CN114369150A - Mptx2蛋白及其治疗耐甲氧西林金黄色葡萄球菌感染相关病症的应用 - Google Patents
Mptx2蛋白及其治疗耐甲氧西林金黄色葡萄球菌感染相关病症的应用 Download PDFInfo
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- CN114369150A CN114369150A CN202210081643.8A CN202210081643A CN114369150A CN 114369150 A CN114369150 A CN 114369150A CN 202210081643 A CN202210081643 A CN 202210081643A CN 114369150 A CN114369150 A CN 114369150A
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Abstract
本发明提供了Mptx2蛋白及其治疗耐甲氧西林金黄色葡萄球菌感染相关病症中的应用。
Description
技术领域
本发明涉及新药物用途领域,具体地,涉及Mptx2蛋白及其治疗耐甲氧西林金黄色葡萄球菌感染相关病症的应用。
背景技术
金黄色葡萄球菌(Staphylococcus aureus)是人类重要的病原菌,是引起人畜感染性疾病的主要原因。金黄色葡萄球菌可以分泌多种毒力因子,引起人体各个组织部位的感染。随着抗生素广泛使用,金黄色葡萄球菌出现了耐药基因,产生了耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)。
MRSA通过改变抗生素作用靶点、产生修饰酶等方式,使MRSA对多种抗生素产生了不同程度的耐药性。MRSA的检出率逐年增高,MRSA占金黄色葡萄球菌的比例在很多地区甚至超过了80%,已经成为院内感染重要的病原菌之一。
MRSA是儿童感染性疾病的常见病原菌之一,是儿童重症监护病房(NICU)内医疗相关感染的主要病原菌,可引起儿童重症感染如脓毒血症、坏死性肺炎等,甚至死亡。2020年全国细菌耐药监测报告显示,儿童MRSA的检出率为29.9%,40%-50%的新生儿在出生后前8周内被检测出MRSA鼻腔定植。
目前,抗生素治疗是治疗MRSA感染的主要手段,但由于MRSA对许多抗生素有多重耐药,治疗效果较差,临床上十分棘手的难题之一。而且儿童由于免疫系统发育不完善,免疫功能弱,导致体内抗生素残留较高,且有多种抗生素残留,对于儿童生长发育极其不利。因此,目前迫切需要找到不易产生耐药性、活性高、安全可靠的新型抗MRSA感染的药物。
发明内容
本发明旨在克服上述缺陷,提供了一种抗生素替换品,即、Mptx2蛋白,及其治疗耐甲氧西林金黄色葡萄球菌感染相关病症上的应用。
本发明提供了一种活性成分,其特征在于:
上述活性成分为Mptx2蛋白;
上述Mptx2蛋白的序列如SEQ ID NO.1所示。
SEQ ID NO.1:Asp Leu ThrArg Pro Tyr Ser Ile Phe Ser Tyr Ser ThrArg ThrLys Asp Asn Glu Ile Leu Leu Phe Val Glu Lys Arg Gly Glu Tyr Met Leu Tyr ValGlyAsn Ser Gly Val Ser Phe Lys Ala Pro ThrAsn Leu Pro Asp Pro Val Arg IleCysVal Asn Trp Glu Ser Gly Ser Gly Ile Ala Glu Phe Trp Leu Asn Gly Lys AlaPheGlyArg Lys Gly Leu Lys Lys Gly Tyr Val Val Gly Gly Asp Ala Lys Ile Ile LeuGlyGln Glu Gln Asp Ser Phe Gly Gly Lys PheAsp Val Lys Gln Ser Phe Val GlyGluIle Trp Asp Val Ser Leu Trp Asn Tyr Val Val Pro Ile Lys Glu Val His AspSer CysAsn Asn GlyAsn Ile Ile Asn Trp Gln Ala Leu Ile His Glu Asp Arg Gly TyrVal ValThr Lys Pro Lys Leu Trp Thr
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述活性成分为如下物质中的一种:
A.Mptx2蛋白,上述Mptx2蛋白的序列如SEQ ID NO.1所示;
B.选自SEQ ID NO.1所示序列中的部分;
B.与SEQ ID NO.1所示序列相差不超过三个片段的序列;
上述用途包括:用于制备治疗或减轻MRSA感染的药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述用途包括:用于制备抑制MRSA细菌生长的药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述用途包括:用于制备缓解MRSA引起的脏器病理性损伤的药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述用途包括:用于制备缓解MRSA引起的肝脏、脾脏和肾脏病理性损伤的药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述用途包括:用于制备的改善MRSA诱导的肝脏和肾脏炎症反应药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述用途包括如下中的至少一种:
用途A.用于制备减低肝脏组织中IFN-γ表达水平的药物;
用途B.用于制备减低肝脏组织中IL-10表达水平的药物;
用途C.用于制备减低肝脏组织中NF-κB1A表达水平的药物;
用途D.用于制备减低肝脏组织中CXCL-2表达水平的药物;
用途E.用于制备减低肝脏组织中IL-6表达水平的药物;
用途F.用于制备减低肝脏组织中CCL-17表达水平的药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述用途包括:用于制备减缓由MRSA引起的小鼠体重丢失的药物。
进一步地,本发明提供的一种活性成分的用途,其特征在于:
上述药物的有效剂量不低于10-8/ml。
此外,本发明提供了一种MRSA实验模型,其特征在于:向实验模型添加MRSA菌液。
本发明的作用和效果:
抗菌肽(antimicrobial peptide)是生物体内经诱导而产生的有抗菌活性的小分子多肽,是机体先天性免疫系统的关键组成部分。抗菌肽是抗生素的理想代替品之一。天然抗菌肽和常规的抗生素不同,是某个特定基因编码的蛋白产物,因此具有独特的抗菌机制和广谱抗细菌、真菌、病毒、螺旋体、寄生虫的活性,且具有不易产生耐药性的特性。
粘膜穿透素(mucosal pentraxin,Mptx)蛋白2是机体表达的分泌性蛋白,属于穿透素(pentraxin,PTXs)家族,可能具有抗菌肽活性。穿透素家族(PTXs)是参与炎症急性期和免疫应答反应过程中的一类高度保守的蛋白亚家族。PTXs家族蛋白表面均含有保守的钙结合位点,其表面与磷脂表面保守结合。电镜下,该家族是由5个相同亚单位形成环状五聚体结构,结构高度稳定。根据亚单位其分子量大小,分为短穿透素蛋白(25kDa)和长穿透素蛋白(>45kDa)。短的穿透素蛋白中有2个经典分子,C反应蛋白(CRP)和血清淀粉样P组分(SAP)。穿透素家族在机体受到感染或者组织损伤时增加,能激活补体和与巨噬细胞杀伤致病细胞,也可以参与细胞碎片的识别,结合和清除。
但,目前尚未有研究发现Mptx2蛋白在感染过程中的作用,尤其是当MRSA感染时,能否代替传统抗生素起到治疗MRSA感染的作用。
本发明通过体外诱导表达和纯化了Mptx2蛋白;通过体内、外抑菌实验均发现Mptx2蛋白能够有效治疗MRSA引起的脓毒血症。
由此,在本发明中公开了Mptx2蛋白作为新型抗菌肽用于治疗MRSA感染引起的脓毒血症的方法,为临床上治疗MRSA感染相关脓毒血症提供新的治疗策略,为临床上治疗MRSA感染导致的其他相关性疾病提供潜在治疗方案。
附图说明
图1.Mptx2 PCR扩增产物电泳图和考马斯亮蓝染色图
其中,图1A:Mptx2全长PCR扩增产物的琼脂电泳图。
图1B:纯化的Mptx2蛋白考马斯亮蓝染色图(M:Marker;1:未诱导菌体;2:诱导菌体;3:菌体裂解液;4:过滤液;5:第1次清洗液;6:第2次清洗液;7:第1次洗脱液;8:第2次洗脱液)。
图2.Mptx2蛋白抑制MRSA生长
其中,图2A为MRSA细菌生长动力学曲线(****,MRSA+PBS vs.MRSA+10μg/ml,p<0.0001)。
图2B为Mptx2蛋白和MRSA细菌共同孵育第4h OD600测量结果。
图2C为Mptx2蛋白和MRSA细菌共同孵育第24h细菌平板测定结果。
图2D为Mptx2蛋白和MRSA细菌共同孵育第24h细菌数目测量结果。
图3.MRSA感染小鼠流程图和小鼠体重变化
其中,图3A为MRSA感染小鼠流程图。
图3B为MRSA感染小鼠体重变化。
图4.小鼠肝脏、脾脏和肾脏病理变化和评分
其中,图4A为肝脏、脾脏和肾脏HE染色光镜(各组放大倍数:×200)。
图4B为各组小鼠肝脏病理学评分。
图4C为各组小鼠肾脏病理学评分。
图4D为各组小鼠脾脏病理学评分。
图5.小鼠血液及腹腔灌洗液中MRSA细菌含量检测结果
其中,图5A为Mptx2对血液和腹腔灌洗液中MRSA细菌含量的影响。
图5B为各组小鼠血液中MRSA细菌含量的比较。
图5C为各组小鼠腹腔灌洗液中MRSA细菌含量的比较。
图6.小鼠肝脏、肾脏和脾脏中MRSA细菌含量检测结果
其中,图6A为Mptx2蛋白对肝脏、肾脏和脾脏中MRSA细菌含量的影响。
图6B为各组小鼠肝脏中MRSA细菌含量的比较。
图6C为各组小鼠肾脏中MRSA细菌含量的比较。
图6D为各组小鼠脾脏中MRSA细菌含量的比较。
图7.小鼠肝脏相关炎症因子表达水平检测
其中,图7A为小鼠肝脏组织中IFN-γ的表达水平。
图7B为小鼠肝脏组织中IL-10的表达水平。
图7C为小鼠肝脏组织中NF-κB1A的表达水平。
图7D为小鼠肝脏组织中CXCL-2的表达水平。
图7E为小鼠肝脏组织中IL-6的表达水平。
图7F为小鼠肝脏组织中CCL-17的表达水平。
图8.小鼠肾脏相关炎症因子表达水平检测
其中,图8A为小鼠肾脏组织中IFN-γ的表达水平。
图8B为小鼠肾脏组织中IL-10的表达水平。
图8C为小鼠肾脏组织中NF-κB1A的表达水平。
图8D为小鼠肾脏组织中CXCL-2的表达水平。
图8E为小鼠肾脏组织中IL-6的表达水平。
图8F为小鼠肾脏组织中CCL-17的表达水平。
具体实施方式
实施例1.Mptx2蛋白表达与纯化
1.1主要试剂
(1)胰蛋白胨:ST800,上海碧云天生物技术有限公司
(2)酵母膏:ST968,上海碧云天生物技术有限公司
(3)氯化钠:1249KG001,
(4)高纯度低电渗琼脂糖:G5056-100G,武汉塞维尔生物科技有限公司
(5)pET-28载体:上海吉凯基因化学技术有限公司
(6)BL21(DE3)化学感受态细胞::C504-03,南京诺唯赞生物科技股份有限公司
(7)卡那霉素:E004000-5G,上海元象医疗器械有限公司
(8)培养皿100×20mm TC表面PS(聚苯乙烯)材质:430167,康宁显示科技有限公司。
(9)异丙基-β-D-硫代半乳糖苷(IPTG)/Isopropylβ-D-thiogalactoside:MB3026,上海源祉生物科技有限公司
(10)Ni-NTA Sefinose(TM)Resin Kit:C600332-0001,生工生物工程(上海)股份有限公司
(11)Vivaspin 20,3kDa MWCO PES:28932358,上海柏根生物科技有限公司
(12)PD MidiTrap G-25:28918008,上海柏根生物科技有限公司
(13)NUPAGE 4×LDS SAMPLE BUFFER:NP0008,英潍捷基(上海)贸易有限公司
(14)考马斯亮蓝快速染液(免脱色):PS111,上海熠晨生物科技有限公司
(15)PIERCE BCA PROTEIN ASSAY:23227,英潍捷基(上海)贸易有限公司
1.2试剂准备
1、液体Luria-Bertani(LB)培养基配置:称取胰蛋白胨10g,酵母提取物5g,氯化钠10g,加入1L双蒸水,摇动容器制止溶质完全溶解。高压蒸汽灭菌。配置好的液体LB培养基可于4℃冰箱保存1个月。
2、LB固体培养基配置:在1L液体LB培养基加入15g琼脂糖(1.5%),高压蒸汽灭菌。
3、含50μg/ml卡那霉素固体LB琼脂培养板:高压灭菌后,将融化的LB固体培养基置于55℃的水浴中,待培养基温度降到55℃时(手可触摸)加入卡那霉素(终浓度为50μg/ml),以免温度过高导致抗生素失效,并充分摇匀。15~20ml固体LB培养基倒一个培养皿。待液体凝固后,打开紫外灯,灭菌30min。
4、100mM IPTG:称取2.38g IPTG溶于100ml双蒸水中。配置完成后用0.22μm滤膜过滤,-20℃保存。
5、50mg/ml卡那霉素配置∶称取500mg卡那霉素粉末融于10ml双蒸水中,0.22μm滤膜过滤,分装,-20℃保存。
6、含20mg/ml溶菌酶的Binding buffer溶液:称取200mg溶菌酶粉末加入10mlBinding buffer中。
1.3方法
1.3.1pET-28-Mptx2(NM_001205011)载体构建
设计Mptx2(NM_001205011)全长上下游引物,进行PCR扩增,获得Mptx2(NM_001205011)cDNA,在pET-28质粒两端分别引入BamHI和XhoI限制性内切酶的酶切位点,连接pET-28载体,构建质粒并转化大肠杆菌DH5α,酶切鉴定并测序。
1.3.2获得含重组表达质粒的表达菌种
(1)从-80℃冰箱中取100μl BL21化学感受态细胞悬液,迅速置于冰上融化,解冻后立即置冰上。
(2)加入5μl质粒DNA溶液(浓度为100ng/μl,设置阴性对照组),轻弹管壁混匀(避免用枪吹打),冰上放置30分钟后。
(3)42℃水浴90s,水浴后迅速置于冰上静置2min,切勿摇动离心管。
(4)向管中加入800μl LB液体培养基(不含卡那霉素),混匀后37℃摇床220rpm培养45min,使细菌恢复正常生长状态,并表达质粒编码的抗生素抗性基因(卡那霉素)。
(5)将上述菌液摇匀后取100μl涂布于含50μg/ml卡那霉素琼脂培养板上,正面向上放置30min,待菌液完全被培养基吸收后倒置培养皿,放置于37℃培养箱中培养12-16h。
(6)剩余菌液置于4℃冰箱保存。
1.3.3Mptx2蛋白诱导表达及鉴定
(1)挑取卡那霉素琼脂培养基平板上的单菌落至装有5ml LB液体培养基(含卡那霉素50μg/ml)的试管中,于37℃摇床培养过夜(220rpm,16h)。
(2)按照1:100接种量将上述中试管中的菌体接种至装有50ml液体LB培养基的三角瓶中,6层灭菌纱布封口,37℃摇至OD600值约为0.6。
(3)取部分液体作为未诱导的对照组,余下的加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度1mM作为实验组,两组继续放置于于37℃摇床上(220rpm,4h)。
(4)4℃离心(3000rpm,15min),取上清。加入5μl LDS sample buffer,70℃金属浴10min。
(5)收获沉淀,用PBS重悬沉淀,4℃离心(8000rpm,10min)。
(6)重复步骤5三次,收集菌体。取细菌沉淀悬浮液,加入4×LDS samplebuffer,70℃孵育10min。
(7)取细菌沉淀,用1ml含20mg/ml溶菌酶的Binding buffer溶液重悬沉淀。冰上孵育30min,使溶菌酶充分反应。
(8)置于冰浴中,使用超声破碎仪破碎菌体,功率100W,工作0.8s,间歇1.2s,时间1min。
(9)4℃离心(14000×g,30min),将上清液吸至1.5ml离心管中,沉淀用300μL PBS重悬。
(10)上清液和重悬后沉淀溶液中加入4×LDS sample buffer,放入70℃金属浴中煮沸(300rpm,10min)
(11)上清和细菌沉淀分别进行SDS-PAGE电泳,与未诱导表达的菌体比较,考马斯亮蓝染色、脱色后观察蛋白的表达情况并拍照记录。
1.3.4Mptx2蛋白纯化
(1)取出收集到的细菌沉淀,冰上解冻15min,用10ml含20mg/ml溶菌酶的Bindingbuffer溶液重悬细菌沉淀。
(2)冰上孵育30min。通过轻轻旋转细胞悬液混合2-3次。
(3)置于冰浴中,使用超声破碎仪破碎菌体,功率100W,工作0.8s,间歇1.2s,时间1min。
(4)4℃离心(14000×g,30min),轻轻吸取上清,将所有上清收集到15ml离心管中(上清液中含有重组蛋白的可溶性部分)。收集的上清经0.22μm滤膜过滤。
(5)取出镍柱,轻轻地将快速镍柱倒置几次,使其树脂重新悬浮。加入含上清的15ml离心管中。将15ml离心管置于摇床上慢摇(70rpm,30min,)使镍与His标签蛋白充分结合。
(6)打开镍柱出口的密封件,打开螺帽,让存储缓冲液排出。用2ml Bindingbuffer平衡镍柱。
(7)将步骤4中的细菌裂解物上清液加到镍柱上,每次2ml,缓慢过柱。所有上清液过柱2次。
(8)取出Binding bufer,加入浓盐酸调节pH为6.3,该溶液记为Washing buffer;取出Elution buffer,加入浓盐酸调节pH为4.5,该溶液记为Elution buffer。
(9)用Washing buffer(pH为6.3)洗涤镍柱,每次加入2ml,多次洗涤,去除杂蛋白。
(10)用1ml Elution buffer(pH为4.5)洗脱结合的目的蛋白,第一次洗脱的最后一滴取180μl,取60μl 4×LDS sample buffer添加到180μl洗脱液中,标记为洗脱液1;再加1ml Elution buffer反复洗脱;第三个1m l的最后一滴取180μl,取60μl 4×LDS loadingbuffer添加到180μl洗脱液中,标记为洗脱液2。
(11)将收集的洗脱液1,洗脱液2蛋白于70℃金属浴煮沸(300rpm,10min)。蛋白-20℃储存,以进行SDS-PAGE分析。
1.3.5Mptx2蛋白纯度鉴定
(1)用SDS-PAGE分析所有组分,跑胶顺序:阴性组、诱导组、洗脱液2。
(2)小心拆下玻璃板和垫片并取下凝胶。将凝胶置于纯水中漂洗5分钟去处杂质,以降低背景
(3)弃去纯水,加入适量考马斯亮蓝快速染液,以覆盖凝胶为宜,室温下水平摇床上染色30min。
(4)从考马斯亮蓝快速染液中取出凝胶,并在去污液中轻轻搅动染色凝胶,直到背景变得清晰。
(5)观察蛋白的表达情况并拍照记录。
1.3.6蛋白脱盐
(1)取出PD-10柱,将储存液以自然流速流出。用1ml Binding buffer平衡PD-10柱。
(2)上样:在柱子平衡后,将样品加到柱子上。
(3)洗脱:样品全部进入柱子内后,柱顶加入1ml Binding buffer洗脱残留样品。
(4)在蛋白浓缩前,用BCA法测定脱盐后蛋白浓度。
1.3.7超滤法浓缩蛋白
(1)将经过脱盐得到的蛋白溶液加入超滤管中。
(2)4℃离心(4000rpm,1h),直至溶液变为1ml左右。可根据需要选择不同的离心时间,以达到不同的浓缩倍数。
(4)BCA试剂法测定蛋白浓度,当蛋白浓度大于至1mg/ml时,收集蛋白。将溶液从超滤管中收集到1.5ml离心管中,标记好储存液的成分,蛋白名称,浓缩后蛋白浓度,制备日期。
1.4结果
1.4.1 Mptx2原核表达质粒构建
扩增662bp的Mptx2(NM_001205011)全长基因(图1A)。经双酶切的质粒GV296与胶回收的Mptx2(NM_001205011)全长片段连接并转化大肠杆菌DH5α后,双酶切阳性质粒得到的片段约662bp,与目的基因大小一致。测序结果表明,pET-28质粒中成功插入Mptx2(NM_001205011)全长目的基因。
Mptx2质粒信息如下所示:
GGATCCATGGAGAAGCTTATTGTGGGCATCCTGTTTCTCTCTGTTCTTTCAGGAAGTGTAGCACAAACAGACATGAAGGGAAAGGCATTTATTTTCCCTCAAGAATCATCCACTGCCTATGTGTCCCTGATACCGAAGGTGAGGAAGTCACTGCAGAACTTCACTCTGTGTATAAAGGCCTTCACAGACCTGACTCGCCCTTATAGCATCTTCTCCTACAGCACAAGAACTAAGGACAATGAGATTCTTCTCTTTGTGGAAAAGAGAGGAGAATATATGCTCTATGTTGGGAATTCGGGAGTCAGTTTCAAAGCACCCACAAATCTTCCTGATCCAGTCCGTATCTGTGTGAACTGGGAGTCTGGCTCTGGGATTGCAGAATTCTGGCTGAATGGAAAGGCATTTGGGAGAAAAGGCTTGAAGAAGGGATACGTGGTGGGGGGTGATGCAAAGATCATTCTAGGACAAGAACAGGATTCCTTTGGGGGAAAATTTGATGTAAAACAATCCTTTGTTGGGGAAATATGGGATGTTTCCTTGTGGAACTATGTGGTCCCCATAAAAGAGGTGCATGACAGCTGTAACAATGGCAATATTATAAACTGGCAAGCTCTTATTCATGAAGACAGGGGCTATGTGGTGACTAAGCCCAAACTGTGGACTTAACTCGAG
1.4.2 Mptx2蛋白在大肠杆菌BL21菌株中的表达
取对照组(未添加IPTG诱导剂)和诱导组细菌沉淀经SDS-PAGE电泳,考马斯亮蓝染色分析(图1B)。结果显示Mptx2蛋白可在大肠杆菌BL21菌株中表达。考马斯亮蓝染色结果显示在洗脱液中检测到重组Mptx2蛋白。在洗脱液中,可以看到相对分子质量约为25.5kDa的条带,其大小均与预期蛋白大小相符。取第2次洗脱液进行脱盐和超滤浓缩,直至重组蛋白浓度大于1mg/ml。最终,重组Mptx2蛋白浓度为1.1mg/ml。
实施例2.Mptx2蛋白在体外抑制MRSA生长实验
2.1主要材料
(1)金黄色葡萄球菌:ATCC-6538,北京百欧博伟生物技术有限公司
(2)MRSA:ATCC-43300,北京百欧博伟生物技术有限公司
2.2方法
2.2.1MRSA菌复苏培养
(1)取100μl MRSA菌液加至1L含50μg/ml卡那霉素的液体LB培养基中。放置于摇床上过夜培养(37℃,220rpm,16h)。
(2)培养至对数生长期后,离心收集菌体沉淀(4℃,4000rpm,15min).
(3)用灭菌PBS缓冲液重悬菌体沉淀,使用平板计数法测定菌液菌落总数。-80℃保存备用。
(4)使用前,用灭菌PBS缓冲液将MRSA稀释至终浓度为5×105CFU/ml。
2.2.2 Mptx2蛋白浓度调整
用灭菌PBS缓冲液将药物浓度调整为1mg/ml。所有蛋白溶液在使用前均使用0.22μm滤膜过滤除菌,保存于无菌1.5ml离心管。
2.2.3 Mptx2蛋白与MRSA共孵育
(1)将10μl复苏的MRSA细菌悬液接种到总共200μl含有不同浓度(0,2或10μg/ml)Mptx2蛋白。每2小时通过OD600测量监测细菌的生长。
(2)取3只5ml离心管,分别标记为MRSA组(对照组)、MRSA+1μg/mlMptx2组、MRSA+10μg/ml Mptx2组。第1管加入1980μl液体LB培养基和20μl浓度为5×105CFU/ml的MRSA菌液(作为对照组)。第2管加入1978μl液体LB培养基、20μl浓度为5×105CFU/ml的MRSAμl菌液和2μl重组Mptx2蛋白(作为1μg/ml组),第3管加入1960μl液体LB培养基、20μl菌液和20μl重组Mptx2蛋白(作为10μg/ml组),振荡混匀。于37℃恒温箱中孵育24h。
2.2.4细菌菌落总数测定
(1)将孵育好的菌液用灭菌PBS连续稀释至终浓度为1×107CFU/ml。
(2)取100μl孵育好的菌液均匀涂布于卡那霉素琼脂培养板上。平板平放于超净台上30min,使菌液渗入培养基表层内。将平板倒置于37℃恒温箱中培养24h,24h后计算平板菌落数目,乘于稀释倍数即得出各组菌落数目。
2.3结果
2.3.1细菌菌落总数测定
根据GB 4789.2-2016标准进行细菌平板计数。MRSA菌落总数为1.26×1011CFU/ml。
2.2.2重组Mptx2蛋白抑制细菌生长
将MRSA分别与不同浓度的重组Mptx2蛋白共同孵育,进行OD600测定(图2A-B)。细菌生长动力学曲线结果显示,Mptx2蛋白有效抑制MRSA的生长,且抑菌作用呈现为剂量依赖。将MRSA分别与PBS、1μg/ml和10μg/ml重组Mptx2蛋白共同孵育24h,进行细菌菌落总数测定。结果发现,PBS组细菌总数为5.9×108CFU/ml,给予1μg/ml重组Mptx2蛋白后细菌总数减少至3.4×108CFU/ml(p=0.073),给予10μg/ml重组Mptx2蛋白细菌总数为1.4×108CFU/ml(p<0.0001)。结果表明,重组Mptx2蛋白能有效抑制MRSA的生长。
实施例3.Mptx2蛋白在小鼠MRSA感染相关性脓毒血症中的干预作用
3.1主要材料
(1)C57BL/6小鼠:上海吉辉实验动物饲养有限公司
(2)1ml注射器:YA0550,上海晶旷生物科技有限公司
3.2方法
3.2.1Mptx2蛋白浓度调整
用灭菌PBS缓冲液将药物浓度调整为1mg/ml。所有蛋白在使用前均使用0.22μm滤膜过滤除菌,保存于无菌1.5ml离心管。
3.2.2 MRSA菌液复苏
将20μl MRSA菌液加入20ml液体LB培养基中,所有液体置于无菌50ml离心管中。放置到摇床上摇16h至对数生长期(37℃,220rpm)。离心收集菌液(3000rpm,15min,常温)。用1ml PBS重悬菌液,确定菌液浓度为1.5×108CFU/ml时的OD600值。
3.2.3小鼠急性感染实验
(1)取6周龄野生型(wide type,WT)C57BL/6小鼠作为感染对象,随机分成3组,分组如下:包括WT+PBS组(n=8),WT+MRSA组(n=14),WT+MRSA+10μg/ml Mptx组(n=14)。小鼠雌雄比例为1:1,小鼠随机分组。
(2)WT+MRSA组(n=14)和WT+MRSA+10μg/ml Mptx组(n=14)每只小鼠腹腔注射100μl菌液(1.5×107CFU,即100μl浓度为1.5×108CFU/ml的菌液),WT+PBS组给予等量的灭菌PBS。
(3)腹腔注射菌液2h后,腹腔注射100μl浓度为10μg/ml重组Mptx2蛋白。腹腔注射感染24h后,小鼠麻醉之后眼球取血,脱颈处死,开腹取材。取肝脏、脾脏和肾脏于4%多聚甲醛中固定。收集肝脏、脾脏、肾脏、腹腔灌洗液和血液用于细菌总数测定。
3.2.5器官细菌负荷量测定
(1)用无菌剪刀、镊子取小鼠肝脏、脾脏、肾脏,根据重量加入无菌PBS溶液,无菌环境中进行匀浆处理。
(2)后对匀浆液进行连续梯度稀释,分别吸取100μl匀浆液涂布在含有卡那霉素的LB培养板上,过夜培养,观察菌落状态并计数。
(3)取腹腔灌洗液和血液用无菌PBS溶液连续梯度稀释,分别吸取100μl匀浆液涂布在含有卡那霉素的LB培养板上,过夜培养,观察菌落状态并计数。
3.2.6组织病理学评分
取小鼠肝脏、脾脏和肾脏置于4%多聚甲醛中固定,常规脱水、包埋、切片、HE染色,观察切片。
3.2.7检测肝脏和肾脏炎症反应
使用RNA提取试剂盒提取小鼠肝脏和肾脏组织RNA,定量PCR检测小肝脏和肾脏相关炎症基因的表达水平,包括IFN-γ、IL-10、NF-κB1A、CXCL-2、CCL-17和IL-6。
3.3结果
3.3.1Mptx2蛋白能减缓小鼠体重丢失
小鼠感染MRSA流程如图3A所示,各组小鼠体重丢失情况如图2B所示。未给予MRSA感染的WT+PBS组小鼠体重缓慢上升,第2天体重增长初始体重的100.8%。给予MRSA感染的各组小鼠均出现不同程度的体重丢失,WT+MRSA+PBS组小鼠体重丢失最为严重,感染后第2天体重减少为初始体重的89.2%;与WT+MRSA+PBS组小鼠相比,WT+MRSA+10μg/ml Mptx2组小鼠体重丢失减缓(89.2%vs.90.8%,p=0.0107)。结果表明,给予Mptx2蛋白能减缓MRSA感染相关性脓毒血症小鼠体重丢失。
3.3.2Mptx2蛋白能缓解MRSA引起的脏器病理性损伤
病理检测结果如图4A和B显示,对比各组小鼠肝脏、脾脏和肾脏病理,相比于WT+PBS组小鼠,WT+MRSA+PBS组小鼠肝脏实质内和肝血窦周围可见有大量中性粒细胞浸润,肝细胞肿胀、坏死,核固缩;肾皮质内肾小球损伤、可见大量中心粒细胞浸润;脾脏红髓和白髓界限消失,生发中心出现。而给予Mptx2蛋白治疗后,WT+MRSA+Mptx2组各个脏器损伤有所改善,炎症细胞浸润减少。病理评分也显示,WT+MRSA+PBS组小肝脏、脾脏和肾脏发生严重的炎症,而给予Mptx2蛋白后肝脏、脾脏和肾脏炎症明显减轻。结果表明Mptx2蛋白可有效缓解MRSA造成的肝脏、脾脏和肾脏病理性损伤。
3.3.3 Mptx2蛋白能减轻MRSA感染情况
对各组小鼠血液和腹腔灌洗液中的MRSA细菌负荷进行检测,结果如图5A和B。结果显示,MRSA+Mptx2组血液和腹腔灌洗液中MRSA细菌数量较MRSA组明显减少。对比各组小鼠肝脏、脾脏和肾脏中的MRSA细菌数量,结果如图6A-D。WT+PBS组小鼠各器官中均未检测到MRSA,WT+MRSA+PBS组小鼠各脏器均可见MRSA定植分布。在给予Mptx2蛋白治疗,WT+MRSA+Mptx2组小鼠各脏器的MRSA细菌数目明显减少。以上结果表明,Mptx2蛋白能有效抑制MRSA感染。
3.3.5 Mptx2蛋白能改善MRSA诱导的肝脏和肾脏炎症反应
使用定量PER检测小鼠肝脏组织中相关炎症基因的表达水平,结果如图7。MRSA感染小鼠脾脏组织中IFN-γ、IL-10、NF-κB1A、CXCL-2、IL-6和CCL-17的表达水平均较PBS组小鼠明显升高,给予Mptx2蛋白干预可明显减低小鼠肝脏组织中IFN-γ、IL-10、NF-κB1A、CXCL-2、IL-6和CCL-17的表达水平。使用定量PER检测小鼠肾脏组织中相关炎症基因的表达水平,结果如图8。MRSA感染组小鼠脾脏组织中IFN-γ、IL-10、NF-κB1A、CXCL-2、IL-6和CCL-17的表达水平均较PBS组小鼠明显升高,而在MRSA+Mptx2组小鼠肾脏中这6个炎症基因表达水平明显降低。以上结果表明Mptx2蛋白能改善MRSA诱导的肝脏和肾脏炎症反应。
序列表
<110> 上海市儿科医学研究所
<120> Mptx2蛋白及其治疗耐甲氧西林金黄色葡萄球菌感染相关病症的应用
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Claims (10)
1.一种活性成分,其特征在于:
所述活性成分为Mptx2蛋白;
所述Mptx2蛋白的序列如SEQ ID NO.1所示。
2.一种活性成分的用途,其特征在于:
所述活性成分为如下物质中的一种:
A.Mptx2蛋白,所述Mptx2蛋白的序列如SEQ ID NO.1所示;
B.选自SEQ ID NO.1所示序列中的部分;
B.与SEQ ID NO.1所示序列相差不超过三个片段的序列;
所述用途包括:用于制备治疗或减轻MRSA感染的药物。
3.如权利要求1所述的一种活性成分的用途,其特征在于:
所述用途包括:用于制备抑制MRSA细菌生长的药物。
4.如权利要求1所述的一种活性成分的用途,其特征在于:
所述用途包括:用于制备缓解MRSA引起的脏器病理性损伤的药物。
5.如权利要求1所述的一种活性成分的用途,其特征在于:
所述用途包括:用于制备缓解MRSA引起的肝脏、脾脏和肾脏病理性损伤的药物。
6.如权利要求1所述的一种活性成分的用途,其特征在于:
所述用途包括:用于制备的改善MRSA诱导的肝脏和肾脏炎症反应药物。
7.如权利要求1所述的一种活性成分的用途,其特征在于:
所述用途包括如下中的至少一种:
用途A.用于制备减低肝脏组织中IFN-γ表达水平的药物;
用途B.用于制备减低肝脏组织中IL-10表达水平的药物;
用途C.用于制备减低肝脏组织中NF-κB1A表达水平的药物;
用途D.用于制备减低肝脏组织中CXCL-2表达水平的药物;
用途E.用于制备减低肝脏组织中IL-6表达水平的药物;
用途F.用于制备减低肝脏组织中CCL-17表达水平的药物。
8.如权利要求1所述的一种活性成分的用途,其特征在于:
所述用途包括:用于制备减缓由MRSA引起的小鼠体重丢失的药物。
9.如权利要求1-7任一所述的一种活性成分的用途,其特征在于:
所述药物的有效剂量不低于10-8/ml。
10.一种MRSA实验模型,其特征在于:向实验模型添加MRSA菌液。
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WO2004055052A2 (en) * | 2002-12-17 | 2004-07-01 | Stichting Top-Instituut Voedselwetenschappen | Nucleic acid sequences for use as biomarker for damage to the intestinal epithilum |
CN102657844A (zh) * | 2006-03-10 | 2012-09-12 | 泰克诺根股份公司 | 长五聚环蛋白ptx3在预防或治疗病毒性疾病中的应用 |
CN102675433A (zh) * | 2012-05-04 | 2012-09-19 | 重庆原伦生物科技有限公司 | 耐甲氧西林金黄色葡萄球菌IsdB蛋白活性片段的重组蛋白、及其制备方法和应用 |
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WO2004055052A2 (en) * | 2002-12-17 | 2004-07-01 | Stichting Top-Instituut Voedselwetenschappen | Nucleic acid sequences for use as biomarker for damage to the intestinal epithilum |
CN102657844A (zh) * | 2006-03-10 | 2012-09-12 | 泰克诺根股份公司 | 长五聚环蛋白ptx3在预防或治疗病毒性疾病中的应用 |
CN102675433A (zh) * | 2012-05-04 | 2012-09-19 | 重庆原伦生物科技有限公司 | 耐甲氧西林金黄色葡萄球菌IsdB蛋白活性片段的重组蛋白、及其制备方法和应用 |
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