CN116555319A - 多拷贝整合与高密度发酵合成短吻鳄抗菌肽am-cath的方法 - Google Patents
多拷贝整合与高密度发酵合成短吻鳄抗菌肽am-cath的方法 Download PDFInfo
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Abstract
本发明公开了多拷贝整合与高密度发酵合成短吻鳄抗菌肽AM‑CATH的方法，属于基因工程与生物工程技术领域。本发明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，将短吻鳄抗菌肽AM‑CATH表达产量提高2‑3倍。然后通过构建和优化两拷贝整合方法，并通过发酵工艺优化，实现短吻鳄抗菌肽AM‑CATH的高水平合成，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，尤其涉及多拷贝整合与高密度发酵合成短吻鳄抗菌肽AM-CATH的方法。
背景技术
短吻鳄抗菌肽AM-CATH是从美国短吻鳄（Alligator mississippiensis）发现了的一种抗菌肽，其由一个N端的α-螺旋和一个中心脯氨酸铰链组成。抗菌肽AM-CATH以及其两个截短肽对多种革兰氏阴性菌，包括临床分离的多重耐药鲍曼不动杆菌（Acinetobacter baumannii）和耐碳青霉烯肺炎克雷伯菌（Klebsiella pneumoniae）有较强的抑菌活性。使用溴化乙二胺吸收试验，发现这些肽可透过细菌细胞膜，对盐抑制的敏感性低于许多其他已知的肽链。在3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化铵（MTT）作用24小时后，短吻鳄抗菌肽AM-CATH对绵羊红细胞无溶血作用，对A549人肺上皮细胞无明显细胞毒性。与人类的LL-37相似，短吻鳄抗菌肽cathelicidins可能在短吻鳄的先天免疫反应中发挥重要作用。
目前，无论是通过化学合成或者生物合成方法合成短吻鳄抗菌肽AM-CATH，所获得的短吻鳄抗菌肽AM-CATH产量都无法满足实际工业使用中的需求。本发明采用短吻鳄抗菌肽基因，将其通过酶切酶连形成多拷贝基因，构建酵母表达的重组质粒，建立酵母表达系统用于获取短吻鳄抗菌肽。
发明内容
本发明通过采用基因工程技术，构建串联两拷贝的质粒，以毕赤酵母为宿主，通过发酵工艺优化，实现短吻鳄抗菌肽AM-CATH的高水平合成。
为实现上述目的，本发明采用如下技术方案：
1）将短吻鳄抗菌肽AM-CATH全基因合成并插入到pPICZαA载体上，成功构建单拷贝AM-CATH-pPICZαA重组质粒，分别利用两组限制性内切酶对单拷贝重组质粒进行双酶切，获得两段包含有抗菌肽AM-CATH的基因片段；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，获得两拷贝数的重组表达载体，对连接产物进行鉴定；
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。
1）将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短吻鳄抗菌肽AM-CATH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；短吻鳄抗菌肽AM-CATH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，所述的短吻鳄抗菌肽AM-CATH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。
本发明对提取的重组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，以获得表达短吻鳄抗菌肽AM-CATH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，再对所得到的短吻鳄抗菌肽AM-CATH纯化。
本发明的有益效果在于：
（1）本发明采用酵母表达短吻鳄抗菌肽AM-CATH时，通过串联两拷贝的方式，将短吻鳄抗菌肽AM-CATH表达产量提高2-3倍。
（2）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；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。
（3）本发明相较于短吻鳄抗菌肽AM-CATH的单拷贝表达，使得短吻鳄抗菌肽AM-CATH的产量提高2-3倍，且并未增加生产工序，生产成本与单拷贝表达的成本基本持平。
附图说明
图1为短吻鳄抗菌肽单拷贝基因全合成图；
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，短吻鳄抗菌肽AM-CATH对大肠杆菌（E. coli）的抑菌结果，左图为单拷贝AM-CATH抑菌结果，右图为两拷贝AM-CATH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。
图4为短吻鳄抗菌肽AM-CATH对金黄色葡萄球菌（S. aureus）的抑菌结果，左图为单拷贝AM-CATH抑菌结果，右图为两拷贝AM-CATH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。
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 2.6%，蛋白胨Peptone 2%, 磷酸钾缓冲液(pH=6.0)100mmol/L, YNB 1.2%, 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.短吻鳄抗菌肽单拷贝基因的全合成
根据现有的短吻鳄抗菌肽氨基酸序列，按照毕赤酵母对遗传密码子的偏好性对短吻鳄抗菌肽基因进行密码子优化，全基因合成并插入到pPICZαA载体上。
GGT TTG TTC AAG AAG TTG AGA AGA AAG ATT AAG AAG GGT TTC AAG AAG ATTTTC AGA AAG TTG CCA CCA ATT GGT GTT GGT GTT TCT ATT CCA TTG GCT GGA AAG AGA
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短吻鳄抗菌肽基因替换，末尾加TAA终止，再连接载体TGA之后的序列。短吻鳄抗菌肽单拷贝基因全合成图如图1所示。
实施例2
两拷贝载体的构建
1）分别利用两组限制性内切酶Bgl II-Age I和Xcm I-BamH I对AM-CATH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，获得两段包含有抗菌肽AM-CATH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，获得两拷贝的重组表达载体，随后将表达载体送往生工公司进行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
重组酵母菌的诱导表达及检测
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
两拷贝表达产量与单拷贝表达产量的比较
分别取不同拷贝数的短吻鳄抗菌肽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。不同拷贝数短吻鳄抗菌肽产量对比如下表：
表1不同拷贝数短吻鳄抗菌肽AM-CATH产量对比
| 拷贝数 | 1 | 2 |
| 产量 | 18mg/L | 31 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重组表达的短吻鳄抗菌肽样品，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。得出重组表达的短吻鳄抗菌肽AM-CATH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。
Claims (3)
1.多拷贝整合与高密度发酵合成短吻鳄抗菌肽AM-CATH的方法，其特征在于包括以下步骤：
A、构建重组短吻鳄抗菌肽AM-CATH多拷贝表达载体
1）将短吻鳄抗菌肽AM-CATH全基因合成并插入到pPICZαA载体上，成功构建单拷贝AM-CATH-pPICZαA重组质粒，分别利用两组限制性内切酶对单拷贝重组质粒进行双酶切，获得两段包含有抗菌肽AM-CATH的基因片段；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，PCR扩增，获得两拷贝数的重组表达载体，对重组表达载体进行鉴定；
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；
B、重组短吻鳄抗菌肽AM-CATH菌株的制备
1）将重组表达载体转入毕赤酵母中，筛选重组酵母表达菌株；
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短吻鳄抗菌肽AM-CATH蛋白。
2.根据权利要求1所述的多拷贝整合与高密度发酵合成短吻鳄抗菌肽AM-CATH的方法的步骤A中（3），提取重组质粒后对测序鉴定后的重组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，以获得表达短吻鳄抗菌肽AM-CATH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，再对所得到的短吻鳄抗菌肽AM-CATH纯化。
3.根据权利要求1所述的多拷贝整合与高密度发酵合成短吻鳄抗菌肽AM-CATH的方法的步骤B中（1）中诱导表达的BMMY培养基的配制：酵母提取物Yeast extract 2.6%，蛋白胨Peptone 2%, 磷酸钾缓冲液(pH=6.0)100mmol/L, YNB 1.2%, 硫酸铵 1%，加水配置成100ml的培养基。
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