CN113304247B - Reg4蛋白及其在抵抗肠炎伤寒沙门氏菌感染中的应用 - Google Patents
Reg4蛋白及其在抵抗肠炎伤寒沙门氏菌感染中的应用 Download PDFInfo
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- CN113304247B CN113304247B CN202110355131.1A CN202110355131A CN113304247B CN 113304247 B CN113304247 B CN 113304247B CN 202110355131 A CN202110355131 A CN 202110355131A CN 113304247 B CN113304247 B CN 113304247B
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Abstract
本发明提供了一种活性因子的用途，其特征在于：所述活性因子为Reg4，或与其相差不多于三个氨基酸序列的物质；所述用途包含如下用途中的至少一种：用途A.用于制备治疗和/或缓解基于伤寒沙门氏菌感染引发的病症的药物；用途B.用于制备治疗和/或缓解基于伤寒沙门氏菌感染引发的并发症的药物。
Description
技术领域
本发明涉及一种活性因子，具体地，涉及Reg4蛋白及其在抵抗肠炎伤寒沙门氏菌感染中的应用。
背景技术
沙门氏菌是重要的人畜共患传染病原菌，其感染是引起世界性胃肠疾病的主要因素，临床上主要以伤寒沙门氏菌(Salmonella typhimurium)和副伤寒沙门氏菌为主。沙门氏菌是引起儿童出现细菌感染性腹泻的最主要致病菌。文献报道显示，沙门氏菌感染在腹泻患儿粪便病原学检测中阳性率高达5.5％。沙门氏菌感染极易导致患儿出现发热、呕吐和腹泻等全身不适症状，严重影响患儿身心健康。沙门氏菌感染具有传染性，可能呈流行性爆发趋势。使用抗生素是治疗肠炎沙门氏菌感染的重要手段。但近年来，随着抗生素的大量使用和滥用，沙门氏菌耐药问题愈发严峻。环丙沙星是临床上治疗沙门氏菌感染的主要药物之一，但目前已经存在大量沙门氏菌对环丙沙星产生耐药性。而且，抗生素残留与年龄存在相关性，由于儿童免疫系统发育不完善，免疫功能弱，导致抗生素在儿童体内残留较高，并且残留抗生素的种类繁多，对儿童的生长发育非常不利。因此，目前迫切需要开发不易产生耐药性、活性高、安全可靠的新型抗沙门氏菌感染的药物。
抗菌肽是抗生素的理想代替品之一。抗菌肽是一类小分子多肽，在动植物体内分布广泛，是机体先天性免疫系统的重要组成部分。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，而且具有不易产生耐药性的特性。
发明内容
本发明经研究表明Reg4蛋白能够有效治疗肠炎伤寒沙门氏菌感染，故而，提出了Reg4蛋白作为抗菌肽用于治疗伤寒沙门氏菌感染所致急性胃肠炎的方法。
本发明提供了一种活性因子的用途，其特征在于：
上述活性因子选自如下S1-S2中的一种或几种：
S1.上述活性因子为重组人Reg4，其氨基酸序列如SEQ ID NO:1所示；
S2.上述活性因子为重组鼠Reg4，其氨基酸序列如SEQ ID NO:2所示；
上述用途包含如下用途中的至少一种：
用途A.用于制备治疗和/或缓解基于伤寒沙门氏菌感染引发的病症的药物；
用途B.用于制备治疗和/或缓解基于伤寒沙门氏菌感染引发的并发症的药物。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述用途包括如下用途中的至少一种：
用途1.用于制备治疗伤寒沙门氏菌感染所致急性胃肠炎的药物；
用途2.用于制备缓解伤寒沙门氏菌感染引起体重降低情况的药物；
用途3.用于制备提高基于伤寒沙门氏菌感染致死的存活率的药物；
用途4.用于制备治疗和/或缓解伤寒沙门氏菌引起的肠道损伤的药物；
用途5.用于制备治疗和/或缓解伤寒沙门氏菌引起的肠道损伤的药物；
用途6.用于制备治疗和/或缓解伤寒沙门氏菌对各脏器的侵袭的药物。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述活性因子的用量为药物总重量的10-8-100％。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述药物的剂型为经胃肠道以外给药途径剂型或经胃肠道给药剂型。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述药物的给药途径剂型选自注射给药剂型、呼吸道给药剂型、滴鼻剂、皮肤给药剂型、黏膜给药剂型或腔道给药剂型、散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂；
上述注射给药剂型的输注方式选自静脉内输注、腹膜内注射、皮下输注、肌肉输注。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述Reg4蛋白的表达与纯化方法如下所示：
S1.pET-28-Reg4(NM_026328-6His)和pET-28-Reg4(NM_001159352-6His)载体构建；
S2.获得含重组表达质粒的表达菌种；
S3.Reg4蛋白诱导表达及鉴定；
S4.Reg4蛋白纯化；
S5.蛋白脱盐；
S6.超滤法浓缩蛋白。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述人Reg4的原核表达质粒如SEQ ID NO.3所示。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述鼠Reg4的原核表达质粒如SEQ ID NO.4所示。
进一步地，本发明提供的一种活性因子的用途，其特征在于：
上述Reg4蛋白在大肠杆菌BL21菌株中进行表达。
本发明的作用和效果：
本发明公开的Reg4蛋白抗菌活性较强，具有分子质量小、抗蛋白酶降解、抗菌谱广、抗菌机制异于传统抗生素，对抗生素产生的超级耐药菌株具有较好的抗菌活性等特性。
本发明公开的Reg4蛋白属于人体抗菌肽中的一种，不会引起机体排异反应，且无药物残留等风险。
本发明公开的Reg4蛋白有望替代传统抗生素，成为今后治疗肠炎伤寒沙门氏菌感染的最佳药物。
附图说明
图1.琼脂电泳图；
其中，图A为鼠Reg4全长RCR扩增产物的琼脂电泳图；
图B为人Reg4全长PCR扩增产物的琼脂电泳图。
图2.考马斯亮蓝染色图；
其中，图A为纯化的重组人Reg4蛋白考马斯亮蓝染色图；
图B为纯化的鼠Reg4蛋白考马斯亮蓝染色图。
图3.体外抑菌实验菌落数目测定结果；
其中，图3A和图3B为重组鼠Reg4蛋白抑制鼠伤寒沙门氏菌生长的作用；
图3C和图3D为重组人Reg4蛋白抑制肠沙门氏菌亚种肠炎血清型生长的作用。
图4A.小鼠体重变化结果；
图4B.存活率变化结果；
图5.盲肠HE病理及评分；
其中，图5A为盲肠HE染色光镜(各组放大倍数：×100)；
图5B为各组小鼠盲肠炎症病理学评分。
图6.盲肠及粪便中鼠伤寒沙门氏菌菌落检测结果；
其中，图6A为重组鼠Reg4蛋白对盲肠和粪便中伤寒沙门氏菌含量的影响；
图6B为各组小鼠盲肠组织中鼠伤寒沙门氏菌含量的比较；
图6C为各组小鼠盲肠组织中鼠伤寒沙门氏菌含量的比较。
图7.鼠伤寒沙门氏菌在体内脏器定值分布数量检测结果；
其中，图7A为各组小鼠肝脏、脾脏和肠系膜淋巴结中鼠伤寒沙门氏菌的含量；
图7B为各组小鼠肝脏中鼠伤寒沙门氏菌含量的比较；
图7C为各组小鼠脾脏中鼠伤寒沙门氏菌含量的比较；
图7D为各组小鼠肠系膜淋巴结中鼠伤寒沙门氏菌含量的比较。
具体实施方式
1.Reg4蛋白表达与纯化
1.1主要试剂
(1)pET-28载体：CV205，上海吉凯基因化学技术有限公司
(2)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
(3)卡那霉素：E004000-5G，上海元象医疗器械有限公司
(4)异丙基-β-D-硫代半乳糖苷(IPTG)/Isopropylβ-D-thiogalctoside：MB3026，上海源祉生物科技有限公司
(5)NUPAGE 4X LDS SAMPLE BUFFER：NP0008，英潍捷基(上海)贸易有限公司
(6)Ni-NTA Fast Start Kit(6):30600，上海翊瑞生物科技有限公司
(7)Vivaspin 20,3kDa MWCO PES：28932358，上海柏根生物科技有限公司
(8)PD MidiTrap G-25：28918008，上海柏根生物科技有限公司
(9)雅酶PS111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
(10)PIERCE BCA PROTEIN ASSAY:23227，英潍捷基(上海)贸易有限公司。
1.2方法
1.2.1pET-28-Reg4(NM_026328-6His)和pET-28-Reg4(NM_001159352-6His)载体构建
设计Reg4(NM_026328-6His)和Reg4(NM_001159352-6His)载体全长上下游引物，进行PCR扩增，获得Reg4(NM_026328-6His)cDNA和Reg4(NM_001159352-6His)cDNA，在pET-28质粒两端分别引入HindⅢ和BamHⅠ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。
其中，鼠Reg4质粒引物序列如下：
| ID | seq | |
| SEQ ID NO.5 | Reg4(55744-1)-P1 | AAGAAGGAGATATACCATGGCTTCCAAAGGCGTGCG |
| SEQ ID NO.6 | Reg4(55744-1)-P2 | CGAGTGCGGCCGCAAGCTTCTAATGATGATGATGATGATGTGTC |
人Reg4质粒引物序列如下：
1.2.2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30min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200rpm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，37℃培养12-16h。
(6)剩余菌液置于4℃冰箱保存。
1.2.3Reg4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，37℃摇床培养过夜。
(2)按照1:100接种量将上述中试管中的菌体接种至装有50ml 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后。
(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-硫代半乳糖苷至终浓度1mM作为实验组，两组继续37℃摇床200rpm养4h。
(4)离心4000rpm，10min，取上清，加入5μl的4×LDS sample buffer，70℃孵育10min。
(5)收获沉淀，用PBS重悬沉淀，4℃，4000rpm离心10mi n，重复三遍，收集菌体。取细菌沉淀悬浮液，加入4×LDS sample buffer，70℃孵育10min。
(6)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。
(7)-20℃下冷冻并储存细菌沉淀过夜。
1.2.4Reg4蛋白纯化
(1)细菌沉淀冰上解冻15min，在10ml裂解缓冲液重悬。(确保100μl溶菌酶和10μl核酸酶已添加到裂解缓冲液中)。
(2)室温孵育1hh。通过轻轻旋转细胞悬液混合2-3次。
(3)在4℃下离心14000rpm，30min。分别收集上清和沉淀。
(4)取上清18μl，加6μl 4×LDS loading buffer；细菌沉淀用PBS重悬，取18μl，加6μl 4×LDS loading buffer，70℃孵育10min。
(5)轻轻地将快速镍柱倒置几次，使其树脂重新悬浮。
(6)打开镍柱出口的密封件，打开螺帽，让存储缓冲液排出。
(7)将步骤4中的细菌裂解物上清液加到镍柱上。
(8)收集滤过液，将6μl 4×LDS loading buffer添加到18μl滤过液中，-20℃储存，以进行SDS-PAGE分析。
(9)用4ml清洗缓冲液洗涤镍柱2次。收集两次洗涤液。将6μl 4×SDS loadingbuffer添加到18μl洗涤液中，-20℃储存，以进行SDS-PAGE分析。
(10)用1ml洗脱缓冲液洗脱结合的目的蛋白，2次。
(11)在单独的试管中收集每个洗脱部分。将6μl 4×LDS loading buffer添加到18μl洗脱液中，-20℃储存，以进行SDS-PAGE分析。
1.2.5Reg4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、上清、过滤液、清洗液1、清洗液2、洗脱液1、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5min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。
1.2.6蛋白脱盐
(1)使用灭菌双蒸水平衡GE PD MidiTrap G-25离心式脱盐柱。
(2)上样:在柱子平衡后，将样品加到柱子顶上。
(3)洗脱:样品全部进入柱子内后，柱顶加入灭菌双蒸水。
1.2.7超滤法浓缩蛋白
(1)将蛋白溶液加入超滤管中。
(2)在4℃下离心4000rpm。
(3)加入需要的蛋白储存液至4ml，离心。
(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EP管中，标记好储存液的成分，蛋白名称，蛋白浓度(浓缩后)，制备日期。
1.3结果
1.3.1Reg4原核表达质粒构建
扩增527bp的Reg4(NM_026328-6His)全长基因(图1A)和530bp的Reg4(NM_001159352-6His)全长基因(图1B)。经双酶切的质粒CV205与胶回收的Reg4(NM_026328-6His)和Reg4(NM_001159352-6His)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527bp和530bp，与目的基因大小一致。测序结果表明，pET-28质粒中成功插入Reg4(NM_026328-6His)全长目的基因或Reg4(NM_001159352-6His)全长目的基因。
1.3.2Reg4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。结果显示重组人Reg4蛋白和重组鼠Reg4蛋白均可在大肠杆菌BL21菌株中的表达，且为包涵体表达(图2A和图2B)。分别取未诱导菌体、诱导菌体、菌体裂解液、过滤液、第1次清洗液、第2次清洗液、第1次洗脱液和第2次洗脱液经SDS-PAGE分离。考马斯亮蓝结果显示在洗脱液中检测到重组人Reg4蛋白和重组鼠Reg4蛋白。在洗脱液中，可以看到相对分子质量约为17kDa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，重组人Reg4蛋白浓度为1.30mg/ml，重组鼠Reg4蛋白浓度为1.02mg/ml。
2体外抑菌实验
2.1主要材料
(1)鼠伤寒沙门氏菌(带红色荧光)：bio-1089980，北京百欧博伟生物技术有限公司
(2)肠沙门氏菌亚种肠炎血清型：BNCC186357，北纳创联生物技术有限公司
2.2方法
2.2.1沙门氏菌培养
分别取100μl鼠伤寒沙门氏菌(Salmonella typhimurium，S.Tm)和肠沙门氏菌(Salmonella enterica subsp.Enterica serovar Enteritidis，S.Enteritidis)菌液加至1000ml含50μg/ml卡那霉素的LB培养基中，37℃，过夜培养，培养至对数生长期后，4000rpm，20min，离心收集菌体，用PBS缓冲液重悬，使用平板计数法测定菌液菌落总数，-80℃保存备用。使用前，用灭菌PBS缓冲液将鼠伤寒沙门氏菌和肠沙门氏菌稀释至终浓度为5×105CFU/ml。
2.2.2Reg4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 EP管。
2.2.3Reg4蛋白与沙门氏菌共孵育
取15ml离心管4支，排成一排，第1管加入1980ul LB培养基和20μl稀释好的鼠伤寒沙门氏菌菌液(作为对照组)，第2管加入1978μl LB培养基、20μl菌液和2μl重组鼠Reg4蛋白(作为1μg/ml组)，第3管加入1960μl LB培养基、20μl菌液和20μl重组鼠Reg4蛋白(作为10μg/ml组),混匀。盖好盖子，置37℃恒温箱中孵育24h。同理，将重组人Reg4蛋白与肠炎沙门氏菌共孵育24hh。
2.2.4细菌菌落总数测定
将孵育好的菌液用灭菌水连续稀释至终浓度为1×107CFU/ml。取100μl孵育好的菌液均匀涂抹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。鼠伤寒沙门氏菌菌落总数为5.5×109CFU/ml，肠沙门氏菌菌落总数为1.86×108CFU/ml。
2.2.2重组Reg4蛋白抑制细菌生长
将鼠伤寒沙门氏菌分别与PBS、1μg/ml和10μg/ml重组鼠Reg4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9.71×109CFU/ml,给予1μg/ml重组鼠Reg4蛋白后细菌总数减少至5.87×109CFU/ml(p＝0.026)，给予10μg/ml重组鼠Reg4蛋白细菌总数则减少约200倍，总数为4.75×107CFU/ml(p<0.0001)。结果表明，重组鼠Reg4蛋白能有效抑制鼠伤寒沙门氏菌的生长。同样地，将肠沙门氏菌亚种肠炎血清型细菌分别与PBS、1μg/ml和10μg/ml重组人Reg4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1.06×1010CFU/ml,给予1μg/ml重组人Reg4蛋白后细菌总数减少至7.25×109CFU/ml，给予10μg/ml重组人Reg4蛋白细菌总数则减少约6倍，总数为1.67×109CFU/ml(p＝0.0008)。结果表明，重组人Reg4蛋白能有效抑制肠沙门氏菌亚种肠炎血清型细菌的生长。
3体内抑菌实验
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)灌胃针8号：GWZ-8-45，上海晶旷生物科技有限公司
3.2方法
3.2.1Reg4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 EP管。
3.2.2鼠伤寒沙门氏菌菌液稀释
用灭菌PBS缓冲液将伤寒沙门氏菌稀释至终浓度为1×109CFU/ml。
3.2.3小鼠急性感染实验
取7-8周龄野生型(wide type,WT)C57BL/6小鼠和Reg4基因敲除小鼠(Reg4ΔIEC)小鼠作为感染对象，随机分成6组，分组如下：包括WT+PBS组(n＝10)，WT+S.Tm+PBS组，WT+S.Tm+1μg/ml Reg4组(n＝10)，WT+S.Tm+10μg/ml Reg4组(n＝10)，Reg4ΔIEC+S.Tm+PBS组(n＝9)和Reg4ΔIEC+S.Tm+10μg/ml Reg4组(n＝9)。WT组小鼠雌雄比例为1:1，Reg4ΔIEC组小鼠雌雄比例为4:5。
在细菌感染前一天先给予每只小鼠灌胃20mg卡那霉素溶液(100μl，浓度为200mg/ml)。给予每只小鼠灌胃100μl鼠伤寒沙门氏菌菌液(细菌总量为1×108CFU)，对照组给予灌胃100ul灭菌PBS，给药组经灌胃给予100μl浓度为1μg/ml或10μg/ml重组鼠Reg4蛋白，WT+PBS组、WT+S.Tm+PBS组和Reg4ΔIEC+S.Tm+PBS组给予等量100μl灭菌PBS溶液。灌胃之前均需要用75％酒精消毒灌胃针。
每日定时记录小鼠体重，在感染第3天进行取材。小鼠麻醉之后眼球取血，脱颈处死，开腹取材。取部分盲肠和结肠置于4％多聚甲醛中固定。收集肝脏、脾脏、肠系膜淋巴结、部分盲肠和小鼠粪便用于细菌总数测定。刮取回肠末端和结肠肠粘膜。
3.2.4小鼠慢性感染实验
取7-8周龄野生型C57BL/6小鼠(雌雄各半)作为慢性感染实验对象，随机分成3组，包括WT+PBS组(n＝10)，WT+S.Tm+PBS组(n＝10)，WT+S.Tm+10μg/ml鼠Reg4组(n＝10)。
在细菌感染前一天先给予每只小鼠灌胃20mg卡那霉素溶液(100μl，浓度为200mg/ml)。给予每只小100μl鼠伤寒沙门氏菌菌液(细菌总量为1×108CFU)，WT+S.Tm+10μg/mlReg4组第一天灌胃100μl浓度为10μg/ml鼠Reg4蛋白，之后每天经腹腔注射给予100μl 10μg/ml鼠Reg4蛋白；WT+PBS组和WT+S.Tm+PBS组给予第一天给予灌胃100μl灭菌PBS溶液，之后每日给予腹腔注射100μl灭菌PBS。灌胃之前均用75％酒精消毒灌胃针。每日观察小鼠体重变化和存活情况。
3.2.5脏器细菌负荷量测定
用无菌剪刀、镊子取小鼠肝脏、脾脏、肠系膜淋巴结、盲肠和粪便，根据重量加入无菌PBS溶液，无菌环境中进行匀浆处理。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。
3.2.6肠道组织病理学评分
取小鼠盲肠和结肠置于4％多聚甲醛中固定，常规脱水、包埋、切片、HE染色，观察切片。
3.3结果
3.3.1Reg4蛋白能减缓小鼠体重丢失和提高存活率
各组小鼠体重丢失情况如图4A所示，未给予S.Tm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102.7％，而给予S.Tm感染的各组小鼠均出现不同程度的体重丢失。Reg4ΔIEC+S.Tm+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91.2％，其次是WT+S.Tm+PBS组(94.46％)、Reg4ΔIEC+S.Tm+10μg/ml Reg4组(96.04％)、WT+S.Tm+1μg/ml Reg4组(96.4％)和WT+S.Tm+10μg/ml Reg4组(100.2％)。在感染后第2天体重中，与WT+S.Tm+PBS组小鼠相比，WT+S.Tm+10μg/ml Reg4组小鼠体重丢失减缓(100.2％vs.94.46％，p＝0.0005)；与Reg4ΔIEC+S.Tm+PBS组小鼠相比，Reg4ΔIEC+S.Tm+10μg/ml Reg4组小鼠体重丢失减缓(96.04％vs.94.46％，p＝0.0137)。结果表明，给予Reg4蛋白能减缓小鼠体重丢失。
在慢性感染实验中，各组小鼠存活情况如图4B所示。WT+S.Tm+PBS组小鼠在感染后第7天全部死亡，而WT+S.Tm+10μg/ml Reg4组小鼠与WT+S.Tm+PBS组小鼠相比死亡率有所下降(p<0.05)。
3.3.2Reg4蛋白能缓解鼠伤寒沙门氏菌引起的肠道损伤
病理检测结果如图5显示，相比于WT+S.Tm组小鼠盲肠，WT+S.Tm+PBS组和Reg4ΔIEC+S.Tm+PBS组小鼠盲肠肠绒毛均发生严重的破损、脱落，粘膜脱落至肠腔内，肠腔内可见纤维素渗出，大部分腺体被破坏，粘膜肌层和粘膜下层可见有大量中性粒细胞浸润。而Reg4蛋白给药组，包括WT+S.Tm+1μg/ml Reg4组、WT+S.Tm+10μg/ml Reg4组和Reg4ΔIEC+S.Tm+PBS组小鼠盲肠损伤均有所改善，肠粘膜脱落减轻，固有层炎症细胞浸润减少。肠道病理评分也显示，WT+S.Tm+PBS组和Reg4ΔIEC+S.Tm+PBS组小鼠发生严重的炎症，而给予Reg4蛋白后肠道炎症明显减轻。结果表明Reg4蛋白科研有效缓解鼠伤寒沙门氏菌造成的肠道病理损伤。
对各组小鼠粪便和盲肠中的鼠伤寒沙门氏菌荷菌数进行检测，结果如图6。
WT+PBS组盲肠和粪便中均未检测到鼠伤寒沙门氏菌，而WT+S.Tm+PBS组小鼠盲肠和粪便均检测到大量鼠伤寒沙门氏菌。在给予Reg4蛋白治疗后，WT+S.Tm+1μg/ml Reg4组小鼠盲肠和粪便中的鼠伤寒沙门氏菌数目明显减少，且随着Reg4蛋白给药剂量增加之后，WT+S.Tm+10μg/ml Reg4组小鼠各脏器鼠伤寒沙门氏菌数目进一步减少(p<0.05)。在Reg4ΔIEC小鼠中，给予灌胃鼠伤寒沙门氏菌后，Reg4ΔIEC+S.Tm+PBS组盲肠和粪便中鼠伤寒沙门氏菌数目较WT+S.Tm+PBS组小鼠进一步增多，给予Reg4蛋白治疗后，Reg4ΔIEC+S.Tm+10μg/ml Reg4组小盲肠和粪便中鼠伤寒沙门氏菌数量减少。以上结果均表明Reg4蛋白能减少盲肠和粪便中伤寒沙门氏菌含量。
3.3.3Reg4蛋白能减轻鼠伤寒沙门氏菌对小鼠脏器组织的侵袭情况
对各组小鼠肠系膜淋巴结、脾脏和肝脏中的鼠伤寒沙门氏菌荷菌数进行检测，结果如图7。WT+PBS组各器官中未检测到鼠伤寒沙门氏菌，WT+S.Tm+PBS组小鼠各脏器均可见鼠伤寒沙门氏菌定植分布。在给予Reg4蛋白治疗后，WT+S.Tm+1μg/ml Reg4组小鼠各脏器的鼠伤寒沙门氏菌数目明显减少，且随着Reg4蛋白给药剂量增加之后，WT+S.Tm+10μg/mlReg4组小鼠各脏器鼠伤寒沙门氏菌数目进一步减少。在Reg4ΔIEC小鼠中，给予灌胃鼠伤寒沙门氏菌后，Reg4ΔIEC+S.Tm+PBS组各脏器鼠伤寒沙门氏菌数目较WT+S.Tm+PBS组小鼠进一步增多，给予Reg4蛋白治疗后，Reg4ΔIEC+S.Tm+10μg/ml Reg4组小鼠各脏器鼠伤寒沙门氏菌定植减少。以上结果均表明Reg4蛋白能有效抑制鼠伤寒沙门氏菌侵袭其他器官组织。
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序列表
<110> 上海市儿科医学研究所
<120> Reg4蛋白及其在抵抗肠炎伤寒沙门氏菌感染中的应用
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Cys Gly Ala Gly Thr Gly Cys Gly Gly Cys Cys Gly Cys Ala Ala Gly
1 5 10 15
Cys Thr Thr Cys Thr Ala Ala Thr Gly Ala Thr Gly Ala Thr Gly Ala
20 25 30
Thr Gly Ala Thr Gly Ala Thr Gly Thr Gly Gly Thr Cys Gly Gly Thr
35 40 45
Ala Cys Thr Thr Gly Cys Ala Cys Ala Gly Gly
50 55
Claims (5)
1.一种活性因子的用途，其特征在于：
所述活性因子选自如下S1-S2中的一种或几种：
S1.所述活性因子为重组人Reg4，其氨基酸序列如SEQ ID NO:1所示；
S2.所述活性因子为重组鼠Reg4，其氨基酸序列如SEQ ID NO:2所示；
所述用途为用于制备治疗和/或缓解基于伤寒沙门氏菌感染引发的病症的药物。
2.一种活性因子的用途，其特征在于：
所述活性因子选自如下S1-S2中的一种或几种：
S1.所述活性因子为重组人Reg4，其氨基酸序列如SEQ ID NO:1所示；
S2.所述活性因子为重组鼠Reg4，其氨基酸序列如SEQ ID NO:2所示；
所述用途包括如下用途中的至少一种：
用途1.用于制备治疗伤寒沙门氏菌感染所致急性胃肠炎的药物；
用途2.用于制备缓解伤寒沙门氏菌感染引起体重降低情况的药物；
用途3.用于制备提高基于伤寒沙门氏菌感染致死的存活率的药物；
用途4.用于制备治疗和/或缓解伤寒沙门氏菌引起的肠道损伤的药物；
用途5.用于制备治疗和/或缓解伤寒沙门氏菌对各脏器的侵袭的药物。
3.如权利要求1所述的一种活性因子的用途，其特征在于：
所述药物的剂型为经胃肠道以外给药途径剂型或经胃肠道给药剂型。
4.如权利要求1所述的一种活性因子的用途，其特征在于：
所述药物的给药途径剂型选自注射给药剂型、呼吸道给药剂型、滴鼻剂、皮肤给药剂型、黏膜给药剂型或腔道给药剂型、散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂；
所述注射给药剂型的输注方式选自静脉内输注、腹膜内注射、皮下输注、肌肉输注。
5.如权利要求1所述的一种活性因子的用途，其特征在于：
所述人Reg4/鼠Reg4蛋白在大肠杆菌BL21菌株中进行表达。
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| CN116236563A (zh) * | 2023-01-12 | 2023-06-09 | 佛山病原微生物研究院 | Asb17在制备用于预防或治疗鼠疫伤寒沙门氏菌导致炎症的药物中的用途 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101951945A (zh) * | 2007-11-07 | 2011-01-19 | 健泰科生物技术公司 | 用于治疗微生物病症的组合物和方法 |
| CN102370966A (zh) * | 2010-08-13 | 2012-03-14 | 上海伍洋生物医药技术有限公司 | Reg4的用途及其药物组合物 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101951945A (zh) * | 2007-11-07 | 2011-01-19 | 健泰科生物技术公司 | 用于治疗微生物病症的组合物和方法 |
| CN102370966A (zh) * | 2010-08-13 | 2012-03-14 | 上海伍洋生物医药技术有限公司 | Reg4的用途及其药物组合物 |
Non-Patent Citations (2)
| Title |
|---|
| Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut;Houbao Qi等;COMMUNICATIONS BIOLOGY;第3卷;483 * |
| Reg4 protects against Salmonella infection- associated intestinal inflammation via adopting a calcium-dependent lectin-like domain;Weipeng Wang等;Int Immunopharmacol;第113卷;109310 * |
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