CN104447976B - 一种猪源抗菌肽cecropin P1突变体及其制备方法和应用 - Google Patents
一种猪源抗菌肽cecropin P1突变体及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种新型猪源抗菌肽cecropin P1突变体及其制备方法和应用。本发明通过生物软件对猪源抗菌肽cecropin P1的氨基酸序列进行空间结构分析，将其31个氨基酸中的2处氨基酸进行突变(R10L、K12N)，并在C末端添加天冬酰胺以利于C末端的酰胺化，获得一种新型猪源抗菌肽突变体，使其抗菌效力获得显著提高。在此基础上，选用毕赤酵母偏爱密码子，人工合成新型猪源抗菌肽cecropin P1突变体基因，克隆于毕赤酵母中表达，获得新型抗菌肽重组酵母菌株，并将发酵规模放大到发酵罐水平，实现抗菌肽产品的高密度发酵和高效表达。发酵液进一步纯化后可制成粉剂、液体等抗菌肽制剂用于畜禽疾病的预防和治疗。
Description
技术领域
本发明属于功能多肽筛选技术领域，具体涉及一种新型猪源抗菌肽cecropin P1突变体及其制备方法和应用。
背景技术
抗生素的滥用不仅导致了药物残留等公共卫生问题，而且由于耐药菌株和新的致病亚型菌株的出现，使得细菌性疾病的防治再次成为困扰人类的难题。所以开发新的抗菌药物成为一个越来越重大的研究课题。抗菌肽(Antibacterial peptide，ABP)又叫抗微生物肽(Antimicrobial peptide)、抗生素肽(Antibiotics peptide)，是生物体经诱导产生的一类具有多种生物活性的小分子多肽，是宿主免疫防御系统的一个重要组成部分，具有广谱抗菌、无耐药性及毒副作用等优点。目前为止，已经有千余种抗菌肽被分离，它们广泛分布于哺乳动物、鱼类、昆虫、两栖动物、鸟类、植物和细菌等生物体内。
抗菌肽cecropin P1是Lee等(1989)首先从猪小肠分离出得到的，其含有31个氨基酸残基，分子量是3339Da，不含半胱氨酸，不能形成分子内二硫键，有强碱性的N端和强疏水性的C端，其中酰胺化的C端对其广谱抗菌作用极为重要。cecropin P1氨基酸序列与昆虫cecropin A有64％相似性，与cecropin B有75％的相似性。对其二级结构的理论预测及CD谱和二维核磁共振数据表明，其分子内含有两亲水性α-螺旋和疏水性α-螺旋，中间是形成柔性弯曲的谷氨酸-甘氨酸(Glu-Gly)卷曲序列。研究表明，其螺旋-卷曲-螺旋结构对保持高抗菌活性具有特殊的重要性。cecropin P1对革兰氏阴性菌和一部分革兰氏阳性菌都有抗菌活性。
与传统抗生素相比，抗菌肽虽有较多优势，并可能成为抗生素的替代品，但仅有少数抗菌肽进入临床试验，其他均由于各种原因停止于各期临床试验阶段，而且失败的原因尚不清楚据推测可能是由于全身或局部毒性、不稳定性以及耐药性有关。
发明内容
本发明的目的是提供一种新型猪源抗菌肽突变体，使其抗菌效力获得显著提高，从而弥补现有技术的不足。
本发明首先提供一种新型猪源抗菌肽，其编码蛋白的氨基酸序列为SEQID NO:1；
上述蛋白的一种核苷酸序列为SEQ ID NO:2；
本发明的抗菌肽用于制备饲料添加剂。
本发明还提供一种猪源抗菌肽的重组毕赤酵母的制备方法，其制备步骤如下：
1)根据猪源抗菌肽的氨基酸序列，选用毕赤酵母偏好密码子，合成抗菌肽基因序列，连入重组酵母表达载体中，构建成表达重组质粒；
2)将构建的表达重组质粒电转化宿主酵母菌，构建能表达猪源抗菌肽cecropinP1突变体的重组基因工程酵母菌；用该重组基因工程菌高密度发酵表达出猪源抗菌肽cecropin P1突变体。
本发明利用基因工程技术构建了能表达一种新型猪源抗菌肽cecropin P1突变体的重组酵母菌株。将重组猪源抗菌肽cecropin P1突变体制备成抗菌肽制剂，其抑菌效价高、抑菌谱广，具有广阔的市场前景和开发价值。
具体实施方式
下面结合具体实施方式来进一步描述本发明，但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，这些修改和替换均落入本发明保护范围内。
实施例1新型猪源抗菌肽cecropin P1突变体及其基因的获得
1、猪源抗菌肽cecropin P1(GenBank登录号：AB186032)是由31个氨基酸组成的具有双亲性α螺旋的抗菌肽，对细菌有很强的抑菌活性。为了进一步提高其抑菌活性，通过生物软件对猪源抗菌肽AB186032的氨基酸序列进行空间结构分析，选择亲水端α螺旋的两个氨基酸(10L、12N))用亲水性更强的精氨酸(R)和赖氨酸(K)进行替换，目的是为了进一步提高亲水性和其与细胞膜的结合能力，同时在抗菌肽的C末端增加天冬酰胺以促进C末端的酰胺化，最后获得一种新型猪源抗菌肽cecropin P1突变体，其氨基酸序列为SEQ ID NO:1。
2、根据获得的新型猪源抗菌肽cecropin P1突变体的氨基酸序列SEQ ID NO:1，按照毕赤酵母基因密码子偏好性进行重新设计，获得编码新型猪源抗菌肽cecropin P1突变体的核苷酸序列SEQ ID NO:2。并在其N端引入Kex2酶切位点。两端引入XhoI和XbaI酶切位点，以便于克隆入毕赤酵母表达载体中。
实施例2基因工程猪源抗菌肽cecropin P1突变体表达载体的构建与工程菌的获得
1、将含抗菌肽基因的载体和酵母表达载体均用XhoI和XbaI双酶切，酶切产物回收并连接，进行PCR鉴定、测序。
2、阳性质粒经SacI单酶切线性化后加入毕赤酵母感受态细胞悬液中。电转化后均匀涂布于含100μg/mL Zeocin的YPDS选择平板上，30℃孵育3-5天。待YPDS平板上的阳性转化子生长较大，将各转化子依次点种至含Zeocin 200μg/mL、500μg/mL、1000μg/mL的YPDS选择平板，以在高浓度Zeocin平板上正常生长的菌落为可能高拷贝重组菌株。
3、将筛选到的阳性重组菌单菌落接种于含100μg/mL Zeocin的YPD培养液中，28℃振摇培养18个小时。取此菌液按4％体积比转接于5ml BMGY培养基中，28℃摇振培养18-24h左右，OD600值约为5-6。培养物直接转接于25ml BMMY培养基中，28℃继续摇振培养。为了维持诱导表达，每隔24h补加100％甲醇使终浓度达1％。48h后，4℃5000r/min离心10min，收集上清，进行抑菌活性测定。能产生抑菌活性的重组酵母菌株即为能产生新型猪源抗菌肽cecropin P1突变体的阳性菌株。
实施例3发酵、纯化新型猪源抗菌肽cecropin P1突变体的制备
1、发酵工艺
1)将筛选得到的阳性重组子活化后按1％-10％接种量接种于三角瓶，28-30℃，200r/min摇床培养16-24h后以5％-20％接种量接入10L发酵罐(实装培养基6L)，温度28-30℃，转速500-1500r/min，培养基pH值5.0-6.0，通气量0.1-1.0VVM(1L发酵液1min通入的氧气量)，溶氧>20％情况下进行发酵，在培养18-24h后流加50％甘油4h，待溶氧突然升至100％时流加甲醇至发酵结束，整个发酵持续48-72h。
2)发酵结束后原罐蒸汽100℃灭菌10-20min，放料，5000r/min离心10min，收集发酵上清即为抗菌肽半成品。
3)新型抗菌肽制剂
抗菌肽半成品经微滤、超滤、喷雾干燥、冻干等生产的粉剂和以生化方法精制、纯化后得到液体制剂。
上述基因工程抗菌肽可做成畜禽饲料添加剂或畜禽疾病的预防和治疗制剂。
实施例4新型猪源抗菌肽cecropin P1突变体的最小抑菌浓度测定(MIC)
1、试验菌株：金黄色葡萄球菌(Cowan I)、猪金黄色葡萄球菌肠毒素75-1、大肠杆菌K12D31、猪大肠杆菌O78、猪大肠杆菌O157-H71和猪副伤寒沙门氏菌C782
2、菌株处理：将菌种复苏，在固体LB培养基中划线，在培养箱中37℃过夜培养。将过夜培养的细菌挑单菌落于含有25ml液体MHB培养基的三角瓶中，将三角瓶放于摇床37℃培养12-18h。将培养后的菌液在OD600的条件下测其吸光度值，用MHB培养基调节菌液浓度，使其处于0.6-0.8之间。之后将菌液稀释成浓度为5×105CFU/mL，依次取90μl加入到96孔板的各孔内。
3、抗菌肽定量后用MHB培养基依次做倍比稀释。将稀释好的抗菌肽各取10μl依次加入到96孔板的各个孔中，此时每个孔的反应体系为100μl。96孔板盖盖后，37℃振荡培养24h后，观察并用酶标仪在600nm处测量并记录实验结果。抗菌肽样品经过二倍稀释后，成一系列浓度，以抑制细菌生长的最小浓度来定义最小抑菌浓度(MIC)。利用菌液和MHB培养基分别用来做阴性和阳性对照，各自代表抑菌率为0和100％。同时设立猪源抗菌肽cecropinP1标准品试验组作为对照，用于观察抗菌肽改造前后的抑菌活性变化。
4、用微量稀释法测定重组新型猪源抗菌肽cecropin P1突变体对多种细菌的最低抑菌浓度，结果表明其对革兰氏阳性菌和阴性菌均有显著的抑制效果，改造后的新型猪源抗菌肽cecropin P1突变体与猪源抗菌肽cecropin P1标准品相比，对革兰氏阴性菌的抑菌能力得到大大提高，也提高了对革兰氏阳性菌的抑菌活性。
表1抗菌肽对多种细菌的最低抑菌浓度
实施例5新型猪源抗菌肽cecropin P1突变体的最低溶血浓度测定(MHC)
抗菌肽对血红细胞的溶血活性是衡量其对真核细胞毒性的最主要方法。本试验目的即是验证新型猪源抗菌肽cecropin P1突变体是否具有细胞毒性。
1、将重悬于PBS中的8％猪红细胞100μl加入96孔板中，再加入PBS系列稀释的抗菌肽100μl，使各孔中抗菌肽的浓度分别为100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.12μg/mL、1.56μg/mL和0.78μg/mL。阳性对照孔加入100μl 0.2％Triton X-100，阴性对照孔加100μl PBS，37℃孵育1h后，3000rpm离心5min后，从各孔吸取100μl上清到另一96孔板中，550nm波长测定OD值，计算溶血百分比＝[(实验孔OD值-阴性孔OD值)/(阳性孔OD值-阴性孔OD值)]×100。同时设立猪源抗菌肽cecropin P1作为对照。
2、结果表明：新型猪源抗菌肽cecropin P1突变体与猪源抗菌肽cecropin P1一样，对猪红细胞基本无溶血活性，是一种安全的抗菌肽。
Claims (4)
1.一种猪源抗菌肽cecropin P1突变体，其特征在于，所述的猪源抗菌肽cecropin P1突变体的氨基酸序列为SEQ ID NO:1。
2.一种核苷酸片段，其特征在于，所述的核苷酸片段编码权利要求1所述的猪源抗菌肽cecropin P1突变体。
3.如权利要求2所述的核苷酸片段，其特征在于，所述的核苷酸片段的序列为SEQ IDNO:2。
4.权利要求1所述的猪源抗菌肽cecropin P1突变体在制备畜禽饲料添加剂或免疫增强剂中的应用。
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