CN106967740A - 一种大肠杆菌融合表达菌丝霉素、其制备方法及应用 - Google Patents
一种大肠杆菌融合表达菌丝霉素、其制备方法及应用 Download PDFInfo
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- CN106967740A CN106967740A CN201710085950.2A CN201710085950A CN106967740A CN 106967740 A CN106967740 A CN 106967740A CN 201710085950 A CN201710085950 A CN 201710085950A CN 106967740 A CN106967740 A CN 106967740A
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- plectasin
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- fusion protein
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Abstract
本发明公开了一种融合表达菌丝霉素、其制备方法和应用，该重组菌丝霉素的氨基酸序列为SEQ ID NO:1；按照大肠杆菌偏爱密码子设计的核苷酸序列为SEQ ID NO:2。本发明利用原核表达载体构建了能够表达菌丝霉素‑硫氧还蛋白融合蛋白的大肠杆菌BL21(DE3)宿主菌。将该菌株扩增培养，通过异丙基‑β‑D‑硫代半乳糖苷诱导表达，离心收获菌体，裂解菌体后离心收获上清，经过亲和层析纯化获得菌丝霉素融合蛋白，无需通过酶切去除硫氧还蛋白即可以显著抑制革兰氏阳性金黄色葡萄球菌、肺炎链球菌等的生长。本发明无需通过酶切去除融合蛋白即可有显著的抑菌作用，大大降低了生产成本。
Description
背景技术
抗菌肽是一类广泛存在于自然界生物体中的小肽类物质，它是机体先天性免疫系统的重要组成部分。由于抗菌肽对细菌、真菌、寄生虫、病毒、肿瘤细胞等有着广泛的抑制作用，并且随着越来越多的抗生素耐药微生物的出现，使得抗菌肽在医药行业和食品添加剂等领域有良好的应用前景。菌丝霉素是一种真菌防御素，属于抗菌肽的一种。菌丝霉素的成熟功能片段有40个氨基酸,其分子量为4.4kD,其生物学活性主要表现为对革兰氏阳性菌有较强的杀菌作用,尤其是对肺炎链球菌、脓性链球菌、金黄色葡萄球菌等,有与青霉素、万古霉素相当的抗菌作用。研究表明菌丝霉素在体外抑菌试验、动物试验及一些临床试验中表现出无细胞毒性优势,且不产生耐药性,因此作为抗生素替代物具有相当大的治疗潜力。
目前国内外对菌丝霉素基因工程表达已有了一些研究，但是由于菌丝霉素抗菌肽具有抗菌作用，表达宿主如大肠杆菌中表达的菌丝霉素抗菌肽会反馈性抑制宿主细胞活性，影响菌丝霉素抗菌肽的进一步表达，因此在菌丝霉素抗菌肽基因工程研究中常采用酵母菌表达，或者大肠杆菌中加上大的融合蛋白一起表达后采用酶切去除融合蛋白标签纯化后制得，但两种不同的表达方式成本均较高，不适合广泛用于动物饲料添加剂、抗菌药物制剂、保健品或防腐剂，因此市场上尚无成熟的菌丝霉素抗菌肽产品。而本发明采用大肠杆菌融合表达的菌丝霉素无需酶切去除融合蛋白标签，对革兰氏阳性菌有较强的杀菌作用，但对宿主大肠杆菌无明显毒性，解决了菌丝霉素产业化的瓶颈问题。
发明内容
本发明的目的是提供一种融合表达菌丝霉素的制备及应用方法，该方法利用大肠杆菌表达系统表达菌丝霉素融合蛋白，所制备的菌丝霉素抗菌肽可应用于治疗、预防革兰氏阳性菌感染或作为畜禽饲料添加剂。
本发明首先提供一种菌丝霉素融合蛋白，由菌丝霉素抗菌肽和硫氧还蛋白融合表达获得，其氨基酸序列为SEQ ID NO:1。
上述菌丝霉素融合蛋白的一种基因编码核苷酸序列为SEQ ID NO:2。
上述菌丝霉素融合蛋白的表达和纯化的方法，包括以下步骤：
1)根据菌丝霉素和硫氧还蛋白氨基酸序列按照大肠杆菌密码子偏好设计融合基因DNA序列；
2)人工合成步骤1)中融合基因，连接入原核表达载体中，构建成表达重组质粒；
3)将构建的表达重组质粒转化宿主菌，构建能表达菌丝霉素融合蛋白的重组基因工程菌；
4)对该重组基因工程菌进行增值培养和诱导表达培养，收集菌体；
5)将步骤4)所得菌体，进行裂解，离心，收集裂解上清；
6)对步骤5)中所得菌体裂解上清进行Ni-NTA亲和层析纯化，即得菌丝霉素融合蛋白。
本发明的有益效果是：
本发明利用大肠杆菌原核表达系统制备了一种新的菌丝霉素融合蛋白，将菌丝霉素抗菌肽与硫氧还蛋白融合表达，有利于抗菌肽中巯基的形成，实现可溶性表达，同时也有利于菌丝霉素发挥抑菌作用，不必通过蛋白酶切除硫氧还蛋白也可以发挥菌丝霉素抑菌作用，大大降低了制备成本，解决了产业化的瓶颈问题。
附图说明
图1为菌丝霉素融合蛋白的模拟空间构象图；
图2为双酶切鉴定菌丝霉素表达质粒的凝胶电泳图；其中M：DNA分子量标准；1：表达质粒未酶切；2：表达质粒双酶切后；
图3为菌丝霉素融合蛋白重组菌诱导表达后的SDS-PAGE鉴定图；其中M：蛋白质分子量标准；1：表达硫氧还蛋白的宿主菌；2：表达菌丝霉素融合蛋白的宿主菌破碎后离心上清；3：表达菌丝霉素融合蛋白的宿主菌破碎后离心沉淀；
图4为菌丝霉素融合蛋白的HPLC纯度分析图。
具体实施方式
申请人根据菌丝霉素和硫氧还蛋白氨基酸序列设计融合基因DNA序列，通过化学合成的方式获得相应的DNA序列并插入原核表达载体，转化大肠杆菌BL21(DE3)，构建重组基因工程菌，通过对工程菌的诱导、超声破碎、蛋白纯化制备出具有显著抑制金黄色葡萄球菌和肺炎链球菌等革兰氏阳性菌的菌丝霉素融合蛋白。
下面结合具体的实施方式对本发明做进一步的详细说明，所述内容是对本发明的解释而非限定。本领域技术人员应该理解的是，在不偏离本发明的技术方案的情况下可以对本发明的技术方案的细节和形式进行修改或替换(如更换原核表达载体或宿主菌等)，这些修改和替换均落入本发明保护范围内。
实施例1菌丝霉素融合蛋白的制备工艺流程
(1)菌丝霉素融合蛋白基因的设计、合成
根据已发表的文献(Mygind PH，et al.Nature,2005；437(13):975-980)中报道的腐生子囊菌菌丝霉素的氨基酸序列和Genbank中硫氧还蛋白(Trx)的氨基酸序列，再参考大肠杆菌偏好密码子设计菌丝霉素融合蛋白基因，菌丝霉素与Trx之间用GGGGS柔性Linker连接，设计的基因序列如下：
CATATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTGGCGGTGGTAGTATGGGCTTTGGCTGTAATGGTCCGTGGGATGAAGATGATA TGCAGTGCCATAATCATTGTAAATCTATCAAAGGCTACAAAGGTGGTTATTGCGCGAAAGGCGGTTTTGTGTGCAAA TGTTATCTCGAG(其中波浪下划线部分为Noc I和Xho I酶切位点，粗下划线部分为柔性Linker，细下划线部分为菌丝霉素基因，其它部分为硫氧还蛋白基因。)由上海捷瑞生物工程有限公司按照设计的DNA序列合成基因并测序鉴定。其模拟空间构象如图1所示。
(2)菌丝霉素融合蛋白原核表达载体的构建
将合成的菌丝霉素融合蛋白基因和原核表达载体pET30b(+)经Noc I和Xho I双酶切，用1.5％琼脂糖凝胶电泳后用DNA凝胶回收试剂盒回收目的基因和载体基因片段。通过T4DNA连接酶将菌丝霉素融合蛋白基因通过Noc I和Xho I酶切位点插入pET30b(+)载体，将构建好的重组质粒转化大肠杆菌BL21(DE3)感受态细菌，接种至含有100μg/mL卡那霉素的LB平板培养基上，次日挑取单菌落在含100μg/mL卡那霉素的液体LB培养基中扩增培养，通过质粒小量提取试剂盒提取质粒，经Noc I和Xho I双酶切后用1.5％琼脂糖凝胶电泳，在450bp左右出现条带，结果如图2所示。同时将质粒进行DNA测序鉴定。
(3)重组菌的诱导表达
选取鉴定正确的重组菌接种于含氨苄青霉素(100μg/mL)的液体LB培养基中少量扩增，随后接种于新的液体LB培养基(含氨苄青霉素100μg/mL)中放大培养2～3h后，测OD值在达到0.6时，加入终浓度为0.05M的IPTG，32℃诱导表达5h，收集细菌，破碎后离心，经SDS-PAGE电泳检测在菌体破碎上清中于22kDa处可见优势表达条带，其结果如图3所示。
(4)菌丝霉素融合蛋白的纯化
将菌体破碎、离心后上清液与Ni-NTA树脂混合，4℃结合1h装入层析柱，用pH7.4磷酸盐缓冲液配制的50mmol/L，100mmol/L，500mmol/L咪唑依次进行洗涤，流速2mL/min，收集500mmol/L咪唑洗脱液；通过Sephadex G25脱盐层析柱，用pH6.5的50mMTris-HCl缓冲液洗脱，收集洗脱峰；再用阳离子交换层析CM柱进一步纯化，用pH5.0的PB洗脱，收集流穿峰，再依次用0.3M NaCl和0.5M NaCl洗涤，收集0.5M NaCl洗脱峰，即为纯化后的菌丝霉素融合蛋白。经液相色谱(HPLC)检测纯度大于95％，其结果如图4所示。
实施例2菌丝霉素融合蛋白的抑菌、杀菌实验
(1)纸片法检测菌丝霉素融合蛋白的抑菌效果
测试菌种分别为临床耐甲氧西林金黄色葡萄球菌MRSA15471114、MRSA15471118和耐青霉素肺炎链球菌PRSP31355，其中以金黄色葡萄球菌MRSA15471114和MRSA15471118用M-H培养基培养，耐青霉素肺炎链球菌PRSP31355用含5％绵羊血M-H培养基培养，37℃下培养至OD600＝0.5，取100μL菌液均匀涂布在琼脂平板上。在琼脂平板表面均匀放置高压灭菌过的纸片，在纸片上分别滴加30μL 1mg/mL菌丝霉素和10μL 1mg/mL头孢氨苄。以滴加30μLddH2O的纸片作为阴性对照。结果表明，菌丝霉素融合蛋白对三种临床耐药菌均有抑菌效果。
(2)最小抑菌浓度(MIC)和最小杀菌浓度(MBC)实验鉴定菌丝霉素融合蛋白的抑菌杀菌活性
细菌的培养同步骤(1)。将菌液用生理盐水调节至在625nm波长检测吸收值为0.09～0.10，然后在试管中用相应的培养基对菌液进行倍比稀释，再加入菌丝霉素融合蛋白使其终浓度分别为1000μg/mL、100μg/mL、10μg/mL、1μg/mL、0.5μg/mL，在37℃下培养12小时判断最小抑菌浓度结果。在产生抑菌效果的孔中取10μL菌液接种在琼脂培养基上，37℃培养12小时用以判断最小杀菌浓度结果。实验均以氨苄西林为对照，并设三组平行。结果表明：菌丝霉素对临床耐甲氧西林金黄色葡萄球菌MRSA15471114的MIC为10μg/mL，MBC为10μg/mL；对MRSA15471118的MIC为10μg/mL，MBC为10μg/mL；对耐青霉素肺炎链球菌PRSP31355的MIC为1μg/mL，MBC为1μg/mL，具有较好的抑菌及杀菌效果。
实施例3菌丝霉素融合蛋白在小鼠体内的急性毒性试验
本实验的目的是观察菌丝霉素融合蛋白对小鼠有无毒性影响。采用健康的BALB/c小鼠40只，雌雄各半，体重22±0.31g。菌丝霉素融合蛋白1mg/mL，每日肌肉注射1次，0.1mL/次，连续注射7天，观察小鼠的毒性反应。实验结果表明，在试验过程中小鼠无异常反应，饮食及活动正常，40只小鼠全部存活。处死小鼠，观察其心、肝、肺、脾、肾、胃肠等脏器均无异常。证明菌丝霉素融合蛋白对动物无毒性作用。
实施例4菌丝霉素融合蛋白在治疗母牛乳腺炎中的应用
选择诊断为乳腺炎的泌乳期4～8岁青壮年黑白花奶牛，共15头，随机分为3组，每组5头。第1组为空白对照组，不给予药物；第2组为菌丝霉素组，将菌丝霉素融合蛋白(0.1mg/mL)给予乳房灌注，每天给药2次，每次给药20mL；第3组为药物治疗组，按青霉素G钾10万单位，进行乳房内注射给药，每天给药2次，连续给药7d。
观察指标为痊愈和有效：痊愈指临床乳腺炎的症状全部消失，乳房组织柔软，牛奶中没有凝块，牛奶颜色较白。有效指临床乳腺炎的症状减轻，或者不再恶化，牛奶正常，但乳房肿胀没有完全消失。结果如表1所示，菌丝霉素组痊愈3头，有效2头；药物治疗组痊愈2头，有效2头，无效1头；空白对照组5头全部无效。可见菌丝霉素融合蛋白对治疗奶牛乳腺炎100％有效，而抗生素治疗有效率仅80％，提示菌丝霉素融合蛋白对耐药菌有很好的抑制作用，能够用于治疗奶牛乳腺炎且不会造成抗生素残留。
表1菌丝霉素治疗母牛乳腺炎的疗效观察
实施例5菌丝霉素融合蛋白对肉仔鸡生长性能的影响
选择1日龄白羽肉仔鸡120羽，随机分成3组，每组40羽，对照组饲喂基础日粮，黄霉素组饲喂基础日+5mg/kg黄霉素，菌丝霉素组饲喂基础日粮+2.5mg/kg菌丝霉素融合蛋白冻干粉。试验期间仔鸡自由采食，自由饮水，饲养管理和免疫按常规方法进行。试验期42d(前期0～21d，后期22～42d)。饲养期间每日记录各组的采食量及鸡只的死淘情况，并于21日龄和41日龄时称重，计算日增重比和料重比。结果显示，试验前期(0～21d)，黄霉素组、菌丝霉素组日增重比对照组分别提高了1.33％和2.14％，料重比分别降低了1.77％和3.01％；与黄霉素组相比，菌丝霉素组日增重提高了0.81％，料重比降低了1.24％。试验后期(22～42d)，黄霉素组、菌丝霉素组日增重比对照组分别提高了3.46％和6.32％,料重比分别降低了6.55％和13.41％；与黄霉素组相比，菌丝霉素组日增重提高了2.86％，料重比降低了6.86％。总体来看(0～42d)，黄霉素组、菌丝霉素组日增重比对照组分别提高了2.04％和6.05％，料重比分别降低了7.15％和11.41％；与黄霉素组相比，菌丝霉素组日增重提高了4.01％，料重比降低了4.26％。实验结果表明菌丝霉素融合蛋白可以显著提高白羽肉鸡的生长性能，提高饲料利用率，且效果优于黄霉素。
SEQUENCE LISTING
<110> 芜湖天明生物技术有限公司
<120> 一种大肠杆菌融合表达菌丝霉素、其制备方法及应用
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 159
<212> PRT
<213> 菌丝霉素
<400> 1
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<210> 2
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<212> DNA
<213> 菌丝霉素
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ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180
atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240
ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300
aaagagttcc tcgacgctaa cctggccggt ggcggtggta gtatgggctt tggctgtaat 360
ggtccgtggg atgaagatga tatgcagtgc cataatcatt gtaaatctat caaaggctac 420
aaaggtggtt attgcgcgaa aggcggtttt gtgtgcaaat gttatctcga g 471
Claims (6)
1.一种大肠杆菌融合表达菌丝霉素，其特征在于，所述融合表达菌丝霉素由菌丝霉素抗菌肽和硫氧还蛋白融合表达获得。
2.根据权利要求1所述的一种大肠杆菌融合表达菌丝霉素，其特征在于，所述融合表达菌丝霉素氨基酸序列为序列表中SEQ ID NO:1。
3.根据权利要求2所述的一种大肠杆菌融合表达菌丝霉素，其特征在于，所述融合表达菌丝霉素的基因编码核苷酸序列为SEQ ID NO:2。
4.根据权利要求1任意一项所述的大肠杆菌融合表达菌丝霉素，其特征在于，所述大肠杆菌包括JM109、BL21、DH5α、Rosetta或Top10菌株。
5.一种权利要求1所述大肠杆菌融合表达菌丝霉素的制备方法，其特征在于，所述的制备方法包括：
1)将菌丝霉素抗菌肽和硫氧还蛋白串联，得到融合抗菌肽的氨基酸序列；
2)根据大肠杆菌密码子的偏爱性，设计融合抗菌肽的重组DNA序列；
3)将重组DNA序列构建到重组质粒表达载体上；
4)将表达载体导入大肠杆菌进行融合菌丝霉素抗菌肽的表达。
6.权利要求1-5任意一项所述的大肠杆菌融合表达菌丝霉素在制备饲料添加剂、抗菌药物制剂、保健品、防腐剂中的应用。
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