CN116333067A - 抗菌肽突变体Sub168-QC/R、重组菌株及其应用 - Google Patents
抗菌肽突变体Sub168-QC/R、重组菌株及其应用 Download PDFInfo
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Abstract
本发明涉及一种抗菌肽突变体Sub168‑QC/R、重组菌株及其应用,属于基因工程及生物技术领域,所述的抗菌肽突变体Sub168‑QC/R的氨基酸序列如SEQ IDNO.1所示,核苷酸序列如SEQ ID NO.2所示。本发明还提供包含SEQ ID NO.2的重组载体和重组菌株。本发明抗菌肽突变体Sub168‑QC/R对大肠杆菌和金黄色葡萄球菌等多种致病菌的抑制活性较原抗菌肽Sub168均有提升,且对胰蛋白酶的稳定性也得到提升。此外,抗菌肽突变体Sub168‑QC/R也显示出良好的热稳定性、pH稳定性和胃蛋白酶稳定性。本发明涉及的突变体在制备新型抗菌剂及其应用方面具有很大的优势及潜力。
Description
技术领域
本发明属于基因工程及生物技术领域,具体涉及抗菌肽突变体Sub168-QC/R、重组菌株及其应用。
背景技术
抗菌肽是一类具有广谱抗菌活性的小分子肽类物质,抗菌肽具有的多种抗菌机制使其具有不易产生耐药性的优点,在作为抗生素替代品的新型抗菌剂开发方面具有很大的优势及应用潜力。抗菌肽Sublancin168(Sub168)是枯草芽孢杆菌生长过程中的一种代谢产物,对革兰氏阴性菌及革兰氏阳性菌均有一定的抗菌活性。但该抗菌肽对致病菌的抗菌活性还不足以满足作为抗菌剂的应用,有待于通过分子生物学手段进行改造得到活性提升的突变体。且抗菌肽作为新型抗菌剂的开发,除了抗菌活性外,热稳定性、pH稳定性及消化酶稳定性也是影响其应用的关键性因素,在研究过程中上述关键特性的评价也极为必要。其中,由于动物胃肠道中含有多种消化酶,抗菌肽的消化酶稳定性是决定抗菌肽能否真正通过动物胃肠道从而实现对动物的生长起到促进作用的关键性因素。
抗菌肽Sublancin168已被证明能够抑制多种革兰氏阴性菌及革兰氏阳性菌的生长,但该抗菌肽想要作为抗菌剂在多领域中应用,还存在抗菌活性低等问题,此外,消化酶稳定性低也是影响该抗菌肽实际应用的重要因素。
为了降低生产成本,目前主要通过微生物宿主异源表达的方法对抗菌肽进行生产,以满足应用抗菌剂的高产量低成本需求。里氏木霉是一种近年来被广泛研究和应用的微生物表达宿主,具有表达量高、胞外分泌能力强等优点。且该宿主菌株被美国FAO认定为安全(GRAS)菌株,该菌株分泌生产的代谢产物被赋予的安全性特征也具有更广的应用范围。因此,利用里氏木霉对抗菌肽进行异源表达是生产抗菌肽的优势方法。
发明内容
本发明要解决的技术问题在于通过对抗菌肽Sublancin168进行突变,筛选出抗菌效果好的突变体。
本发明是通过如下技术方案来实现的:
一种抗菌肽突变体Sub168-QC/R,所述抗菌肽突变体Sub168-QC/R的氨基酸序列是SEQ ID NO.1,所述抗菌肽突变体Sub168-QC/R的核苷酸序列为SEQ ID NO.2。
本发明的再一目的是提供包含上述抗菌肽突变体Sub168-QC/R基因的木霉重组表达载体。
本发明的再一目的是提供包含上述抗菌肽突变体Sub168-QC/R的重组菌株,所述菌株为里氏木霉Tu6。
本发明还提供一种抗菌剂,所述抗菌剂中包含所述的抗菌肽突变体Sub168-QC/R。
本发明还提供所述抗菌肽突变体Sub168-QC/R作为新型抗菌剂的应用。
本发明所述抗菌肽突变体Sub168-QC/R的氨基酸序列是在原抗菌肽Sub168的基础上,将34位点上的Q和36位点上的C同时突变为正电氨基酸R。
编码SEQ ID NO.1所述氨基酸序列的核苷酸序列如SEQ ID NO.2所示,所述SEQ IDNO.2是在原始氨基酸序列SEQ ID NO.3的抗菌肽Sub168基础上,翻译为编码的核苷酸序列SEQ ID NO.4,并进行该核苷酸片段的人工合成,对该人工合成的片段进行PCR反应获得的突变体核苷酸序列。
SEQ ID NO.1:
GLGKAQCAALWLQCASGGTIGCGGGAVACQNYRRFRR
SEQ ID NO.2:
GGATTGGGTAAGGCTCAATGTGCTGCTTTGTGGTTGCAATGTGCTTCAGGA
GGTACTATTGGATGTGGAGGTGGAGCTGTTGCTTGTCAAAACTACAGAAGA
TTTAGAAGA
SEQ ID NO.3:
GLGKAQCAALWLQCASGGTIGCGGGAVACQNYRQFCR
SEQ ID NO.4:
GGATTGGGTAAGGCTCAATGTGCTGCTTTGTGGTTGCAATGTGCTTCAGGA
GGTACTATTGGATGTGGAGGTGGAGCTGTTGCTTGTCAAAACTACAGACAA
TTTTGTAGA
本发明所述的一种抗菌肽突变体Sub168-QC/R的制备方法,具体为:选择正电荷增加的策略,在原抗菌肽Sub168氨基酸序列的基础上,将34位点上的Q和36位点上的C同时突变为正电氨基酸R。抗菌肽Sub168的核苷酸序列,设计突变体制备所需的引物,使用PCR扩增的方式获得突变体的核苷酸序列,将PCR产物使用琼脂糖凝胶电泳验证,之后通过体外同源重组插入里氏木霉表达载体,构建抗菌肽Sub168突变体QC/R的里氏木霉表达载体Sub168-QC/R-pCBHG并对其进行测序验证。将构建的重组表达载体使用聚乙二醇介导的原生质体转化法转入到宿主里氏木霉Tu6中,经过转化子筛选及验证后,接入摇瓶进行发酵表达获得抗菌肽突变体Sub168-QC/R。
本发明与现有技术相比的有益效果:
通过正电荷增加的策略及PCR扩增的方法得到了一种抗菌肽突变体Sub168-QC/R的PCR片段,构建了该突变体的重组表达载体。
本发明将抗菌肽突变体Sub168-QC/R在里氏木霉Tu6中异源表达,获得突变体的重组菌株及发酵表达产物,具备抗菌肽突变体Sub168-QC/R在里氏木霉Tu6中的制备方法。
本发明表征了抗菌肽突变体Sub168-QC/R的抗菌活性,其对大肠杆菌、沙门氏菌、金黄色葡萄球菌和产气角膜梭菌的活性较原抗菌肽Sub168有一定的提升。
本发明所述的抗菌肽突变体Sub168-QC/R的胰蛋白酶稳定性较原抗菌肽Sub168有一定的提升。
本发明所述的抗菌肽突变体Sub168-QC/R具有良好的热稳定性、pH稳定性和胃蛋白酶稳定性。
本发明所述抗菌Sub168肽突变体-QC/R的特性有利于其作为新型抗菌剂的应用。
附图说明
图1为抗菌肽突变体Sub168-QC/R热稳定性示意图;
图2为抗菌肽突变体Sub168-QC/R的pH稳定性示意图;
图3为抗菌肽突变体Sub168-QC/R的胃蛋白酶稳定性示意图;
图4为抗菌肽突变体Sub168-QC/R的胰蛋白酶稳定性示意图。
具体实施方式
实施例1PCR制备抗菌肽突变体Sub168-QC/R的核苷酸片段
以GenBank:ACE07988.1中公布的抗菌肽Sub168的氨基酸序列为原始序列,使用Vector NTI 13.5软件翻译为编码该氨基酸序列的核苷酸序列,并进行该核苷酸片段的人工合成。本实施例采用抗菌肽正电荷增加的改造策略,选择将原抗菌肽Sub168氨基酸序列中第34位的负电氨基酸Q和第36位中的不带电氨基酸C同时突变为正电氨基酸R。设计抗菌肽突变体Sub168-QC/R的PCR扩增引物,前引物为:5’-gtcaaaactacagaagatttagaagacatcat-3’,后引物为:5’-aagcaacagctccacctcca-3’,Tm值分别为60.6和60。以原抗菌肽Sub168的核苷酸序列为模板,使用高保真扩增酶进行PCR扩增,本实施例所采用的PCR扩增体系为:/>Buffer 10μL;dNTP 4μL;/>0.5μL;前引物2μL;后引物2μL;原抗菌肽Sub168核苷酸片段1μL;ddH2O 30.5μL。所采用的PCR退火温度为56℃,根据/>的反应说明书依次设置变性、退火和延伸的反应条件,完成PCR反应,获得抗菌肽突变体Sub168-QC/R的PCR扩增产物。使用琼脂糖凝胶电泳对突变体的PCR扩增产物进行验证,最终得到抗菌肽突变体Sub168-QC/R的核苷酸片段。
实施例2抗菌肽突变体Sub168-QC/R重组表达载体Sub168-QC/R-PCBHG的构建。
使用前引物:5’-gctccgtggcgaaagcct-3’和后引物:5’-agcacgagctgtggccaag-3’通过PCR反应将木霉表达载体进行线性化,以高保真扩增酶Super-FidelityDNAPolymerase为基础的PCR反应体系(50μL)为:Buffer 25μL;dNTP 1μL;/>0.5μL;前引物2μL;后引物2μL;模板1μL;ddH2OμL;18.5μL。PCR反应条件根据该酶的说明书依次进行设置,退火温度根据引物的Tm值设置。使用琼脂糖核酸凝胶电泳检验PCR产物,并对产物使用Dpn1酶进行模板消化,使用Cycle Pure Kit PCR纯化试剂盒对消化产物回收纯化,获得线性化载体片段。将实施例1中的突变体核苷酸片段与线性化载体基于ExnaseⅡ进行体外同源重组连接,连接体系根据所用酶的说明书进行设置。连接产物通过热激法转化至大肠杆菌DH5α中,涂布后置于37℃下培养14-16h,对单菌落转化子进行菌落PCR及测序验证,筛选阳性转化子以完成抗菌肽突变体Sub168-QC/R重组表达载体Sub168-QC/R-PCBHG的构建。
实施例3抗菌肽突变体Sub168-QC/R的重组表达载体的木霉转化
将传代培养好的里氏木霉菌株Tu6接种至YEG液体培养基,于30℃、180rpm的恒温摇床中培养20h作为转化宿主。将生长成熟的YEG液体培养基中的宿主菌株进行酶解,酶解条件为30℃、80rpm,酶解时间为2h。酶解过程中每隔30min使用显微镜观察原生质体制备的情况,将酶解液冷冻离心后收集制备的原生质体。
将10μL构建的抗菌肽突变体Sub168-QC/R的重组质粒与100μL木霉原生质体在50μL 25%PEG6000溶液存在的条件下进行混合,混合产物冰浴25min后再于含有2mL 25%PEG6000的体系下室温孵育20min。最终的孵育产物与木霉转化上层培养基(1%葡萄糖、0.6硫酸铵、0.6%磷酸二氢钾、1%硫酸镁、10%山梨醇、0.3%琼脂糖)混匀,并一同倒入木霉转化的下层固体培养基(1%葡萄糖、0.6硫酸铵、0.6%磷酸二氢钾、1%硫酸镁、2%琼脂粉),置于30℃培养5-6天待转化子长出。
实施例4抗菌肽突变体Sub168-QC/R木霉转化子的发酵表达
将生长的木霉转化子依次进行初筛、复筛以及提取基因组进行验证,将筛选出的阳性木霉转化子接种至木霉发酵培养基(1%葡萄糖、0.6硫酸铵、0.6%磷酸二氢钾、1%硫酸镁、10%山梨醇、0.1%氯化钙、0.1%柠檬酸、0.4%磷酸氢二铵)中,于30℃、180rpm的条件下发酵表达。将发酵液进行离心,去除菌体,获得发酵产物。并进一步的验证筛选和分离,获得抗菌肽突变体Sub168-QC/R。
实施例5抗菌肽突变体Sub168-QC/R的抗菌活性测定
选取常见的革兰氏阴性菌大肠杆菌、沙门氏菌及革兰氏阳性菌金黄色葡萄球菌、单增李斯特菌、产气荚膜梭菌为被测致病菌,测定抗菌肽突变体Sub168-QC/R对上述五种致病菌的最小抑菌浓度(MIC)来表征其抗菌活性。
MIC的测定方法为微量连续二倍稀释法,具体为:将五种被测细菌使用其液体培养基培养至生长对数期,并收集各菌体稀释至菌体浓度为105CFU/mL备用。将纯化收集的抗菌肽突变体Sub168-QC/R溶液在96孔板中依次进行连续二倍稀释,呈浓度梯度。并在96孔板浓度梯度的样品中均加入等量的稀释后的菌悬液,并于37℃培养箱中孵育16h,培养之后肉眼观察各孔洞的浑浊程度,其中能使孔洞澄清的最小浓度为抗菌肽突变体Sub168-QC/R对各细菌的MIC。
结果如表1所示,抗菌肽突变体Sub168-QC/R对五种被测细菌的活性较原抗菌肽Sub168均有提升,其对大肠杆菌、沙门氏菌、金黄色葡萄球菌、单增李斯特菌和产气荚膜梭菌的MIC分别为25、25、6.25、12.5和6.25μg/mL。
表1抗菌肽突变体Sub168-QC/R对不同细菌的最小抑菌浓度
实施例6抗菌肽突变体Sub168-QC/R的热稳定性
本实施例中所使用的被测指示菌为大肠杆菌,将抗菌肽突变体Sub168-QC/R和原抗菌肽Sub168均置于沸水浴中加热处理不同时间后测定其对大肠杆菌的抑制率,以表征抗菌肽突变体Sub168-QC/R的热稳定性。结果如图1所示,抗菌肽突变体Sub168-QC/R经过沸水浴处理180min后仍能对大肠杆菌保持较高的抑制作用,说明所述抗菌肽突变体Sub168-QC/R具有良好的热稳定性。
实施例7抗菌肽突变体Sub168-QC/R的pH稳定性
本实施例中所使用的被测指示菌为大肠杆菌,将抗菌肽突变体Sub168-QC/R和原抗菌肽Sub168均置于不同pH的缓冲液中孵育后测定其对大肠杆菌的抑制率,以表征抗菌肽突变体Sub168-QC/R的pH稳定性。结果如图2所示,抗菌肽突变体Sub168-QC/R在酸性条件下较原抗菌肽Sub168对大肠杆菌的抑制率较高,在碱性条件下二者对大肠杆菌的抑制率相当,说明抗菌肽突变体Sub168-QC/R具有良好的pH稳定性。
实施例8抗菌肽突变体Sub168-QC/R的消化酶稳定性
对抗菌肽突变体Sub168-QC/R进行消化酶稳定性测定,评价其对消化酶的耐受性。本实施例中所使用的指示菌为大肠杆菌。本实施例使用的消化酶为胃蛋白酶(使用pH为2.0的Gly-HCl缓冲液稀释胃蛋白酶至3000U/mL)和胰蛋白酶(使用pH为8.0的Tris-HCl缓冲液稀释胰蛋白酶至250U/mL)。抗菌肽突变体Sub168-QC/R分别使用稀释至一定活性后的胃蛋白酶及胰蛋白酶溶液稀释至其对大肠杆菌的MIC。将使用消化酶溶液稀释的抗菌肽突变体Sub168-QC/R溶液置于37℃下孵育不同时间(30-180min)作为待测样品。测定并计算待测样品对大肠杆菌的抑制率来表征其对消化酶的耐受性。
抗菌肽突变体Sub168-QC/R的胃蛋白酶稳定性如图3所示,处理不同时间后突变体及原抗菌肽Sub168均能对大肠杆菌有明显的抑制活性,说明抗菌肽突变体Sub168-QC/R具有良好的胃蛋白酶稳定性。抗菌肽突变体Sub168-QC/R的胰蛋白酶稳定性如图4所示,经胰蛋白酶溶液处理90min之前,抗菌肽突变体Sub168-QC/R和原抗菌肽Sub168均对大肠杆菌有明显的抑制作用,抑制率均高于85%。而处理时间超过90min时,原抗菌肽Sub168对大肠杆菌的抑制率明显下降,而抗菌肽突变体Sub168-QC/R经胰蛋白酶溶液处理180min后对大肠杆菌的抑制率仍能保持在60%以上,说明抗菌肽突变体Sub168-QC/R较原抗菌肽Sub168的胰蛋白酶稳定性有一定的提升。
Claims (5)
1.一种抗菌肽突变体Sub168-QC/R,其特征在于,所述抗菌肽突变体Sub168-QC/R的氨基酸序列是SEQ ID NO.1,所述抗菌肽突变体Sub168-QC/R的核苷酸序列为SEQ ID NO.2。
2.一种木霉重组表达载体,其特征在于,所述载体包含权利要求1所述抗菌肽突变体Sub168-QC/R基因。
3.一种重组菌株,其特征在于,所述重组菌株包含权利要求1所述抗菌肽突变体Sub168-QC/R的基因,所述菌株为里氏木霉Tu6。
4.一种抗菌剂,其特征在于,所述抗菌剂中包含权利要求1所述的抗菌肽突变体Sub168-QC/R。
5.一种抗菌肽突变体Sub168-QC/R的应用,其特征在于,所述应用为权利要求1所述抗菌肽突变体Sub168-QC/R在制备新型抗菌剂中的应用。
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