTTTATTGGAATTTGTCACCGCTGCAGGAATAACTCACGGCATGGACGAGCTGTACAAG。
（2）重组融合蛋白CalTx-GFP的构建
采用限制性内切酶SphI、StuI双酶切的pPink-MF-HC-GFP（本实验室构建）。胶回收后的产物与合成的CalTx基因用T4 DNA连接酶连接，转入感受态E.coli DH5α。挑选单克隆，采用PCR及测序进行质粒阳性鉴定。
实施例3
重组酵母菌的诱导表达及检测
将80 ul prb1和pep4双蛋白酶缺陷型毕赤酵母感受态细胞和5µg EcoN 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1ml YPDS，上下摇动混匀。28℃培养2h。混均后，吸取300µl 菌液涂布在PAD平板上，在28℃培养3～7d。挑取3-8白色且菌落大的单克隆，再一次在PAD平板面划线，28℃培养3～7 d，将构建表达质粒转入PichiaPink Strain 4。挑取单菌落接入，30℃，280rpm振荡培养。培养菌体浓度至OD600=2~6，更换为BMMY培养基。每隔24h，补加甲醇至终浓度为0.5wt%。发酵120h，收集的上清液，-80℃保存备用。菌体采用荧光显微镜观察经甲醇诱导后的酵母。酵母细胞发出绿色荧光，表明酵母成功表达融合蛋白CalTx-sfGFP融合蛋白（图1）。
采用SDS-PAGE检测发酵液上清液融合蛋白。跑胶结束后，切除溴酚蓝条带。采用GBox Chemi XT4荧光化学发光成像系统（Gene company limited）观察胶上荧光（图2）。五天内，发酵液上清中均观察到荧光，酵母成功分泌表达GFP融合蛋白至胞外。随着发酵时间延长，融合蛋白表达量增加，在84h～96h左右，达到高峰。然后将胶放入转膜缓冲液，进行Western-blot检测（图3）。结果同样验证了融合蛋白成功分泌至发酵液中，且随着发酵时间延长，融合蛋白表达量增加。
实施例4
蛋白的纯化及分子量测定
收集发酵液的上清液，采用GFP单克隆柱进行纯化，0.1M Gly(pH3.0)洗脱，收集洗脱液。纯化后蛋白，送往上海生物工程有限公司。采用基质辅助激光解吸电离飞行时间质谱（5800 MALDI-TOF/TOF）对样品的相对分子质量进行测定。MALDI-TOF/TOF测定纯化获得蛋白相对分子质量为29568.36Da，峰单一，与CalTx-GFP预测结果一致。这表明CalTx-GFP融合蛋白在酵母中成功表达（图4）。因此，酵母表达小肽CalTx的快速筛选方法成功建立。
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。
SEQUENCE LISTING
<110> 福建农林大学
<120> 一种酵母生物合成芋螺毒素的方法
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Asn Cys Pro Ala Gly Cys Arg Ser Gln Gly Cys Cys Met
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tacttagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180
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Claims (1)
1.一种在毕赤酵母中表达芋螺毒素的方法,其特征在于，包括以下步骤：
1）GenBank 中检索获得芋螺毒素小肽序列或成熟肽基因,按照毕赤酵母对遗传密码子的偏爱性对反翻译芋螺毒素成熟肽基因进行密码子优化；
2）对pPink-HC质粒进行改造：首先加入分泌信号a-mating facter，将质粒改造成分泌表达质粒pPink-HC-MF；其次加入sfGFP，作为筛选信号，成功构建pPink-HC-MF-GFP；最后将芋螺毒素成熟肽基因克隆至pPink-HC-MF-GFP；
3）重组表达载体转入毕赤酵母,筛选重组酵母表达菌；
4）对阳性克隆子进行诱导表达，荧光检测外源蛋白表达，获得表达芋螺毒素的毕赤酵母工程菌；
5）纯化后，采用质谱测定准确分子量验证芋螺毒素与GFP融合蛋白表达；
所述芋螺毒素小肽序列为NCPAGCRSQGCCM，采用的引物序列为：CaITx-P：CAACTGTCCAGCTGGTTGTAGATCTCAAGGTTGTTGTATGAGG，CaITx-R：CCTCATACAACAACCTTGAGATCTACAACCAGCTGGACAGTTGCATG。
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