CN115558019A - 一种眼镜王蛇抗菌肽oh-cath30的基因工程制备方法 - Google Patents
一种眼镜王蛇抗菌肽oh-cath30的基因工程制备方法 Download PDFInfo
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Abstract
本发明公开了一种眼镜王蛇抗菌肽OH‑CATH30的基因工程制备方法,具体为眼镜王蛇抗菌肽OH‑CATH30的表达载体pPIC9K‑OH‑CATH30的构建、诱导表达产物和纯化。具体操作为:载体pPIC9K中插入了用于重组表达序列为SEQIDNo.1的OH‑CATH30基因序列,构建表达载体pPIC9K‑OH‑CATH30,表达载体在宿主细胞毕赤酵母中进行诱导表达,获得表达产物重组多肽OH‑CATH30。本发明利用基因工程表达方法生产获得的抗菌肽OH‑CATH30,具有明显的抑菌活性,降低生产成本,缩短生产周期,增加产量,能够在水产养殖业、医药行业中产业化应用。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种眼镜王蛇抗菌肽OH-CATH30的基因工程制备方法。
背景技术
抗菌肽是存在于生物体内能抵抗外界微生物侵害的一类小分子多肽,它是先天免疫系统的重要组成部分,多数抗菌肽具有抗微生物活性,对革兰氏阳性和革兰氏阴性细菌、真菌和病毒都有抑杀作用,有些抗菌肽还具有抗菌特异性。抗菌肽抑菌机理主要是作用于细菌的细胞膜,破坏其完整性并产生穿孔现象,造成细胞内容物溢出胞外而死亡,不易产生耐药性,因此抗菌肽的产业化应用是解决抗生素抗药性问题的一条有效途径。
目前抗菌肽的产业化应用研究还处在起步阶段,常用的生产方法主要有生物提取法、化学合成法和基因工程表达。天然抗菌肽在生物体内含量极低,靠天然物提取应用是不可能的,且生物提取法的生产成本高,不能满足规模化生产的需求。化学合成法是利用已知氨基酸序列的抗菌肽来化学合成,但合成的大多为3-9个氨基酸序列组成的线性短肽,且化学合成毒性大,成本高,产量低,周期长,合成1kg具备20个氨基酸的抗菌肽的成本约为2000万元,严重制约抗菌肽的产业化发展。
眼镜王蛇抗菌肽OH-CATH30由34个氨基酸序列组成,对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌和产气肠杆菌等多种革兰氏阳性菌和革兰氏阴性菌均具有较好的抑菌效果。但由于眼镜王蛇抗菌肽OH-CATH30天然提取产量少,成本高;化学合成的眼镜王蛇抗菌肽OH-CATH30毒性大,成本高,产量低,周期长,严重限制了眼镜王蛇抗菌肽OH-CATH30的产量和产业化应用。
发明内容
本发明的目的是提供一种眼镜王蛇抗菌肽OH-CATH30的基因工程制备方法,利用基因工程表达方法生产获得抗菌肽OH-CATH30,具有明显的抑菌活性,降低生产成本,缩短生产周期,增加产量,解决眼镜王蛇抗菌肽OH-CATH30的产量瓶颈和产业化应用的问题。
针对上述目的,本发明的技术方案如下:
眼镜王蛇抗菌肽OH-CATH30的制备方法,包括以下步骤:
S01.眼镜王蛇抗菌肽OH-CATH30表达载体pPIC9K-OH-CATH30的构建,具体操作如下:PCR扩增眼镜王蛇抗菌肽OH-CATH30基因序列,获得抗菌肽OH-CATH30的目的基因片段,将OH-CATH30基因片段与pPIC9K载体连接,得到pPIC9K-OH-CATH30重组载体;
S02.将步骤S01获得的重组载体转化入宿主细胞,并对宿主细胞进行诱导表达,获得表达产物;
S03.对步骤S02获得的表达产物进行分离纯化,获得重组多肽OH-CATH30。
进一步的,步骤S02中所述宿主细胞为毕赤酵母GS115。
进一步的,步骤S03中所述分离纯化的方法具体特征在于:先对表达产物进行离心,收集上清液,进行透析后,最后进行亲和层析法纯化。
进一步的,所述眼镜王蛇抗菌肽OH-CATH30具有SEQIDNo.1所示的基因序列。
进一步的,所述眼镜王蛇抗菌肽OH-CATH30具有SEQIDNo.2所示的氨基酸片段序列。
本发明优点:本发明利用基因工程表达方法生产获得重组多肽OH-CATH30,重组多肽OH-CATH30具有明显的抑菌活性,降低生产成本,缩短生产周期,增加产量,可满足产业化的需求。
附图说明
附图1为OH-CATH30 PCR电泳图谱;M为DNA Marker DL2000,1为OH-CATH30 PCR片段。
附图2为pPIC9K载体酶切前后电泳图;M为DNA Marker DL10000,1为pPIC9K载体酶切前片段,2为pPIC9K载体酶切后片段。
附图3为pPIC9K-OH-CATH30重组质粒凝胶电泳图;M为DNA Marker DL10000,1为pPIC9K-OH-CATH30重组质粒片段。
附图4为pPIC9K-OH-CATH30表达载体构件图。
附图5为重组多肽OH-CATH30的液相色谱分析图。
具体实施方式
下面结合附图、具体实施例对本发明作进一步说明。
实验试剂和材料
1.眼镜王蛇抗菌肽OH-CATH30,来自眼镜王蛇(Ophiophagus hannah)的毒腺。
2.载体及宿主细胞:pPIC9K质粒和毕赤酵母GS115菌株均购自于淼灵质粒平台。E.coliDH5α菌株购自生工生物工程(上海)股份有限公司。
3.酶类及必要生化试剂:PrimeSTAR酶,EcoRⅠ、Not Ⅰ和Sac Ⅰ限制性内切酶均购自宝日医生物技术(北京)有限公司。
4.培养基:LB培养基:蛋白胨,酵母浸膏,氯化钠以及固体培养基所需琼脂粉均购自国药集团。
YPD培养基:酵母浸膏,蛋白胨,葡萄糖以及固体培养基所需琼脂粉均购自国药集团。
MD平板:无氨基酵母氮源购自北京索莱宝科技有限公司,葡萄糖和琼脂粉均购自国药集团。
BMGY培养基:酵母浸膏,蛋白胨,磷酸氢二钾,磷酸二氢钾,甘油均购自国药集团,无氨基酵母氮源购自北京索莱宝科技有限公司,生物素购自生工生物工程(上海)股份有限公司。
BMMY培养基:酵母浸膏,蛋白胨,磷酸氢二钾,磷酸二氢钾,甲醇均购自国药集团,无氨基酵母氮源购自北京索莱宝科技有限公司,生物素购自生工生物工程(上海)股份有限公司。
咪唑购自国药集团,氨苄青霉素购自生工生物工程(上海)股份有限公司。
5. AXYGEN凝胶回收试剂盒,AXYGEN质粒提取试剂盒,AXYGEN PCR产物回收试剂盒均购自爱思进生物技术(杭州)有限公司,HisTrap预装柱购自GE通用电气公司。
6.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。
实施例1重组多肽OH-CATH30的制备方法
1.眼镜王蛇抗菌肽OH-CATH30重组载体pPIC9K-OH-CATH30的构建
1)PCR扩增OH-CATH30基因序列、PCR产物的双酶切
根据pPIC9K载体多克隆位点和眼镜王蛇抗菌肽OH-CATH30基因序列,设计扩增OH-CATH30基因的上、下游引物,以眼镜王蛇抗菌肽OH-CATH30的cDNA为模板进行PCR扩增,分别以OH-CATH30-F和OH-CATH30-R为上、下游引物,利用PrimeSTAR酶扩增OH-CATH30基因的目的片段,混合均匀,反应体系为:
无菌MiliQ水 | 22μl |
PrimeSTARMaxPremix | 25μl |
OH-CATH30cDNA | 1μl |
上游引物(20pmol/μl) | 1μl |
下游引物(20pmol/μl) | 1μl |
总反应体积 | 50μl |
PCR反应条件为:94℃ 3min;94℃ 0.5min;57℃ 0.5min;72℃ 1min;72℃10min;16℃ 5min;30个循环;如图1所示,PCR产物长度为142bp。
上游引物OH-CATH30-F:5′-GGGGAATTCAAGAGATTCAAGAAATTTTTCAAGA-3′;
下游引物OH-CATH30-R:
5′-CATGCGGCCGCTTAATGGTGATGGTGATGATGGAAGGGGATGGAGACTC-3′;
取PCR产物1µg,用EcoR Ⅰ和Not Ⅰ限制性内切酶双酶切,37℃孵育4h,反应体系为:
PCR回收产物 | 5μl |
10×H酶反应缓冲液 | 12μl |
0.1%BSA | 12μl |
0.1%Tritox-100 | 12μl |
<i>EcoR</i> Ⅰ限制性内切酶 | 1μl |
<i>Not</i> Ⅰ限制性内切酶 | 1μl |
无菌MiliQ水 | 77μl |
总反应体积 | 120μl |
反应结束后用AXYGEN凝胶回收试剂盒回收除去酶及核苷酸碎片,得到具有EcoRⅠ和Not Ⅰ的粘性末端的OH-CATH30片段。
2)pPIC9K载体的提取、双酶切
将pPIC9K载体转化至E.coliDH5α感受态细胞中,涂布于含有50μg/ml氨苄青霉素的LB琼脂平板上,37℃培育12小时。挑取单克隆菌落,接种于20ml含50µg/ml的氨苄青霉素的LB液体培养基中,180 rpm/min,37℃培养8h至OD600值达到0.5,离心收集菌体,使用AXYGEN质粒提取试剂盒小量提取pPIC9K质粒。
取1µg上述纯化的pPIC9K质粒,用EcoRⅠ和Not Ⅰ限制性内切酶双酶切,37℃孵育4h,反应体系为:
pPIC9K质粒 | 10μl |
10×H酶反应缓冲液 | 12μl |
0.1%BSA | 12μl |
0.1%Tritox-100 | 12μl |
<i>EcoR</i> Ⅰ限制性内切酶 | 1μl |
<i>Not</i> Ⅰ限制性内切酶 | 1μl |
无菌MiliQ水 | 72μl |
总反应体积 | 120μl |
如图2所示,反应结束后,用AXYGEN PCR产物回收试剂盒除去酶及核苷酸碎片,得到具有EcoRⅠ和Not Ⅰ的粘性末端的pPIC9K载体。
3)pPIC9K-OH-CATH30载体的连接、转化和鉴定
pPIC9K载体和OH-CATH30片段经过EcoRⅠ和Not Ⅰ双酶切后具有相同的粘性末端,将二者连接,反应体系为:
pPIC9K载体 | 2.9μl |
OH-CATH30片段 | 2.1μl |
Ligation Solution Ⅰ | 5μl |
总反应体积 | 10μl |
16℃孵育12个小时;次日,将10µl连接产物转化至E.coliDH5α感受态细胞中,涂布于含有50μg/mL氨苄青霉素的LB琼脂平板上培养12个小时。次日挑取单菌落,用上游引物、下游引物进行菌液PCR反应,得到阳性克隆菌,随后将阳性克隆菌送往生工生物公司进行测序,如图3和图4所示。
上游引物5′AOX primer:5′-GACTGGTTCCAATTGACAAGC-3′
下游引物3′AOX primer:5′-AGGATGTCAGAATGCCATTTGCC-3′
2.pPIC9K-OH-CATH30重组质粒在毕赤酵母GS115中的诱导表达
1)pPIC9K-OH-CATH30的线性化
将测序正确的pPIC9K-OH-CATH30菌株划线培养,挑取单克隆,用AXYGEN质粒小提中量试剂盒提取pPIC9K-OH-CATH30质粒。取3µg质粒由Sac Ⅰ限制性内切酶线性化,反应体系为:
重组质粒pPIC9K-OH-CATH30 | 60μl |
<i>Sac</i> Ⅰ限制性内切酶 | 4μl |
10×L酶反应缓冲液 | 12μl |
无菌MiliQ水 | 44μl |
总反应体积 | 120μl |
AXYGEN凝胶回收试剂盒回收线性化后的pPIC9K-OH-CATH30质粒。采用电击法将线性化后的pPIC9K-OH-CATH30质粒转化至毕赤酵母GS115感受态细胞中,涂布于MD平板,28℃培养48h。
2)重组多肽OH-CATH30的诱导表达
从MD平板上挑取单克隆菌落,接种于10ml生长培养基(BMGY)中,设置摇床的温度为28℃,转速为 230 rpm/min,将上述培养基在摇床中培养16小时至OD600值达到2,离心倒掉培养基收集菌体,加入10ml表达培养基(BMMY),用0.5%甲醇诱导表达24小时。
选用毕赤酵母GS115作为宿主细胞,具有以下特点:采用AOX1启动子,酵母信号肽α-factor因子引导重组多肽OH-CATH30的分泌表达,C端添加6×His-Tag便于亲和层析纯化目的多肽。
.亲和层析法纯化眼镜王蛇抗菌肽OH-CATH30重组多肽
1)、上柱前样品的制备
上述实施例2中pPIC9K-OH-CATH30重组菌株诱导表达24h后,12,000 rpm/ min 4℃低温离心30min,收集上清液。PBS透析液4℃冷藏,透析液是50mM磷酸缓冲液和50mMNaCl,透析液pH=8.0,透析上述收集的上清液2~3次,随后12,000 rpm/ min 4℃低温离心30min,再次收集上清液,经0.45μm滤膜过滤,待上柱纯化。
2)、亲和层析纯化OH-CATH30重组多肽
(1)、准备层析试剂,用镍离子螯合亲和层析柱亲和纯化,层析试剂为:
溶液A:20mM磷酸缓冲液、50mM NaCl、10mM咪唑,pH=8.0;
溶液B:20mM磷酸缓冲液、500mM NaCl、1M咪唑,pH=8.0。
(2)、上柱纯化OH-CATH30
①、用5~10个柱体积MilliQ水清洗预装柱(HisTrapTMFFCrude5ml),5~10个柱体积溶液A平衡HisTrap层析柱;
②、将准备好的上清液全部上柱,流速为2ml/min;
③、用5~10个柱体积的溶液A洗去未结合的杂蛋白,用35%溶液B洗脱目的多肽蛋白,35%溶液B含350mM咪唑,并收集洗脱组分。
OH-CATH30重组多肽表达产物能够特异性的与镍离子螯合亲和层析柱结合,在较高浓度的咪唑溶液竞争洗涤,洗脱掉杂蛋白后,用35%溶液B洗脱目的多肽,得到目的多肽,测得纯度>95%。
(3)、透析OH-CATH30重组多肽
将多肽在磷酸盐缓冲液中透析,随后透析入MilliQ水中。
(4)、利用液相色谱进行OH-CATH30重组多肽对比分析
最后以抗菌肽OH-CATH30的合成肽为标准品(纯度≥98%),将透析后获得的目的多肽利用液相色谱进行分析。
结果显示:重组表达获得的目的多肽与抗菌肽OH-CATH30的合成肽保留时间一致,如图5所示。
实施例2眼镜王蛇抗菌肽OH-CATH30的抑菌活性测定
(1)将由上述方法所获得的眼镜王蛇抗菌肽OH-CATH30多肽样品过滤除菌后,以眼镜王蛇抗菌肽OH-CATH30的合成肽为标准品应用绘制蛋白浓度标准曲线,根据公式计算出重组多肽OH-CATH30的浓度。
(2)将多肽溶液倍比稀释至1-32μg/ml。
(3)进行MIC(Minimum Inhibitory Concentration,最小抑菌浓度)的测定,所述MIC测定在96孔细胞培养板上进行。具体操作如下:
①革兰氏阳性菌金黄色葡萄球菌和革兰氏阴性菌大肠埃希氏菌两种菌悬液的准备:取被测细菌划线于MH平板,培养12个小时;
③每种被测菌按照以下操作设置空白对照组、阳性对照组和待测样品实验组,每组设置2个平行样,每种菌重复3次:
Ⅰ阳性对照组:加入50μl磷酸钠盐缓冲液和50μl菌悬液;
Ⅱ空白对照组:加入50μl待测多肽样品和50μl磷酸钠盐缓冲液;
Ⅲ样品实验组:加入50μl待测多肽样品和50μl菌悬液;
④细菌培养24~48h后,观察MIC结果。
抑菌实验结果显示,眼镜王蛇抗菌肽OH-CATH30具有广谱的抑菌活性,能够有效的抑制革兰氏阳性菌如金黄色葡萄球菌(MIC 2~4μg/ml),革兰氏阴性菌大肠埃希氏菌(MIC 2~4μg/ml)的生长。结果如表1
表1 眼镜王蛇抗菌肽OH-CATH30的抑菌活性
CGMCC No.:中国微生物菌种保藏编号;MIC:最小抑菌浓度,用(a)-(b)表示,(a)表示肉眼可见菌体生长的最高浓度,(b)表示不见菌体生长的最小浓度。
从表1数据可知,重组表达获得的眼镜王蛇抗菌肽OH-CATH30对革兰氏阳性菌金黄色葡萄球菌和革兰氏阴性菌大肠杆菌均具有较好的抑菌效果。
实施例3眼镜王蛇抗菌肽OH-CATH30产量的测定
实验表明,通过基因重组表达最后得到的眼镜王蛇抗菌肽OH-CATH30的产量达到约1000mg/kg,生产周期为4天,每生产1kg的眼镜王蛇抗菌肽OH-CATH30的成本大约在3800元。
序列表
<110> 江苏亢钧生物科技有限公司
<120> 一种眼镜王蛇抗菌肽OH-CATH30的基因工程制备方法
<130> 20220401
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<213> 眼镜王蛇(Ophiophagus hannah)
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ttcttcaaga agccgagggt catcggagtc tccatcccct tctaa 105
<210> 2
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<212> PRT
<213> 眼镜王蛇(Ophiophagus hannah)
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Lys Arg Phe Lys Lys Phe Phe Lys Lys Leu Lys Asn Ser Val Lys Lys
1 5 10 15
Arg Ala Lys Lys Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile
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Pro Phe His His His His His His
35 40
Claims (5)
1.一种眼镜王蛇抗菌肽OH-CATH30的基因工程制备方法,其特征在于包括以下步骤:
S01.眼镜王蛇抗菌肽OH-CATH30表达载体pPIC9K-OH-CATH30的构建,具体操作如下:PCR扩增眼镜王蛇抗菌肽OH-CATH30基因,获得抗菌肽OH-CATH30的目的基因片段,将OH-CATH30基因片段与pPIC9K载体连接,得到pPIC9K-OH-CATH30重组载体;
S02.将步骤S01获得的重组载体转化入宿主细胞,并对宿主细胞进行诱导表达,获得表达产物;
S03.对步骤S02获得的表达产物进行分离纯化,获得重组多肽OH-CATH30。
2.如权利要求1所述的一种眼镜王蛇抗菌肽OH-CATH30的基因工程制备方法,其特征在于在所述S02步骤中,所述宿主细胞为毕赤酵母GS115。
3.如权利要求1所述的一种眼镜王蛇抗菌肽OH-CATH30的基因工程制备方法,其特征在于在步骤S03中,所述分离纯化的方法具体特征在于:先对表达产物进行离心,收集上清液,进行透析后,最后进行亲和层析法纯化。
4.一种眼镜王蛇抗菌肽OH-CATH30,其特征在于所述眼镜王蛇抗菌肽OH-CATH30的表达载体用于表达序列为SEQIDNo.1的眼镜王蛇抗菌肽OH-CATH30基因序列。
5.如权利要求4所述的一种眼镜王蛇抗菌肽OH-CATH30,其特征在于所述眼镜王蛇抗菌肽OH-CATH30的氨基酸片段序列为SEQIDNo.2。
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CN116200420A (zh) * | 2022-12-15 | 2023-06-02 | 江苏亢钧生物科技有限公司 | 一种高效表达眼镜王蛇抗菌肽oh-cath30的串联多拷贝重组表达载体及其应用 |
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CN116200420A (zh) * | 2022-12-15 | 2023-06-02 | 江苏亢钧生物科技有限公司 | 一种高效表达眼镜王蛇抗菌肽oh-cath30的串联多拷贝重组表达载体及其应用 |
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