CN111018960B - 一种抗菌肽id13及其制备方法和应用 - Google Patents
一种抗菌肽id13及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种抗菌肽ID13(序列如SEQ ID No.1所示)及其制备方法和应用。利用基因工程技术,实现ID3在毕赤酵母中的重组表达。本发明的抗菌肽ID13对金黄色葡萄球菌、肺炎链球菌、猪链球菌等革兰氏阳性菌具有很好的杀菌活性并对小鼠红细胞的溶血活性以及对鼠源巨噬细胞的细胞毒性很低。
Description
技术领域
本发明涉及基因工程领域,具体地说,涉及一种抗菌肽ID13及其制备方法和应用。
背景技术
抗菌肽(AMPs)是多细胞生物为保护宿主免受病原微生物侵害而产生的内源性多肽,由于它们在构成先天性免疫系统中起着至关重要的作用。通常具有广泛的抗菌活性,包括病毒,细菌,原生动物和真菌(Zasloff等, 2002);多种杀菌靶位点,杀菌模式与现有抗生素不同以及作为免疫调节剂的广泛活性,因此AMPs被认为是未来抗菌药物最有力的候选物之一(Ali Adem Bahar等,2013)。但是天然抗菌肽存在抗菌活性不强、合成成本较高、对真核生物有一定的毒性等不足。因此如何提高其活性和最大程度降低其毒性成为目前抗菌肽药物开发难点和希望所在。所以,通过改造抗菌肽使其具有更高的抑菌活性,降低毒性已成为现在研究的热点。
抗菌肽DLP4是黑水虻通过金黄色葡萄球菌KCCM 40881进行免疫刺激从而产生的高效抗G+菌活性AMP(Park等,2015)。具有CSαβ构象,对革兰氏阳性菌,尤其是耐甲氧西林金黄色葡萄球菌(MRSA)有较显著抑菌效果,耐甲氧西林金黄色葡萄球菌ATCC 43300在DLP4亚单位作用剂量下连续传代30天,未发现MIC升高等耐药性现象,对照头孢曲松耐药性显著,MIC上升256倍(Li等,2017)。但是DLP4由于抗菌活性和细胞毒性使其在临床应用中受到制约。因此,通过对其进行设计改造,保持并提高抗菌活性,同时能够显著降低溶血活性和细胞毒性,是本发明所要解决的关键技术问题。
发明内容
本发明的目的是提供一种昆种防御素DLP4衍生肽ID13及其制备方法和应用。
为实现本发明,昆种防御素DLP4衍生肽ID13及其制备方法和应用,包括以下步骤:
1、衍生肽序列设计:依据昆种防御素DLP4,依据保守序列、二硫键、电荷、疏水性等关键参数设计衍生肽ID13,序列如SEQ ID No.1所示。
2、密码子优化:对所述编码抗菌肽ID13的DNA序列按照酵母所偏爱的密码子进行优化,在优化后的基因序列(SEQ ID No:2)的5’端添加 XhoI酶切位点和Kex2切割位点,在3’端添加TAA和TAG终止子序列以及XbaI酶切位点,具有如SEQ ID No.3所示的核苷酸序列。
3、表达载体构建:将SEQ ID No:3所示的DNA序列和载体pPICZαA 经XhoI和XbaI双酶切和连接,得到的重组酵母表达载体。
4、基因工程菌制备:重组表达载体线性化后转化毕赤酵母X-33,筛选高表达基因工程菌。
5、上述毕赤酵母X-33基因工程菌的培养方法,包括以下步骤:
1)种子液的制备:从YPD平板挑取酵母转化子单菌落,接种于含100μg/mL zeocin的10ml YPD液体培养基中,29℃,250rmp,摇床培养 18-24h,以1%接种量接种于200mL YPD液体培养基中,29℃,250rmp,摇床培养16-18h,至OD600nm值为6,即得种子液;
2)发酵培养:25℃~29℃下,按10%的接种量将上述种子液加入2L 基础盐培养基中,调pH值至5.0,加入9.6ml PMT1,通气量维持在8vvm,转速为600rpm,溶氧维持在20%以上;
3)饲喂碳源:观测溶氧值开始缓慢下降后突然上升至80%以上,开始流加12‰PMT1的50%葡萄糖溶液,流加速度为12~24mL/L/min,连续流加6~8h,转速上调至1000rpm;
4)甲醇诱导:流加葡萄糖后,饥饿半小时,开始补加100%甲醇,由第一小时的流速1mL/L/min逐渐增加到第六小时的6mL/L/min,转速上调至1100rpm,pH上调至5.5,控制溶氧在20%以上,至发酵结束;
其中,步骤2)中使用的基础盐培养基配方为:45g葡萄糖、50g NH4H2PO4、20g K2SO4、15g MgSO4·7H2O、6g KH2PO4、0.4g CaSO4和1.5g KOH,加水定容至1L。
6、本发明还提供上述毕赤酵母X-33基因工程菌分泌的重组蛋白的纯化方法,其包括对发酵液进行透析脱盐、冷冻干燥、复溶和离子交换层析等步骤。
本发明以昆虫防御素DLP4为模板,设计抗菌肽ID13。通过优化抗菌肽ID13基因序列,构建特异表达载体,实现了抗菌肽ID13在毕赤酵母中的表达,并建立完善的纯化体系,可实现规模化生产,可应用于抗菌药物开发,饲料添加剂开发等领域,具有广阔的应用价值和市场前景。
附图说明
图1为本发明实施例2中PCR扩增ID13基因的产物琼脂糖凝胶电泳检测结果;其中,M:DNA Marker I;1:以ID13产物。
图2为本发明实施例3中重组pPICZαA-ID13载体线性化电泳结果;其中,M:Trans5KDNA marker;1:没有被线性化的重组载体 pPICZαA-ID13;2:线性化的重组载体pPICZαA-ID13。
图3为本发明实施例5中ID13重组酵母菌株诱导发酵120h后不同诱导时间的发酵上清抑菌活性检测结果。
图4为本发明实施例5中ID13重组酵母菌株诱导发酵不同诱导时间发酵上清Tricine-SDS-PAGE电泳检测结果;其中,M:超低分子量蛋白 marker;1-6:分别表示诱导0h、24h、48h、72h、96h、120h发酵液上清电泳法条带。
图5为本发明实施例6抗菌肽ID13 Tricine-SDS-PAGE结果。
图6为本发明实施例6抗菌肽ID13质谱鉴定结果。
图7为本发明实施例8抗菌肽ID13溶血性实验结果。
图8为本发明实施例9抗菌肽ID13细胞毒性实验结果。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用原料均为市售商品。
以下实施例中涉及的培养基和缓冲液配方:
LB培养基:胰蛋白胨10g/L,酵母浸提取物5g/L,NaCl 10g/L;固体LB培养基则加入2%的琼脂糖。
低盐LB培养基:胰蛋白胨10g/L,酵母浸提取物5g/L,NaCl 5g/L;固体低盐LB培养基则加入2%的琼脂粉。
MH培养基:酪蛋白水解物17.5g/L,牛肉浸粉5g/L,淀粉1.5g/L。
MHA培养基:固体MH培养基则加入2%的琼脂粉。
YPD培养基:蛋白胨20g/L,酵母浸提取物10g/L,葡萄糖20g/L;固体YPD培养基则加入2%琼脂粉。
YPDS培养基:蛋白胨20g/L,酵母浸提取物10g/L,山梨醇182.2g/L,葡萄糖20g/L,琼脂粉20g/L。
BMGY培养基(/L):酵母浸提取物10g,蛋白胨20g,甘油10m L, 13.4%无氨基酸酵母氮源(YNB)100m L,0.02%生物素2m L,1mol/L磷酸缓冲液,pH6.0,100m L。
有关LB培养基、低盐LB、MH、YPD、YPDS等培养基的使用参照 Invitrogen毕赤酵母操作手册。
20mM磷酸盐缓冲液(A液):0.4654g Na2HPO4,2.9172g NaH2PO4,加去离子水至950mL,置于磁力搅拌器至完全溶解后调pH 6.7,定容至 1000mL。
1M NaCl 20mM磷酸盐缓冲液(B液):0.4654g Na2HPO4,2.9172g NaH2PO4,58.44gNaCl,加去离子水至950mL,置于磁力搅拌器至完全溶解后调pH 6.7,定容至1000mL。
以下实施例中涉及的基因扩增及转化子鉴定方法为PCR法及DNA测序法。
以下实施例中涉及的蛋白质浓度测定方法为Bradford法。
以下实施例中涉及的蛋白分子量的确定方法为MALDI-TOF MS法。
以下实施例中涉及的蛋白纯化的方法为基于离子层析法。
以下实施例中涉及的发酵方法为高密度发酵法。
以下实施例中涉及的菌种和质粒见表1:
表1供试菌种和质粒
实施例1抗菌肽ID13的设计
在昆种防御素DLP4序列基础上,设计衍生肽ID13。氨基酸序列如SEQ ID NO:1所示。
实施例2抗菌肽ID13基因片段的获得
2.1抗菌肽ID13基因表达序列的优化与设计
对抗菌肽ID13的编码基因进行酵母偏爱密码子优化,在优化后的基因序列的5’端添加XhoI酶切位点和Kex2切割位点,在3’端添加TAA和TAG终止子序列以及XbaI酶切位点,所得基因表达框的核苷酸序列如SEQ ID No:3所示。以上序列由上海生工生物工程股份有限公司完成。
实施例3酵母重组表达载体的构建
3.1将实施例二获得的ID13基因片段经XhoI和XbaI核酸内切酶双酶切后回收纯化片段。同时,用XhoI和XbaI双酶切pPICZαA载体(购自 Invitrogen)。
双酶切体系如下:
以上酶切体系加样完毕后置于PCR仪37℃反应4h,2%琼脂糖凝胶电泳检测,电泳条件:120V,30min。酶切产物用DNA产物回收试剂盒回收。ID13基因和pPICZαA载体经过XbaI和XhoI双酶切消化后,用T4 DNA 连接酶将ID13基因与线性化的pPICZαA载体进行连接。连接体系如下:
连接条件:以上连接体系加样完毕后于PCR仪16℃过夜连接。
3.2将获得的重组载体转化到大肠杆菌DH5α中,转化步骤如下:
1)10μL连接产物加入50μL E.coli DH5α感受态细胞,冰浴30min;
2)42℃热激45s,立即冰浴2~3min;
3)加入450μL 37℃预热的LB低盐培养基,37℃,100rpm恢复培养 1h;
4)重悬菌体后取100-200μL涂布含有25μg/mL Zeocin的LB低盐固体培养基;
6)37℃倒置培养12-16h。
3.3大肠杆菌DH5α阳性转化子鉴定
挑取在低盐LB平板上长出的单菌落接种于10mL LB液体培养基中 (含25μg/mlzeocin),37℃,250rpm过夜培养,通过菌落PCR鉴定阳性转化子。挑取经特异引物验证的阳性转化子接种于10mL低盐LB液体培养基中(含25μg/mL zeocin),37℃,250rpm过夜培养,取500μL 测序验证。
挑取阳性转化子,进行菌液PCR验证转化子正确性,PCR体系、条件如下:
PCR体系:
PCR条件:
PCR产物经2%琼脂糖凝胶电泳检测目的条带(图1)。15%甘油管保存含有重组表达载体的E.coli并提取质粒,为线性化电转P.pastoris准备。
实施例4含ID13基因重组酵母菌株的构建
4.1重组载体pPICZαA-ID13的线性化
利用PmeI对组成型重组表达载体pPICZαA-ID13进行酶切,酶切体系和反应条件如下:
以上酶切体系加样完毕后置于PCR仪37℃反应4h,2%琼脂糖凝胶电泳检测,电泳条件:120V,30min。电泳结果(图2)显示:pPICZαA-ID13 重组载体完全线性化。
4.2毕赤酵母X-33感受态的制备
1)挑取YPD平板上的X-33单菌落,接种至10mL YPD液体培养基, 29℃,250rpm,过夜培养;
2)取毕赤酵母X-33过夜培养液以1%接种量,接种至100mL YPD 液体培养基,29℃,250rpm,培养至OD600吸光值为1.1-1.3;
3)50mL培养物,4℃,4000rpm,5min离心后加入50mL无菌水重悬;
4)4℃,4000rpm,5min离心后,去上清,加入25mL无菌水重悬;
5)4℃,4000rpm,5min离心后,去上清,加入2mL 1M山梨醇重悬;
6)4℃,4000rpm,5min离心后,去上清,加入100μL 1M山梨醇重悬后即为X-33感受态细胞;
4.3电转化
取100μL酵母感受态细胞加入线性化重组质粒冻干粉,轻轻混匀,转至冰预冷的电转杯中,冰上放置5min,电转,参数为1200V,25μF,400Ω。电转后立即加入1ml冰预冷的1M山梨醇溶液,混匀转入2mL离心管中, 29℃,复苏2h,取100μL复苏后的菌液涂布在含有100μg/mL zeocin抗生素的YPDS平板上,29℃倒置培养,直至长出单菌落。
4.4 48孔板诱导筛选阳性转化子
在48孔板中每孔加入500μL BMGY培养基,挑取实例3.3中长出的半个单菌落放入48孔板中。分别设置不加菌的空白对照孔,pPICZαA空质粒阴性对照孔和确定可诱导出来的阳性对照孔。29℃,250rpm振荡培养24h后,每孔加入2.5μL甲醇(甲醇的终浓度为0.5%),记为0h,每隔 24h加一次甲醇,分别记为0h,24h,48h,72h,诱导72h后,分别收集 48孔板中的发酵液于1.5mL离心管中,离心取上清,进行抗菌活性检测。
实施例5重组酵母菌株的高密度发酵
从YPD平板挑取转化子单菌落,接种于装液量为10ml YPD液体培养基(含100μg/mlzeocin)的50mL摇瓶中,29℃,250rmp,18-24h,以1%接种量接种于装液量为200ml YPD种子液培养基的1L摇瓶中,29℃,250rmp,16-18h,OD600nm约为6,作为高密度发酵种子液备用。
采用5L发酵罐进行高密度发酵,发酵过程分为三个阶段:(1)菌体生长阶段:加入2L基础盐培养基,121℃灭菌20min,冷却至29℃,调节pH到5.0,加入9.6mL PMT1,接入200mL菌液(1:10),通气量维持在8vvm,转速为600rpm,溶氧维持在20%以上;(2)流加葡萄糖生长阶段:观测溶氧值开始缓慢下降后突然上升,开始流加50%葡萄糖溶液(12‰ PMT1),流加速度为24mL/L/min,连续流加6h,转速上调至1000rpm,其它发酵条件不变;(3)甲醇过渡诱导阶段:发酵条件变化,流加葡萄糖 6h后,饥饿半小时,开始补加100%甲醇,由第一小时的流速1mL/L/min逐渐增加到第六小时的6mL/L/min,转速上调至1100rpm,pH上调为5.5,控制溶氧在20%以上,其他发酵条件不变,直至发酵结束。
从过渡诱导开始,每隔24h取样,用于蛋白表达情况分析及抗菌活性分析。图3为重组酵母菌株高密度发酵上清抑菌效果,图4为发酵上清蛋白电泳图。
实施例6抗菌肽ID13的纯化
1)阳离子交换层析纯化:
将HiPrep SP FF阳离子交换柱(长度16mm,内径10mm,GE Healthcare)利用A液平衡3-5个柱体积后上样。进样完毕后,先用含有 20mM,pH5.7的磷酸盐洗脱缓冲液(A液)进行洗脱,待穿透峰洗脱完后,用含有0.6M NaCl的20mM,pH6.7的磷酸盐洗脱缓冲液(B液)进行洗脱,收集洗脱峰,UV280nm监测洗脱情况。图5为纯化ID13 Tricine-SDS-PAGE图和图6为纯化ID13质谱检测。
2)1kDa透析袋除盐
收集到的洗脱峰,经1kD截留分子量透析袋4℃透析,每2h换水一次,换水6次。收集透析后的透析液,低温真空冷冻干燥机(-54℃,0.016mba) 冻干,获得抗菌肽ID13冻干粉产品。
实施例7抗菌肽ID13抗菌活性检测
根据实施例5中获得抗菌肽ID13冻干粉,用无菌生理水配制浓度为 1280μg/mL的抗菌肽ID13和万古霉素溶液,2倍倍比稀释至终浓度 1.25μg/mL,将不同浓度的抗菌肽ID13溶液和万古霉素溶液分别加到无菌96孔细胞培养板中,每孔10μL,每个样品三个平行,相同量(10μL)无菌生理盐水作为阴性对照,制备MIC板。菌株采用MH液体培养基培养, 37℃振荡培养至OD600nm=0.4,将菌液制备成浓度相当于0.5麦氏比浊标准的菌悬液,经37℃孵育后的无菌MH液体培养基稀释至105CFU/mL后,向制备好的MIC板样品孔中每孔加90μL菌悬液,37℃恒温孵育16-18h 观察并记录实验结果。ID13对病原菌的最小抑菌浓度(MIC)参照微量肉汤稀释法。结果如表2所示。
表2 ID13对革兰氏阳性菌的抗菌活性
实施例8抗菌肽ID13溶血性实验
根据实施例6中获得的抗菌肽ID13冻干粉,将抗菌肽ID13溶解于无菌生理盐水中,配置成浓度为512μg/mL的母液,2倍倍比稀释终浓度为 2μg/mL。取6周龄SPF级的ICR雌鼠,眼球取血,使用肝素钠抗凝管收集。采集的血液于4℃,1500rpm,离心10min,用10mM PBS(pH7.3) 重复洗涤红细胞三次,至上清无色透明制成8%的红细胞悬浮液。各取 100μL红细胞悬浮液和抗菌肽ID13溶液,加入96孔板,37℃孵育1h, 1500rpm离心5min,吸取上清至ELISA酶标板检测540nm下紫外吸光值。生理盐水和0.1%Triton X-100分别为0%和100%溶血对照实验。溶血程度计算公式如下(参考Jung,Park等,2007):溶血度(%)=[(Abs540nm 抗菌肽ID13-Abs540nm生理盐水)/(Abs540nm 0.1%Triton X-100-Abs540nm生理盐水)]×100%。结果如图7所示。
实施例9抗菌肽ID13的细胞毒性实验
37℃,5%CO2及饱和湿度条件下,RAW264.7细胞培养于DMEM完全培养基中。移液枪吹打细胞,DMEM完全培养基重悬细胞,以2.5×l05 cells/mL密度接种于96孔板中,每孔100μL,设置3个重复。24h后移除培养基,每孔按浓度梯度加入100μL浓度为1、2、4、8、16、32、64、128、256μg/mL样品肽,对照孔加等量的PBS溶液。继续孵育24h后,移除培养基,PBS洗两次,每孔中加入100μL浓度为5mg/mL的MTT(避光操作)。将96孔板移至培养箱继续培养4h。弃MTT液后每孔加入150 μL DMSO,振荡器振荡10min,待孔底结晶完全溶解,570nm波长测各孔吸光度。根据以下公式计算细胞存活率:存活率(%)=处理组OD值/ 对照组OD值×100%(参考Jiao等,2015)。结果如图8所示。
本发明成功优化了编码抗菌肽ID13基因,并构建了pPICZαA-ID13 重组表达载体,经PmeI线性化后成功转化到毕赤酵母X-33中获得重组酵母菌株。对纯化后的抗菌肽ID13进行了抗菌活性检测,结果显示,ID13 抗革兰氏阳性菌有较好的抗菌活性(0.95-1.91μM),活性明显好于DLP4 (1.87-3.75μM),ID13的溶血性实验结果表明,浓度在1-256μg/mL范围内的ID13几乎不对红细胞产生溶血。ID13的细胞毒性实验结果表明,ID13 在浓度256μg/mL时细胞的存活率72%。抗菌肽ID13具有抗菌活性好,毒性低的特点,因此在生产实践中的良好的应用前景。
虽然,上文中已经用一般性说明、具体实施方式及试验,对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
参考文献:
1.Bahar A,Ren D.Antimicrobial Peptides[J].Pharmaceuticals,2013, 6(12):1543-1575.
2.Li ZZ,Mao RY,Teng D,et al.,Antibacterial and immunomodulatoryactivities of insect defensins-DLP2 and DLP4 against multidrug-resistantStaphylococcus aureus[J].Scientific Reports 7:12124.
3.Park SI,Kim JW,Yoe SM(2015)Purification and characterization of anovel antibacterial peptide from black soldier fly(Hermetia illucens) larvae[J].Developmental and Comparative Immunology 52:98–106.
4.Zasloff,Michael.Antimicrobial peptides of multicellular organisms[J].Nature,2002,415(6870):389-395。
Claims (8)
1.一种抗菌肽ID13,其序列如SEQ ID No:1所示。
2.根据权利要求1所述的一种抗菌肽ID13的制备方法。
3.一种含有编码抗菌肽ID13的DNA序列,其特征在于所述编码抗菌肽ID13的DNA序列的5’端添加XhoI酶切位点和Kex2切割位点,在3’端添加TAA和TAG终止子序列以及XbaI酶切位点,所得基因表达框的核苷酸序列如SEQ ID No:3所示。
4.一种含有编码抗菌肽ID13 DNA序列的表达载体,其特征在于权利要求3所述的DNA序列和载体pPICZαA经XhoI和XbaI双酶切和连接,得到的重组酵母表达载体。
5.一种含有权利要求4所述表达载体的基因工程菌,其特征在于权利要求4所述表达载体线性化后转化毕赤酵母X-33,筛选高表达基因工程菌。
6.权利要求5所述基因工程菌的培养方法,包括以下步骤:
1)种子液的制备:从YPD平板挑取酵母转化子单菌落,接种于含100μg/ml zeocin的10mL YPD液体培养基中,29℃,250rmp,摇床培养18-24h,以1%接种量接种于200mL YPD液体培养基中,29℃,250rmp,摇床培养16-18h,至OD600nm值为6,即得种子液;
2)发酵培养:25℃~29℃下,按10%的接种量将上述种子液加入2L基础盐培养基中,调pH值至5.0,加入9.6ml PMT1,通气量维持在8vvm,转速为600rpm,溶氧维持在20%以上;
3)饲喂碳源:观测溶氧值开始缓慢下降后突然上升至80%以上,开始流加含12‰PMT1的50%葡萄糖溶液,流加速度为12~24mL/L/min,连续流加6~8h,转速上调至1000rpm;
4)甲醇诱导:流加葡萄糖后,饥饿半小时,开始补加100%甲醇,由第一小时的流速1mL/L/min逐渐增加到第六小时的6mL/L/min,转速上调至1100rpm,pH上调至5.5,控制溶氧在20%以上,至发酵结束;
其中,步骤2)中使用的基础盐培养基配方为:45g葡萄糖、50g NH4H2PO4、20g K2SO4、15gMgSO4·7H2O、6g KH2PO4、0.4g CaSO4和1.5g KOH,加水定容至1L。
7.一种在重组毕赤酵母中表达抗菌肽ID13的方法,其是利用权利要求5所述获得的重组毕赤酵母经过发酵培养,分泌产生抗菌肽ID13。
8.权利要求1所述的抗菌肽ID13在制备抗病原菌药物中的应用,所述的病原菌为金黄色葡萄球菌、肺炎链球菌、猪链球菌。
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