CN103301477A - 大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的应用 - Google Patents
大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的应用 Download PDFInfo
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- CN103301477A CN103301477A CN2013101946105A CN201310194610A CN103301477A CN 103301477 A CN103301477 A CN 103301477A CN 2013101946105 A CN2013101946105 A CN 2013101946105A CN 201310194610 A CN201310194610 A CN 201310194610A CN 103301477 A CN103301477 A CN 103301477A
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- Prior art keywords
- escherichia coli
- sdhb
- sulfur protein
- succinate dehydrogenase
- iron
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Abstract
本发明属于医药技术领域,具体涉及大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的应用。本发明通过主动免疫和被动免疫试验发现大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB和其相应的抗体对大肠埃希菌临床致病菌的感染具有明显的免疫保护作用。因此,大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB和其相应抗体可用于制备防治动物大肠埃希菌临床致病菌感染所引起的疾病的药物。该药物不会产生耐药性,可有效防治大肠埃希菌临床致病菌感染所引起的疾病。
Description
技术领域
本发明涉及医药技术领域,更为具体地,涉及大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的应用。
背景技术
大肠埃希菌(Escherichia coli )通常称为大肠杆菌,分布在自然界,大多数是不致病的,主要附生在人或动物的肠道里,为正常菌群,少数的大肠杆菌具有毒性,可引起疾病,包括人类和家畜家禽的相关疾病。近年来,大肠杆菌病给养禽业带来的损失越来越大,其难于治愈、死亡率高、极易复发等临床特点,正困扰着基层广大养殖户和兽医工作者。例如,大肠杆菌可以引起的仔猪肠道传染性疾病,称为猪大肠杆菌病。常见的有仔猪黄痢、仔猪白痢和水肿病三种,以发生肠炎、肠毒血症为特征。其中仔猪黄痢是发生在1~7日龄仔猪的一种急性、高度致死性的肠道传染病。
大肠埃希菌所引起的疾病虽然可以采用抗生素进行控制,但该病原菌极易产生耐药性,导致抗生素作用无效。并且长期大量使用抗生素不但影响家畜家禽食品的品质,而且污染环境,影响人类身体健康。因此,控制此类疾病最佳的选择是进行疫苗防治。
研制疫苗需要良好的保护原,才可能有效地激发人体免疫***,产生特异性免疫应答,保护机体免除病原菌的感染。目前,有关大肠埃希菌的疫苗已经研制,但由于大肠埃希菌血清型较多,其效果有待于进一步提高。因此,发现新的高效保护原对疫苗的研制十分重要。
琥珀酸脱氢酶(Succinatedehydrogenase,简称SDH)位于线粒体基质,是线粒体呼吸链的复合物Ⅱ,由 A、B 、C、D 4 个亚单位构成。其中SDHB 编码的铁硫蛋白与 SDHA 编码的黄素蛋白形成酶接触中心。
发明内容
本发明所要解决的技术问题是克服现有技术的不足,提供一种大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因的应用。
本发明的另一个目的是提供一种大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的应用。
本发明的另一个目的是提供一种大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体的应用。
本发明的另一个目的是提供一种药物组合物。
本发明通过主动免疫和被动免疫试验发现大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB和大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体对大肠埃希菌临床致病菌的感染具有明显的免疫保护作用。因此,大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB和大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体可用于制备防治大肠埃希菌临床致病菌感染所引起的疾病的药物。该药物不会产生耐药性,可有效防治大肠埃希菌临床致病菌感染所引起的疾病。
本发明是通过以下技术方案予以实现的:
大肠埃希菌琥珀酸脱氢酶铁硫蛋白基因SdhB在制备防治由大肠埃希菌临床致病菌感染引起疾病的药物中的应用;所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白基因SdhB具有下列核苷酸序列之一;
S1.具有SEQ ID NO:1所示的核苷酸序列;
S2.与序列表中SEQ ID NO:1所限定的核苷酸序列具有95%的同源性,且编码相同生物学功能蛋白质的DNA序列。
大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB在制备防治由大肠埃希菌临床致病菌感染引起疾病的药物中的应用;所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB具有下列氨基酸序列之一;
S1.具有SEQ ID NO:2所示的氨基酸序列;
S2通过将SEQ ID NO:2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而产生的衍生蛋白质的氨基酸序列,所述衍生蛋白质与SEQ ID NO:2的蛋白具有相同的生物学功能。
大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体在制备防治由大肠埃希菌临床致病菌感染引起疾病的药物中的应用;所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的氨基酸序列如SEQ ID NO:2所示。
优选地,所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体包括单克隆抗体和多克隆抗体。
一种药物组合物,包含大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体。优选地,所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体包括单克隆抗体和多克隆抗体。
大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB和大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体,对各种种类的大肠埃希菌临床致病菌的感染,均具有明显的免疫保护作用,所述的大肠埃希菌临床致病菌为所有大肠埃希菌致病菌,包括肠产毒型大肠埃希菌、肠致病型大肠埃希菌、肠侵袭型大肠埃希菌、肠出血型大肠埃希菌和/或肠粘附型大肠埃希菌。大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB及其抗体的作用机理是因为虽然既往大家都认为琥珀酸脱氢酶铁硫蛋白SdhB仅定位于胞浆,但本发明创造性地发现其可以在细菌外膜表面上检出。
本发明具有如下有益效果:本发明所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB和大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体对多数种类的大肠埃希菌临床致病菌感染均具有明显的免疫保护作用,因此可用作疫苗组分或者抗细菌感染制剂,用于防治大肠埃希菌临床菌感染所导致的疾病。而且所述药物不会使致病菌产生耐药性,可有效防治大肠埃希菌临床致病菌感染所引起的疾病。
附图说明
图1.大肠埃希菌SdhB基因PCR扩增产物的琼脂糖凝胶电泳结果;其中M为DNA分子量标准。
图2.含有大肠埃希菌SdhB基因的pET-32a重组质粒双酶切鉴定结果;其中M为蛋白质分子量标准。
图3.大肠埃希菌SdhB蛋白表达的聚丙烯酰胺凝胶电泳检测结果。
图4.纯化得到的SdhB蛋白的聚丙烯酰胺凝胶电泳检测结果;其中M为蛋白质分子量标准。
图5.主动免疫试验中观察14天各组小鼠的生存情况。
图6.被动免疫试验中观察14天各组小鼠的生存情况。
图7.临床致病菌株外膜表面上存在SdhB的情况。
具体实施方式
下面结合附图和实施例进一步阐述本发明。应理解,这些实施例仅用于进一步说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等分子克隆实验室手册(2001 by Cold Spring Harbor Laboratory Press)中所述的条件,或按照制造厂商所建议的条件。
实施例1:大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的制备和纯化
S1.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因的扩增。
S11.引物设计:根据NCBI公布的大肠埃希菌的全基因组序列,设计一对引物:正向引物F的核苷酸序列如SEQ ID NO 3所示5'-CCGGAATTCATGAGACTCGAGTTTTCA-3';反向引物R的核苷酸序列如SEQ ID NO 4所示:5'-CCGAAGCTTTTACGCATTACGTTGCAACA-3'。为便于克隆和表达载体的构建,在引物上分别引入限制性内切酶位点EcoR I和HindⅢ。引物由英潍捷基(上海)贸易有限公司合成。
S12.基因扩增:提取大肠埃希菌的全基因组DNA,全基因组DNA的提取采用北京普博欣生物科技有限责任公司的细菌基因组DNA小量提取试剂盒;以获得的大肠埃希菌的全基因组DNA为模板,进行PCR 反应,反应体系如下:PCR反应缓冲液2.5mL,10mmol/L dNTP共1L,10mol/L正向引物F和反向引物R各1L,模板DNA 1L,用超纯水将反应体系补充至25L。PCR反应循环参数如下:第一步:94℃ 2min;第二步:94℃变性60s,55℃退火60s,72℃延伸60s,30个循环。随后继续在72℃延伸10 min,并在4℃下保存。用琼脂糖凝胶电泳鉴定DNA片段的长度(见图1)并回收DNA片段。
S13.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因的克隆、筛选和鉴定。
用限制性内切酶EcoR I和HindⅢ分别酶切步骤S12得到的DNA片段和pET-32a(Novagen)表达载体,参照Takara公司的DNA连接试剂盒使用说明书进行连接反应,反应体系如下:连接酶溶液5L,经酶切的DNA片段1L,经酶切的pET-32a表达载体0.5L,用超纯水将反应体系补充至10L,于16℃左右保温14~16小时。将重组载体转化大肠埃希菌DH5α感受态,转化后将菌体涂于含氨苄青霉素(50 ug/mL)的LB固体平板上,于37℃培养12~16小时。随机挑取含平板上的重组子,接种于含氨苄青霉素(50 ug/mL)的LB液体培养基上,用碱裂解法分别提取质粒进行双酶切鉴定(见图2)。将有***的重组子送至深圳华大基因科技有限公司进行序列测定。测序结果显示:大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因编码序列如SEQ ID NO:1所示,大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的氨基酸序列如SEQ ID NO:2所示。将得到的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因序列与NCBI中报道的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因序列比对,结果表明与NCBI中报道的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB有100%的同源性,说明得到了正确的重组SdhB基因。
S14.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因的表达
将步骤S13得到的含SdhB基因的重组载体以热激法转化大肠埃希菌BL21表达菌,于37℃培养12~16小时。将单菌落接种于含氨苄青霉素(50 ug/mL)的LB液体培养基中,同时接种含有空pET-32a质粒的大肠埃希菌BL21作为对照,于37℃培养至OD值为0.6,加入0.5mmol/L IPTG,于35℃诱导3小时,离心收集菌体。通过SDS-PAGE检测重组SdhB基因是否表达蛋白以及表达蛋白的分子量大小,结果如图3所示。由图可知,重组SdhB基因表达蛋白的分子量与预计一致。
S15.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的纯化。
大量培育转化菌体,离心后进行超声处理,离心收集上清液用Ni-NTA His Bind亲和柱进行纯化。用冲洗缓冲液Buffer E(8M尿素、0.1M NaH2PO4、10mm Tris-HCl,用HCl调节pH值至6.3)洗涤5次,每次用量为1mL;用洗脱缓冲液Buffer F(8M尿素、0.1MNaH2PO4、10mm Tris-HCl,用HCl调节pH值至5.9)洗脱目的蛋白4次,每次用量为1mL,收集洗脱液A;用洗脱缓冲液Buffer G (8M尿素、0.1M NaH2PO4、10mm Tris-HCl,用HCl调节pH值至4.5)洗脱未结合Ni-NTA His Bind亲和柱的蛋白4次,每次用量为1mL,收集洗脱液B。取收集的洗脱液A和洗脱液B进行SDS-PAGE分析,纯化结果如图4所示。将纯化好的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB分装,冷冻干燥成粉末后,冻存于-80℃冰箱中。
实施例2:大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清的制备和纯化。
S1.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清的制备:
将实施例1纯化的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB免疫昆明鼠,获取SdhB抗血清。具体步骤如下:将大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB冻干粉溶于适量的超纯水中,然后与等体积的弗氏完全佐剂(羊毛脂:石蜡油=1:4 v/v,卡介苗为30 mg/mL)混合均匀,其中蛋白含量约为100 ug,然后超声处理2 min,即制得SdhB抗原,采用腹腔注射;14天后,将大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB与等体积的弗氏不完全佐剂(羊毛脂:石蜡油=1:4 v/v)混合,给昆明鼠进行注射,其中蛋白含量约为50 ug;14天后再注射一次。10天后眼部取血,将获得的血在4℃冰箱中静置过夜,使血清自然析出;4℃ 3000 r/min,离心30分钟,取上清液即得SdhB抗血清。用Wethern blotting方法进行了效价测定,为1:16000。
S2.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清的纯化
饱和硫酸铵法初步纯化抗血清:采集的抗血清加入等体积的生理盐水混匀,然后逐滴加入饱和硫酸铵二份,边加边搅,防止出现沉淀,硫酸铵饱和度达50%,混匀后静置4℃冰箱过夜。离心(12000 g,10min),将上清弃去,取沉淀物溶于少量生理盐水中。将上述生理盐水溶解液用33%饱和硫酸铵沉淀,离心(12000 g,10min),收集沉淀,溶解于pH=7.4的PBS。将提取物装入透析袋,在生理盐水中透析,除去硫酸铵。取出离心,吸出上清即得到粗提的抗血清。
CNBr活化的琼脂糖4B柱亲和纯化抗血清:将约4mg纯化的SdhB蛋白偶连到经CNBr活化的琼脂糖4B柱上,做成纯化抗SdhB抗体的亲和层析柱。再将约5mg粗提的抗血清与10mL PBS混合后加到柱上,4℃放置过夜,用PBS溶液、2mol/L NaCI/PBS溶液和PBS(pH4.5)溶液依次洗柱各约10个柱床体积后,用0.1mol/L的甘氨酸 (pH2.4) 进行洗脱并收集蛋白,每管加适量的1mol/L Tris-HCI(pH8.8)进行中和。
S3. 大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清的定量:用Brandford方法对纯化后抗体含量进行定量(具体方法可参照)。
实施例3:大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清的被动免疫保护作用。
采用约3周左右的昆明鼠作为试验对象,肌肉注射40 微克实施例2制备得到的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清,对照组注射等量未免疫家兔的血清,1小时后用从临床病人体内分离的大肠埃希菌临床耐药致病菌株Y17(1.5×108cfu/只)进行感染。观察72小时后各组昆明鼠的生存情况,并计算免疫昆明鼠的相对免疫保护率(表2)。结果表明,用大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清被动免疫的昆明鼠,其死亡率明显下降,相对保护率为93.75%。
表2 大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB抗血清对昆明鼠的保护效果
实施例4:大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB对病原菌的主动免疫保护作用。
将实施例1所制得的纯化的大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB冻干粉溶于适量的超纯水中,然后与等体积的弗氏完全佐剂(羊毛脂:石蜡油=1:4 v/v,卡介苗为30mg/mL)混合均匀,其中蛋白含量约为100 ug,然后超声处理2分钟,即制得SdhB抗原,肌肉注射实验昆明鼠;10天后,将大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB与等体积的弗氏不完全佐剂(羊毛脂:石蜡油=1:4 v/v)混合,其中蛋白含量约为100 ug,肌肉注射实验昆明鼠。对照组昆明鼠对应肌肉注射同等体积生理盐水。第2次免疫后约7天,对小鼠进行攻毒保护性实验。攻毒菌采用分别从临床病人体内分离的大肠埃希菌临床耐药致病菌株Y17和从猪场病猪体内获得的大肠埃希菌临床耐药致病菌株HY。
临床病人耐药菌的攻毒试验
采用临床病人耐药菌Y17,攻毒剂量为1.5×108cfu/只。然后观察14天各组小鼠的生存情况,并计算免疫小鼠的相对免疫保护率。图5为14天内每天小鼠的生存情况,从图可以看出,对照组即没有免疫组的19只小鼠在病原菌攻毒后,其死亡主要发生在第1天,高达15只,第2天死亡1只,从第3天直至第14天,没有小鼠死亡。而免疫SdhB组小鼠从第1天直至第14天,无小鼠死亡。表3为用临床病人耐药菌攻毒后琥珀酸脱氢酶铁硫蛋白SdhB对昆明鼠的保护效果,其相对保护率为100%。
表3大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB主动免疫对Y17感染的保护效果
猪源致病菌的攻毒试验
采用猪源致病菌HY,攻毒剂量为1.25×109cfu/只。然后观察14天各组小鼠的生存情况,并计算免疫小鼠的相对免疫保护率。图6为14天内每天小鼠的生存情况,从图可以看出,对照组即没有免疫组的20只小鼠在病原菌攻毒后,其死亡主要发生在2天内,高达18只,从第3天直至第14天,没有小鼠死亡。而免疫SdhB组小鼠仅在第1天死亡3只。表4为用猪源致病菌的攻毒后琥珀酸脱氢酶铁硫蛋白SdhB对昆明鼠的保护效果,其相对保护率为83.33%。
表4大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB主动免疫对HY感染的保护效果
综上所述,大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB对于临床致病菌具有明显的免疫保护作用,可用作疫苗组分或者抗细菌感染制剂,用于防治大肠埃希菌临床菌感染所导致的疾病。
实施例5:临床致病菌株检出外膜表面上存在SdhB的普遍性
采用临床分离的大肠杆菌致病菌株进行SdhB抗血清的ELISA实验,如果结果是阳性,说明在细菌的外膜表面上存在SdhB。具体过程如下:
挑取27株分离的大肠杆菌致病菌(Y1-Y20,EPEL, E3-H, E4-H, O157, 100108d,HY2011,G×2011)单克隆分别于5mLLB试管中,37℃ 200rpm过夜培养。按1:100转接入100mL新鲜LB培养基中,于37℃ 200rpm培养至OD600=1.0时离心收菌,用生理盐水洗涤2遍后,加入1%的甲醛生理盐水20mL,80℃灭活90min。用生理盐水洗涤甲醛残留后,再用生理盐水调OD600=0.2,每管1mL分装用于后续的实验。
将每种细菌离心去上清后,加入200μL SdhB(5%牛奶PBS稀释,1:500)抗血清,室温摇动孵育。同时以生理盐水和BSA为对照。作用90min后离心去除血清,再用PBS洗涤2遍。加入200μL二抗(5%牛奶PBS稀释,1:3000)室温震动培养1h,离心去除上清,用PBS洗涤3遍。
加入20μLPBS将沉淀混匀后转移至酶标板中,再加入100μL显色液(50μL双氧水+50μLTMD),37℃避光显色10min,加入终止液(2M H2SO4),于450nm读数,图7为测定结果。从图可以看出在这27株多重耐药菌的表面都检测到了SdhB蛋白。
SEQUENCE LISTING
<110> 中山大学
<120> 大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的应用
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<170> PatentIn version 3.3
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<213> 大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB基因
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atgagactcg agttttcaat ttatcgctat aacccggatg ttgatgatgc tccgcgtatg 60
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cagctaaaag agaaagatcc cagcctgtcg ttccgccgct cctgccgtga aggtgtgtgc 180
ggttccgacg gtctgaacat gaacggcaag aatggtctgg cctgtattac cccgatttcg 240
gcactcaacc agccgggcaa gaagattgtg attcgcccgc tgccaggttt accggtgatc 300
cgcgatttgg tggtagacat gggacaattc tatgcgcaat atgagaaaat taagccttac 360
ctgttgaata atggacaaaa tccgccagct cgcgagcatt tacagatgcc agagcagcgc 420
gaaaaactcg acgggctgta tgaatgtatt ctctgcgcat gttgttcaac ctcttgtccg 480
tctttctggt ggaatcccga taagtttatc ggcccggcag gcttgttagc ggcatatcgt 540
ttcctgattg atagccgtga taccgagact gacagccgcc tcgacggttt gagtgatgca 600
ttcagcgtat tccgctgtca cagcatcatg aactgcgtca gtgtatgtcc gaaggggctg 660
aacccgacgc gcgccatcgg ccatatcaag tcgatgttgt tgcaacgtaa tgcgtaa 717
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<212> PRT
<213> 大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB
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Met Arg Leu Glu Phe Ser Ile Tyr Arg Tyr Asn Pro Asp Val Asp Asp
1 5 10 15
Ala Pro Arg Met Gln Asp Tyr Thr Leu Glu Ala Asp Glu Gly Arg Asp
20 25 30
Met Met Leu Leu Asp Ala Leu Ile Gln Leu Lys Glu Lys Asp Pro Ser
35 40 45
Leu Ser Phe Arg Arg Ser Cys Arg Glu Gly Val Cys Gly Ser Asp Gly
50 55 60
Leu Asn Met Asn Gly Lys Asn Gly Leu Ala Cys Ile Thr Pro Ile Ser
65 70 75 80
Ala Leu Asn Gln Pro Gly Lys Lys Ile Val Ile Arg Pro Leu Pro Gly
85 90 95
Leu Pro Val Ile Arg Asp Leu Val Val Asp Met Gly Gln Phe Tyr Ala
100 105 110
Gln Tyr Glu Lys Ile Lys Pro Tyr Leu Leu Asn Asn Gly Gln Asn Pro
115 120 125
Pro Ala Arg Glu His Leu Gln Met Pro Glu Gln Arg Glu Lys Leu Asp
130 135 140
Gly Leu Tyr Glu Cys Ile Leu Cys Ala Cys Cys Ser Thr Ser Cys Pro
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Ala Ala Tyr Arg Phe Leu Ile Asp Ser Arg Asp Thr Glu Thr Asp Ser
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Arg Leu Asp Gly Leu Ser Asp Ala Phe Ser Val Phe Arg Cys His Ser
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ccggaattca tgagactcga gttttca 27
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ccgaagcttt tacgcattac gttgcaaca 29
Claims (6)
1.大肠埃希菌琥珀酸脱氢酶铁硫蛋白基因SdhB在制备防治由大肠埃希菌临床致病菌感染引起疾病的药物中的应用;所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白基因SdhB具有下列核苷酸序列之一;
S1.具有SEQ ID NO:1所示的核苷酸序列;
S2.与序列表中SEQ ID NO:1所限定的核苷酸序列具有95%的同源性,且编码相同生物学功能蛋白质的DNA序列。
2.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB在制备防治由大肠埃希菌临床致病菌感染引起疾病的药物中的应用;所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB具有下列氨基酸序列之一;
S1.具有SEQ ID NO:2所示的氨基酸序列;
S2通过将SEQ ID NO:2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而产生的衍生蛋白质的氨基酸序列,所述衍生蛋白质与SEQ ID NO:2的蛋白具有相同的生物学功能。
3.大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体在制备防治由大肠埃希菌临床致病菌感染引起疾病的药物中的应用;所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的氨基酸序列如SEQ ID NO:2所示。
4.根据权利要求3所述应用,其特征在于,所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体包括单克隆抗体和多克隆抗体。
5.一种药物组合物,其特征在于,包含大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体。
6.根据权利要求5所述应用,其特征在于,所述大肠埃希菌琥珀酸脱氢酶铁硫蛋白SdhB的抗体包括单克隆抗体和多克隆抗体。
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---|---|---|---|---|
CN103690938A (zh) * | 2013-12-18 | 2014-04-02 | 中山大学 | ATP合成酶β亚基在制备免疫保护剂、疫苗或抗细菌感染剂上的应用 |
CN113604493A (zh) * | 2021-06-13 | 2021-11-05 | 兰州大学 | Ldh、表达ldh的重组质粒、表达ldh的重组益生菌及应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1962868A (zh) * | 2005-11-08 | 2007-05-16 | 余俊龙 | 日本血吸虫sdisp基因及其克隆、表达和应用 |
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---|---|---|---|---|
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Non-Patent Citations (2)
Title |
---|
PETER OWEN 等: ""Identification and Partial Characterization of a Novel Bipartite Protein Antigen Associated with the Outer Membrane of Escherichia coli"", 《JOURNAL OF BACTERIOLOGY》 * |
余俊龙 等: ""日本血吸虫琥珀酸脱氢酶铁硫蛋白全长cDNA的克隆、表达及保护性免疫评价"", 《中国科学》 * |
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CN103690938A (zh) * | 2013-12-18 | 2014-04-02 | 中山大学 | ATP合成酶β亚基在制备免疫保护剂、疫苗或抗细菌感染剂上的应用 |
CN103690938B (zh) * | 2013-12-18 | 2016-01-20 | 中山大学 | ATP合成酶β亚基在制备免疫保护剂、疫苗或抗细菌感染剂上的应用 |
CN113604493A (zh) * | 2021-06-13 | 2021-11-05 | 兰州大学 | Ldh、表达ldh的重组质粒、表达ldh的重组益生菌及应用 |
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