CN114432433B - 牛支原体分泌蛋白MbovP725在制备防治牛支原体感染的药物中的用途 - Google Patents
牛支原体分泌蛋白MbovP725在制备防治牛支原体感染的药物中的用途 Download PDFInfo
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- CN114432433B CN114432433B CN202111645435.8A CN202111645435A CN114432433B CN 114432433 B CN114432433 B CN 114432433B CN 202111645435 A CN202111645435 A CN 202111645435A CN 114432433 B CN114432433 B CN 114432433B
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- mycoplasma bovis
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Abstract
本发明公开了牛支原体(Mycoplasma bovis)分泌蛋白MbovP725在制备防治牛支原体感染的药物中的用途,所述牛支原体分泌蛋白MbovP725的氨基酸序列如SEQ ID NO:2所示。所述牛支原体分泌蛋白MbovP725能以剂量依赖性抑制脂多糖诱导的牛巨噬细胞和牛乳腺上皮细胞IL‑1β,IL‑6,TNF‑α的表达从而发挥抗炎作用,同时还能作为磷酸酶与底物进行酶促反应,从而抑制MAPK信号通路关键蛋白分子的磷酸化表达,在防治牛支原体感染方面具有很好的应用前景。
Description
技术领域
本发明属于分子生物学领域,涉及牛支原体(Mycoplasma bovis)分泌蛋白MbovP725的新用途,尤其是在防治牛支原体感染中的用途。
背景技术
牛支原体是牛呼吸道系统综合症的重要病原菌,主要导致牛肺炎和乳腺炎,此外还引起关节炎、耳膜炎、流产、脑炎等多种症状,该病在世界范围内广泛流行。由于牛支原体具有在牛体内持续存在和条件性致病等特征,目前在临床上缺乏有效的防控手段,既无有效疫苗,也无特效药物,且随着抗生素的不合理使用,使其耐药株出现的频率呈逐年增加趋势,给临床治疗该病增加了困难。因此,明确牛支原体致病机制可为研制高效疫苗和新型药物,用于防控牛支原体病提供理论依据。
牛支原体感染宿主后,持续存在于体内而发展为一种慢性炎性反应,这与牛支原体的免疫逃避有关。呼吸道系统综合症感染初期,呼吸道炎症的发生再加上支原体诱导宿主免疫细胞产生促炎因子,这是一种有害的效应机制而加重感染的发生(Baquero et al.,2021)。研究发现,牛支原体可以逃避免疫反应,具有阻止巨噬细胞的吞噬和抑制关键炎性因子表达的能力(Dudek and Szacawa,2020),这很大程度上得益于牛支原体能够调节宿主的免疫应答,使宿主产生的免疫反应不能有效的清除牛支原体(Maunsell and Chase,2019)。因此,支原体可能进化出相应的策略来抑制机体的炎性应答,寻找相关的靶标分子可能具有治疗药物的潜能。
由于牛支原体缺乏细胞壁,它与宿主环境的交流主要依赖于表面的膜蛋白和分泌蛋白,以及锚定在质膜上的相关蛋白,这些蛋白在营养物质摄入、黏附、调节宿主免疫反应方面具有重要的作用(Christodoulides et al.,2018;Zhang et al.,2021)。其中,分泌蛋白常与病原体毒力和免疫反应相关,因此是寻找特异性分子靶标的优先考虑对象。本申请人通过Label free蛋白组学技术与质谱分析鉴定出MbovP725蛋白存在于牛支原体分泌蛋白组中,证明该分泌蛋白是牛支原体的一种丝氨酸/苏氨酸特异性磷酸酶,进一步证明该蛋白可抑制脂多糖(Lipopolysaccharide,LPS)诱导的宿主细胞炎性因子的产生及MAPK信号通路关键蛋白分子的磷酸化表达。鉴于炎性反应是宿主抵御牛支原体感染的重要过程,因此该蛋白是一种很有前途的分子靶标。
发明内容
本发明的目的在于提供牛支原体分泌蛋白MbovP725的新用途,一方面该蛋白可抑制宿主细胞炎性因子的产生从而帮助宿主抵御牛支原体感染,另一方面可作为磷酸酶与底物进行酶促反应,从而抑制MAPK信号通路关键蛋白分子的磷酸化表达,表明该蛋白在防治牛支原体感染方面具有很好的应用前景。
为实现上述目的,申请人以牛支原体本地分离株HB0801基因组为模板,克隆Mbov_0725基因,将基因按照大肠杆菌密码子偏爱性进行修饰,表达和纯化重组rMbovP725。
所述牛支原体分泌蛋白MbovP725的氨基酸序列如SEQ ID NO:2所示。
编码所述牛支原体分泌蛋白MbovP725的核苷酸序列如SEQ ID NO:1所示。
以磷酸酶通用底物——对硝基苯磷酸二钠(pNPP)为反应底物,与rMbovP725蛋白在不同反应体系(pH、温度、金属离子)中进行酶促反应,确认该蛋白为Mg2+依赖的磷酸酶,其最适酶促反应温度约为42℃,最适pH为8-9。
分别在0h、8h、12h、24h、36h、48h对应时间点收集牛支原体培养上清,离心后过滤浓缩,使用蛋白质免疫印迹(Western blot)的方法,证明该蛋白为一种分泌蛋白,可分泌至胞外发挥磷酸酶的去磷酸化作用,影响牛支原体获得生长必须的营养物质。
使用Triton X-114液相分离法去除重组蛋白溶液中内毒素,将去除内毒素后的rMbovP725蛋白与LPS分布在牛巨噬细胞(Bovine macrophage,BoMac)和牛乳腺上皮细胞(Bovine mammary epithelial cells,MAC-T)进行共孵育,通过荧光定量PCR技术检测并证实rMbovP725蛋白能抑制LPS诱导的细胞炎性因子(IL-1β,IL-6,TNF-α)产生,确认该蛋白是牛支原体的一种可抑制宿主细胞炎性反应的分泌蛋白。
鉴于炎性反应是宿主抵御牛支原体感染的重要过程,因此该蛋白可用于制备药物,发挥抗炎作用,帮助宿主抵御牛支原体感染。
该蛋白作为磷酸酶能与底物进行酶促反应,将底物去磷酸化,因此在该蛋白在生化反应等领域也具有一定的应用前景。
更详细的技术方案参见《具体实施方式》所述。
附图说明
图1:构建的重组质粒pET-30a-Mbov_0725图谱。
图2:纯化的牛支原体rMbovP725蛋白电泳图。M表示蛋白分子量marker;1表示蛋白诱导表达前;2表示蛋白诱导表达后;3表示纯化后rMbovP725蛋白,蛋白分子量为33kDa。
图3:MbovP725多抗血清的效价检测结果。
图4:牛支原体MbovP725蛋白在牛支原体培养物上清中分泌鉴定。泳道1-6分别表示培养0h、8h、12h、24h、36h、48h后收取的牛支原体培养上清,MbovP280是已报道的分泌蛋白,为本实验的阳性对照。
图5:牛支原体rMbovP725以pNPP为底物,在不同酶促反应体系下酶活实验结果图。A:不同时间内rMbovP725降解pNPP的磷酸酶活性检测;B:不同剂量的rMbovP725降解pNPP的磷酸酶活性检测;C:不同金属离子作用下rMbovP725的磷酸酶活性检测;D:不同浓度的Mg2+处理后rMbovP725的磷酸酶活性检测;E:rMbovP725的最适pH分析;F:rMbovP725的最适作用温度分析。
图6:牛支原体rMbovP725抑制BoMac和MAC-T细胞炎性因子mRNA产生的分析图;A:rMbovP725在BoMac细胞中对LPS诱导的细胞炎性因子产生影响的统计分析图;B:rMbovP725在MAC-T细胞中对LPS诱导的细胞炎性因子产生影响的统计分析图,*p<0.05,**p<0.01,***p<0.001。
具体实施方式
下面通过具体实施例对本发明进行详细说明。
实施例1:牛支原体Mbov_0725基因克隆和表达纯化
1.1牛支原体Mbov_0725基因克隆和表达和MbovP725纯化
根据大肠杆菌密码子的偏爱性,将该基因的密码子UGA突变为能在大肠杆菌中表达色氨酸的密码子UGG,得到牛支原体HB0801(基因组GenBank登录号为CP002058)中的Mbov_0725基因的核苷酸序列见SEQ ID NO:1,序列大小为858bp。将SEQ ID NO:1所示的序列送商业基因克隆公司进行合成。原基因序列和突变后的基因序列编码的蛋白质序列相同(序列见SEQ ID NO:2),编码285个氨基酸。
将合成的Mbov_0725基因的扩增产物,用Xho I和BamH Ⅰ酶切,同时将pET-30a质粒(购自默克中国有限公司)用Xho I和BamH Ⅰ进行双酶切。将酶切后的Mbov_0725基因和pET-30a质粒用DNA连接酶(T4 DNA ligase,购自NEB公司)进行连接,得到重组质粒pET-30a-Mbov_0725(图谱见图1)。用该重组质粒pET-30a-Mbov_0725转化大肠杆菌DH5α后,置于37℃摇床在180r/min下培养12h,提取质粒,用通用载体T7经过测序,确定插入序列正确后转化大肠杆菌BL21。将所得的重组大肠杆菌BL21在LB液体培养基中培养至OD=0.6时,取1mL菌液作为诱导前对照,同时加入异丙基硫代半乳糖苷(IPTG)至终浓度为0.8mM,在37℃摇床上诱导表达3h。取1mL菌液进行下一步处理:样品处理方法为12000r/min离心1min后弃掉上清,加入1mL磷酸盐缓冲液即PBS(配方:KCl 0.2g,NaCl 8g,Na2HPO4 1.44g,KH2PO4 0.24g,1000mL蒸馏水,pH=7.6)溶液重悬后再以12000r/min离心1min弃掉上清,然后加入30uL的PBS和30uL上样缓冲液(1M Tris-HCl(pH=6.8)1mL,200mM DDT 0.31g,4%SDS 0.4g,0.2%溴酚蓝0.02g,20%甘油2mL,7mL超纯水)重悬。在100℃沸水中煮沸10min。用12%SDS-PAGE凝胶电泳鉴定是否表达。rMbovP725蛋白大小为33kDa。
1.2.牛支原体rMbovP725的纯化
将重组的大肠杆菌BL21用0.8mM IPTG诱导表达后,取菌液8000r/min离心10min后弃掉上清,用500mL的PBS洗一次后在8000r/min离心10min,再重复用500mL的PBS洗一次。弃掉上清后加入30mL的PBS重悬,加入蛋白酶抑制剂(购自上海罗氏制药有限公司),使用液压破碎仪破碎。破碎后以12000r/min离心30min,取30μL上清加入30μL上样缓冲液,在沸水中煮沸10min作为上清组。取少许沉淀加入30μL的PBS和30μL上样缓冲液煮沸10min作沉淀组。经过12%SDS-PAGE凝胶电泳后,确定rMbovP725大部分表达于上清中。
rMbovP725蛋白具体纯化步骤如下:
(1)向亲和层析柱中加入1mL Ni-NTA金属螯合His蛋白纯化介质填料(购自GE公司);
(2)向亲和层析柱中加入12mL ddH2O洗涤;
(3)加入12mL的binding buffer(20mM Na3PO4,0.5M NaCl,20mM咪唑,pH=7.4)平衡柱子;
(4)加入经过0.45μm孔径滤器过滤的蛋白表达上清;
(5)加入50mL binding buffer缓冲液平衡柱子;
(6)加入50mL washing buffer缓冲液(20mM Na3PO4,0.5M NaCl,60mM咪唑,pH=7.4)洗去杂蛋白;
(7)加入12mL elute buffer缓冲液(20mM Na3PO4,0.5M NaCl,1M咪唑,pH=7.4)洗脱目的蛋白;
(8)将洗脱的目的蛋白中加入5×SDS-PAGE蛋白上样缓冲液处理,煮沸10min,冷却至室温备用;
(9)配置12%SDS-PAGE聚丙酰胺凝胶,将处理好的样品以20μL/每孔上样到SDS-PAGE胶加样孔进行电泳(浓缩胶电泳条件为直流电压为80V,分离胶电泳条件为直流电压为120V)。电泳完成后,取下凝胶用考马斯亮蓝染色过夜。然后脱色,确定得到纯化的目的蛋白。
纯化后的rMbovP725蛋白大小为33kDa,且条带单一(图2)。
实施例2:牛支原体MbovP725分泌性验证
2.1制备MbovP725多抗血清制备及效价检测
将纯化后的rMbovP725蛋白分三次免疫BALB/C小鼠,采血,制备抗rMbovP725抗体并进行效价检测,具体方法如下:
(1)用BCA蛋白定量试剂盒测量纯化后蛋白的浓度,首次免疫,每只小鼠取100μg蛋白与相同体积的弗式完全佐剂混匀,充分乳化。乳化完成后采用多点颈背部皮下注射的方法进行免疫,免疫前小鼠采血,所采血液为阴性对照;
(2)首免2周后二免,再间隔1周后三免。二免、三免采用100μg蛋白与相同体积的弗式不完全佐剂混匀并乳化,并同样多点颈背部皮下注射进行免疫;
(3)三免后一周摘眼球采血,37℃放置1h,于4℃过夜,让血清自然析出,4℃4000r/min离心10min,收集血清,小份分装,保存于-20℃。
ELISA效价检测结果如图3所示。
2.2MbovP725分泌特性鉴定
分别在0h、8h、12h、24h、36h、38h对应时间点收取牛支原体培养上清,经12000rpm离心30min,将上清浓缩到1mL后,使用0.1μm的滤器过滤,加入5×SDS-PAGE蛋白上样缓冲液处理,煮沸10min。冷却至室温后,将蛋白样品直接上样到SDS-PAGE胶加样孔,上层胶电泳电压80V,下层胶电泳电压120V。电泳完成后,使用PVDF膜转印蛋白,本实施例采用湿转法,电压为100V,转膜时间为1h。转膜完毕后,使用5%脱脂牛奶室温封闭5h。使用TBST洗膜3次,每次间隔5min,然后与使用TBST稀释的MbovP725蛋白多抗置于4℃孵育过夜。使用TBST洗膜5次,每次5min。然后与使用TBST稀释的抗小鼠IgG抗体(Southern Biotech)(体积比为1:3000)于室温孵育1h。孵育完成后,用TBST洗膜五次,每次间隔5min,并使用化学发光显色和图像获取。试验结果表明,rMbovP725蛋白可分泌于支原体培养上清中(图4)。
实施例3:牛支原体rMbovP725的磷酸酶活性检测
利用对硝基苯磷酸二钠(p-nitrophenyl phosphate disodium salt,pNPP)作为底物,若蛋白为磷酸酶则可与pNPP反应,形成黄色水溶性反应产物,该产物在405nm处有最大吸光值。本实验以pNPP为反应底物,在酶活反应体系(50mM Tris-Hcl,10mM MgCl2,1.77mM pNPP)中进行酶促反应,以不含rMbovP725蛋白或不含pNPP为阴性对照,通过OD405数值来判断pNPP是否可被rMbovP725降解。试验结果表明,rMbovP725能够以时间依赖性和蛋白剂量依赖性降解pNPP。此外,在不同pH、温度、Mg2+浓度、不同金属离子条件的反应体系中进行酶促反应,试验结果表明,rMbovP725为Mg2+依赖的磷酸酶,最适酶促反应pH为8-9,最适反应温度约为42℃,结果如图5所示。
实施例4:牛支原体rMbovP725抑制宿主细胞炎性反应功能验证
4.1牛支原体rMbovP725蛋白内毒素的去除
在表达纯化后的rMbovP725蛋白溶液中加入1%Triton X-114,4℃缓慢摇转混匀1h。然后于30℃水浴锅中放置40min,期间不时颠倒混匀。经15000g室温离心15min,小心移出上层水相,进行下一次抽提,反复抽提3次(抽提过程中所用器材均为无热源器材)。完成最后一次抽提后,使用厦门鲎试剂内毒素检测试剂盒测定rMbovP725蛋白溶液内毒素含量。根据内毒素含量标准曲线计算得rMbovP725蛋白溶液内毒素含量小于1EU/mg。
4.2.rMbovP725可抑制LPS诱导的炎性细胞因子表达
LPS是一种炎性反应诱导剂,可有效激活宿主细胞炎性因子表达,为检测rMbovP725的抑炎作用,我们在两种不同的细胞(BoMac和MAC-T)中,利用LPS进行炎性刺激,同时加入不同浓度的rMbovP725蛋白,利用荧光定量检测炎性因子IL-1β、IL-6、TNF-α的转录水平。具体的,以每孔1×105个BoMac或MAC-T细胞接种于12孔板中,培养过夜至细胞贴壁,分别加入不同剂量的去除内毒素后的rMbovP0725蛋白(1μg、5μg、10μg、20μg),各孔同时加入1μg/mL LPS,与BoMac或MAC-T细胞共孵育12h后,利用Trizol裂解法提取细胞总RNA,将其反转录为cDNA后进行荧光定量PCR技术检测各孔IL-1β,IL-6,TNF-α的mRNA表达,用于确定rMbovP0725蛋白对LPS诱导的细胞炎性因子产生的影响。试验结果表明,rMbovP725蛋白以剂量依赖性显著抑制LPS诱导BoMac和MAC-T细胞的IL-1β,IL-6,TNF-αmRNA表达(图6)。
对序列表的说明:
序列表SEQ ID NO:1是牛支原体Mbov_0725基因依据大肠杆菌密码子偏爱性通过人工突变的核苷酸序列,序列长度为858bp。
序列表SEQ ID NO:2是牛支原体MbovP725蛋白的氨基酸序列,编码285个氨基酸。
附录:说明书中名词术语说明:
牛支原体MbovP725重组蛋白,以rMbovP725表示。
牛支原体MbovP725蛋白的编码基因,以Mbov_0725表示。
牛支原体本地分离株,以牛支原体HB0801表示。
序列表
<110> 华中农业大学
<120> 牛支原体分泌蛋白MbovP725在制备防治牛支原体感染的药物中的用途
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 858
<212> DNA
<213> 牛支原体(Mycoplasma bovis)
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atgaacaaga tcaacctgaa cgacattaaa tatatcttct tcgacctgga cggcaccctg 60
ctgacgaaag ataaagaaat caccaaagaa gttaccgcaa aaattaaaga actgaataag 120
acccatcagg tggttctgaa taccggcaga ccgtggtata tggctcagaa attttataaa 180
cagctggatc tgaaaacccc gtttctgggg ctgaatggcg cactgatata tgattttcaa 240
gacaaaaagg tgctgtataa aaacccaatt aacattgaaa ccagcctgaa gctgctgaat 300
ctgttcctga aatataatat tgctttcctg atctacctgg atgaagaaat gattggtgtt 360
cagaaattta agcatgaatg gttccagaaa gttgtgtatc cgaccgtgta tccgaagaat 420
gagtttagcg caaaatttac cgaaattaac gatcctgaaa accagctgaa actgaatacc 480
ccggttataa aatttctgct tttaatcgat accctgagcg aaagccaact gaaggaactg 540
cagaaagaga tcacgagcgt gagcgatgaa atatataccg tccgtagcca aaaagcagtt 600
ctggatgtga tgcccaaagg tagcgataaa ggtacagcaa ttgaatggtt tagcaaaaat 660
tacctgaaca atgaaaagat ctacgattac agcctgatgt ttggagacgc agcaaatgat 720
attccggcct tcgaaaaagt tgcatacagt tgcgcactga aaagcagcag cctggaggcc 780
gaaaatgcag caaattatgt tgtgagcagc agcaccgata atggaatcat agaatttttt 840
gtgaagcaca ccaattaa 858
<210> 2
<211> 285
<212> PRT
<213> 牛支原体(Mycoplasma bovis)
<400> 2
Met Asn Lys Ile Asn Leu Asn Asp Ile Lys Tyr Ile Phe Phe Asp Leu
1 5 10 15
Asp Gly Thr Leu Leu Thr Lys Asp Lys Glu Ile Thr Lys Glu Val Thr
20 25 30
Ala Lys Ile Lys Glu Leu Asn Lys Thr His Gln Val Val Leu Asn Thr
35 40 45
Gly Arg Pro Trp Tyr Met Ala Gln Lys Phe Tyr Lys Gln Leu Asp Leu
50 55 60
Lys Thr Pro Phe Leu Gly Leu Asn Gly Ala Leu Ile Tyr Asp Phe Gln
65 70 75 80
Asp Lys Lys Val Leu Tyr Lys Asn Pro Ile Asn Ile Glu Thr Ser Leu
85 90 95
Lys Leu Leu Asn Leu Phe Leu Lys Tyr Asn Ile Ala Phe Leu Ile Tyr
100 105 110
Leu Asp Glu Glu Met Ile Gly Val Gln Lys Phe Lys His Glu Trp Phe
115 120 125
Gln Lys Val Val Tyr Pro Thr Val Tyr Pro Lys Asn Glu Phe Ser Ala
130 135 140
Lys Phe Thr Glu Ile Asn Asp Pro Glu Asn Gln Leu Lys Leu Asn Thr
145 150 155 160
Pro Val Ile Lys Phe Leu Leu Leu Ile Asp Thr Leu Ser Glu Ser Gln
165 170 175
Leu Lys Glu Leu Gln Lys Glu Ile Thr Ser Val Ser Asp Glu Ile Tyr
180 185 190
Thr Val Arg Ser Gln Lys Ala Val Leu Asp Val Met Pro Lys Gly Ser
195 200 205
Asp Lys Gly Thr Ala Ile Glu Trp Phe Ser Lys Asn Tyr Leu Asn Asn
210 215 220
Glu Lys Ile Tyr Asp Tyr Ser Leu Met Phe Gly Asp Ala Ala Asn Asp
225 230 235 240
Ile Pro Ala Phe Glu Lys Val Ala Tyr Ser Cys Ala Leu Lys Ser Ser
245 250 255
Ser Leu Glu Ala Glu Asn Ala Ala Asn Tyr Val Val Ser Ser Ser Thr
260 265 270
Asp Asn Gly Ile Ile Glu Phe Phe Val Lys His Thr Asn
275 280 285
Claims (3)
1. 牛支原体(Mycoplasma bovis)分泌蛋白MbovP725在制备防治牛支原体炎症的药物中的用途,所述牛支原体分泌蛋白MbovP725的氨基酸序列如SEQ ID NO:2所示,所述牛支原体分泌蛋白MbovP725以剂量依赖性抑制脂多糖诱导牛巨噬细胞和牛乳腺上皮细胞IL-1β,IL-6,TNF-α的表达从而发挥抗炎作用,同时还能作为磷酸酶与底物进行酶促反应,从而抑制MAPK信号通路关键蛋白分子的磷酸化表达。
2.如权利要求1所述的用途,其特征在于:编码所述牛支原体分泌蛋白MbovP725的核苷酸序列如SEQ ID NO:1所示。
3.如权利要求1所述的用途,其特征在于:所述牛支原体分泌蛋白MbovP725为重组蛋白。
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CN111856005A (zh) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | 牛支原体分泌蛋白MbovP280的应用 |
CN111856006A (zh) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | 牛支原体分泌蛋白MbovP274的应用 |
CN111850002A (zh) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | 牛支原体分泌蛋白MbovP570的应用 |
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CN111856006A (zh) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | 牛支原体分泌蛋白MbovP274的应用 |
CN111850002A (zh) * | 2020-06-22 | 2020-10-30 | 华中农业大学 | 牛支原体分泌蛋白MbovP570的应用 |
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IN SILICO ANALYSIS OF DIFFERENTIAL PROTEINS CRITICAL TO VIRULENCE BETWEEN MYCOPLASMA BOVIS HB0801 AND ITS ATTENUATED STRAINS;MUHAMMAD ASIF RASHEED;中国博士学位论文全文数据库(农业科技辑);第D055-122卷(第第12期期);全文 * |
牛支原体强弱菌株分泌蛋白的分离和初步鉴定研究;胡古月;中国优秀硕士学位论文全文数据库 农业科技辑》(第2019年第01期期);全文 * |
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