CN110713524B - 一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒 - Google Patents
一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒 Download PDFInfo
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Abstract
本发明涉及一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒及其制备方法。该试剂盒包含有:包被抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体的酶标板、抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体、鲍曼不动杆菌阳性对照、阴性对照、洗涤液、酶标二抗、酶显色底物和终止液。本发明的试剂盒对鲍曼不动杆菌四种特异性表面蛋白进行联合检测,大大提高了鲍曼不动杆菌检测的灵敏度、特异性和可重复性,可用于对鲍曼不动杆菌非诊断性检测及研究工作。
Description
技术领域
本发明属于生物技术及传染病诊断研究领域,涉及一种Elisa检测试剂盒,尤其涉及一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒及制备方法。
背景技术
鲍曼不动杆菌(Acinetobacter baumannii,Ab)为非发酵革兰阴性杆菌,广泛存在于自然界,属于条件致病菌。该菌是医院感染的重要病原菌,主要引起呼吸道感染,也可引发菌血症、***、继发性脑膜炎、手术部位感染、呼吸机相关性肺炎等。对常用抗生素的耐药率有逐年增加的趋势,并引起临床医生和微生物学者的严重关注。国内资料表明,A.baumannii约占临床分离的不动杆菌的70%以上。A.baumannii对第三代和***头孢菌素的耐药率已达63.0%~89.9%,对四种氨基糖苷类(阿米卡星、庆大酶素、奈替米星、妥布霉素)和环丙沙星的耐药率菌达96.3%。我国目前的绝大多数菌株对亚胺培南、美罗培南、头孢派酮/舒巴坦和多黏菌素B保持敏感,但在呼吸道感染的治疗中效果较差。综上所述,鉴于近年鲍曼不动杆菌的耐药率有进一步上升的趋势,这应当引起临床医师及微生物界的高度重视。临床医师应重视获得性鲍曼不动杆菌感染,与临床微生物实验室密切协作,加强感染的监测,有效预防和控制感染。
目前检测呼吸道中该病原体的方法主要以传统方法为主,即分离鉴定法,该方法所需时间长,一般要2-3天,很难满足快速鉴定的需要;近几年发展起来的PCR技术,是一种快速、灵敏、特异性好的技术,但是目前该技术还是依赖于传统方法的前增菌步骤,而增菌液中往往还有PCR抑制剂,从而影响扩增的效果。同时,该技术还需要专业的检测设备,不适合进行床旁检测。此外,由于此项技术过于灵敏,经常会造成假阳性。以抗体为基础的免疫学检测已成为人体病原微生物检测不可或缺的重要技术手段。目前已发展了多种特异性免疫检测技术,如放射免疫分析(RIA)、酶免疫分析(EIA)、荧光免疫分析(FIA)、化学发光免疫分析(CIA)、免疫沉淀反应、免疫凝集反应、ELISA检测试剂盒、免疫胶体金试纸条、免疫乳胶检测试剂等。其中ELISA等多种以抗体为基础的免疫学检测技术,以其简便、快速、灵敏、准确、实用的特点已成为病原微生物检测不可或缺的重要手段。因而,研究开发具有自主知识产权的病原微生物的抗体,是开发拥有自主知识产权的ELISA检测方法的基础。
抗原成分的选择是制备特异性抗体的关键。鲍曼不动杆菌fhuE receptor、OmpA、PilF及PonA蛋白均是位于细胞表面的重要分子,本研究选用具有种间特异性的表面蛋白FhuE receptor、OmpA、PilF及PonA等作为备选抗原,同时通过基因优化,融合表达等技术手段制备特异性良好的多克隆抗体,并将其应用于鲍曼不动杆菌EIisa检测试剂盒的制备。
发明内容
为了解决背景技术中存在的上述技术问题,本发明提供了一种可提高鲍曼不动杆菌检测的灵敏度、特异性及可重复性强、操作简单、成本低廉以及快捷迅速地对鲍曼不动杆菌进行检测的一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒及制备方法。
为了实现上述目的,本发明采用如下技术方案:
一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒,其特征在于:所述一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒包括包被抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)的多克隆抗体的酶标板、抗鲍曼不动杆菌表面蛋白(OmpA及ponA)多克隆抗体、鲍曼不动杆菌阳性对照、阴性对照、洗涤液、酶标二抗、酶显色底物以及终止液;所述鲍曼不动杆菌阳性对照是灭活的鲍曼不动杆菌菌液;所述阴性对照是灭活的大肠杆菌菌液;所述洗涤液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2g/L,氯化钠8.5g/L,Tween-200.5mL/L,所述洗涤液的pH=7.4;所述酶标二抗是辣根过氧化物酶标记的羊抗兔IgG;所述酶显色底物是TMB显色液;所述终止液是1M HCL溶液。
一种一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒的制备方法,其特征在于:所述一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒包括包被抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)的多克隆抗体的酶标板、抗鲍曼不动杆菌表面蛋白(OmpA及ponA)多克隆抗体、鲍曼不动杆菌阳性对照、阴性对照、洗涤液、酶标二抗、酶显色底物以及终止液;所述鲍曼不动杆菌阳性对照是灭活的鲍曼不动杆菌菌液;所述阴性对照是灭活的大肠杆菌菌液;所述洗涤液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2g/L,氯化钠8.5g/L,Tween-200.5mL/L,所述洗涤液的pH=7.4;所述酶标二抗是辣根过氧化物酶标记的羊抗兔IgG;所述酶显色底物是TMB显色液;所述终止液是1M HCL溶液;
所述包被抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)的多克隆抗体的酶标板的制备方法是:
1)抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)多克隆抗体的制备:
1.1)分别获取鲍曼不动杆菌表面蛋白Fhue及表面蛋白Pilf胞外结构域中抗原表位最为丰富的肽段并找到该肽段基因编码序列,优化肽段基因编码序列,并将优化后的肽段基因编码序列用柔性连接肽的编码序列进行连接,形成融合基因;所述鲍曼不动杆菌表面蛋白Fhue及表面蛋白Pilf在NCBI蛋白质数据库中的accession number分别为KMV27515及AJF80497;所述柔性连接肽的序列是ggsggsggsggs;同时在该融合基因的5’引入酶切位点NdeI、3’端引入终止信号TAA和酶切位点BamHI后化学合成全基因序列,记为FhuPil;
所述FhuPil的基因全序列是:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCTTGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCGAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGTAAGGATCC;
所述FhuPil基因编码的蛋白质序列是:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQ;
所述FhuPil基因编码的蛋白质序列为鲍曼不动杆菌表面蛋白Fhue的509-688aa及表面蛋白Pilf的28-184aa;两段蛋白序列中间以柔性连接肽ggsggsggsggs进行连接;
1.2)将FhuPil的基因全序列按常规方法克隆入原核表达载体pET-28a(+),转入E.coli BL21(DE3)菌中,用IPTG诱导重组大肠杆菌表达,用Ni2+亲和层析法纯化重组His-FhuPil蛋白;将该重组蛋白作为免疫抗原,与弗氏佐剂混合后反复人工免疫健康的新西兰大白兔,抽血进行效价测定,分离高效价重组蛋白抗体并进行纯化,最终获得抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)多克隆抗体;
2)抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)多克隆抗体的包被:
用PBS缓冲液将步骤1)制备得到的抗鲍曼不动杆菌表面蛋白(Fhue及Pilf)多克隆抗体稀释至浓度为10μg/mL,按100μL/孔的量包被96孔EIA高效结合酶标板,37℃2小时;取出后用250μL洗涤液洗板三次,甩干;用含有1%BSA的洗涤液作为封闭液,按250μL/孔的量加入酶标板,37℃封闭1小时;取出后用250μL洗涤液洗板3次,每次一分钟,甩干,干燥后密封保存;
其中,所述PBS缓冲液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2g/L,氯化钠8.5g/L;所述PBS缓冲液的pH是7.4;
所述洗涤液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2g/L,氯化钠8.5g/L,Tween-200.5mL/L,所述洗涤液的pH是7.4;
所述封闭液是含1%BSA的洗涤液的水溶液,所述封闭液的pH是7.4。
上述抗鲍曼不动杆菌表面蛋白(OmpA及ponA)多克隆抗体的制备方法是:
1)分别获取鲍曼不动杆菌表面蛋白OmpA及表面蛋白ponA胞外结构域中抗原表位最为丰富的肽段并找到该肽段基因编码序列,优化肽段基因编码序列,并将优化后的肽段基因编码序列用刚性连接肽的编码序列进行连接,形成融合基因;所述鲍曼不动杆菌表面蛋白OmpA及表面蛋白ponA在NCBI蛋白质数据库中的accession number分别为AJF83030及ADX05080;所述刚性连接肽的序列是eaaakeaaak;同时在该融合基因的5’引入酶切位点NdeI、3’端引入终止信号TAA和酶切位点BamHI后化学合成全基因序列,记为OmpPon;
所述OmpPon的基因全序列是:
CATATGCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC;
所述OmpPon基因编码的蛋白质序列是:
MLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYNEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY;
所述OmpPon基因编码的蛋白质序列为鲍曼不动杆菌表面蛋白OmpA的31-173aa及表面蛋白ponA的406-572aa;两段蛋白序列中间以刚性连接肽eaaakeaaak进行连接;
2)将OmpPon的基因全序列按常规方法克隆入原核表达载体pET-28a(+),转入E.coli BL21(DE3)菌中,用IPTG诱导重组大肠杆菌表达,用Ni2+亲和层析法纯化重组His-OmpPon蛋白;将该重组蛋白作为免疫抗原,与弗氏佐剂混合后反复人工免疫健康的新西兰大白兔,抽血进行效价测定,分离高效价重组蛋白抗体并进行纯化,最终获得抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体,用封闭液稀释至终浓度为20μg/mL;
所述封闭液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2g/L,氯化钠8.5g/L,牛血清白蛋白10g/L,所述封闭液的pH是7.4。
本发明相对于现有技术具有如下优点:
(1)本发明采用结构分析,基因优化等方式,构建了两个全新的融合基因,通过可溶性过量表达,首次成功获得了可溶性的重组Fhue/Pilf融合蛋白及OmpA/ponA融合蛋白。该两种融合蛋白表达量高,制备成本低廉,蛋白可溶性好,抗原性强,用其制备抗体效价高,成本低。
(2)本发明首次利用鲍曼不动杆菌表面上的四个蛋白暴露区制备的抗体效价高,抗原结合位点多,捕获力强,不存在位点竞争问题,试剂盒检测灵敏度高。其对鲍曼不动杆菌标准菌株ATCC19606的检测灵敏度达到1×103CFU/mL,明显高于微生物传统检测方法,并且具有快速、高效等优点。
(3)该Elisa试剂盒特异性好,用6株鲍曼不动杆菌菌株和17株非鲍曼不动杆菌标准菌株(包含绝大部分呼吸道常见病原体)进行的特异性实验,结果显示本发明的试纸条具有良好的特异性和稳定性,其可检出所有所试的鲍曼不动杆菌,而与所有非鲍曼不动杆菌标准菌株均无交叉反应,十分适合临床非诊断性应用。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法。
实施例1
抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体的制备:
1.1)鲍曼不动杆菌FhuPil融合基因的克隆
获取鲍曼不动杆菌表面蛋白Fhue及Pilf(其NCBI蛋白质数据库中的accessionnumber分别为KMV27515及AJF80497)胞外结构域中抗原表位最为丰富的肽段并找到其基因编码序列,优化其基因编码序列,并将此二序列用柔性连接肽(ggsggsggsggs)的编码序列进行连接,形成融合基因。同时在该融合基因的5’引入酶切位点NdeI、3’端引入终止信号TAA和酶切位点BamHI后化学合成全基因序列,记为FhuPil。其基因全序列及编码的氨基酸序列如序列表所示。具体的说,FhuPil基因编码的蛋白质序列为鲍曼不动杆菌表面蛋白Fhue的509-688aa及表面蛋白Pilf的28-184aa,两个蛋白序列中间以柔性连接肽(ggsggsggsggs)进行连接。将此基因序列交由南京金斯瑞生物科技有限公司进行全基因化学合成,交货时该人工合成的基因片段连于载体pUC57上。将含有该段人工合成的DNA片段的载体pUC57用NdeI及BamHI进行双酶切后按常规方法回收目的片段,备用。同时采用NdeI及BamHI对载体pET-28a(+)进行双酶切,并按常规分子生物学方法将经双酶切后获得的FhuPil基因连入pET-28a(+)载体中,并转化大肠杆菌TOP10,构建pET-FhuPil表达载体。经酶切和序列测定证实表达载体构建无误。该载体表达重组FhuPil融合蛋白。
1.2)鲍曼不动杆菌FhuPil融合蛋白的表达与纯化
将上述鉴定正确的阳性克隆菌培养后提取质粒,按常规技术转入感受态E.coliBL21(DE3)中,转化完成后将菌液涂布于含50μg/mL卡那霉素的LB平板上,按常规方法筛选表达菌株。挑取pET-FhuPil转化的具有外源蛋白表达能力的单个菌落并接种入100mL LB培养基中,于37℃培养过夜。取出菌液后,按1:100接种于100mL含有50μg/mL卡那霉素的LB培养基中,于30℃培养至OD600=0.6时,加入1mol/L IPTG至终浓度为0.5mmol/L,于18℃摇菌培养,诱导融合蛋白表达。诱导12h后于8000r/min下离心10min收集菌体。将此菌体用50mLBufferA(50mM Na3PO4,0.5M NaCl;pH7.4)洗涤3次并用50mL上样缓冲液(50mM Na3PO4,0.5M NaCl;5mM咪唑,pH7.4)重悬后进行超声破碎,操作条件为:功率50W,工作时间2s,间隔时间3s,报警温度60℃,总时间30min。超声完成后,12000g离心15min分别收集沉淀和上清后进行电泳检测。发现重组FhuPil融合蛋白以溶解形式存在于菌体中。薄层扫描显示,该重组蛋白占细菌总蛋白的30%以上。而将未经序列优化的野生型FhuPil基因按上述同样方式进行表达,表达产物仅占菌体总蛋白的4%左右,表达量微弱,说明基因优化效果良好。将上述获得的超声破碎上清液用0.45μm的滤膜进行过滤后用His Trap affinity columns(GEhealthcare公司产品),按照说明书的方法进行重组FhuPil蛋白的纯化。具体方法如下:
1.2.1)连接层析***,该***包括进样管、蠕动泵(上海沪西分析仪器厂,型号DHL-A)、层析柱(GE healthcare公司产品,商品名His Trap affinity columns)及紫外检测器(上海沪西分析仪器厂,型号HD1),柱体积为2ml,预热紫外检测仪30min左右至读数稳定;
1.2.2)校对T%:调节光亮旋钮至显示100%;
1.2.3)旋转灵敏度至合适位置,一般用0.2A;
1.2.4)以上样缓冲液平衡层析***,至读数稳定后旋转“调零”至显示“000”;
1.2.5)上蛋白样品,流速控制在5ml/min以内,收集穿透液;
1.2.6)用上样缓冲液洗去未结合的蛋白,此过程中记录读数,直到读数不再变化为止,收集洗脱液;
1.2.7)用BufferA+10mM咪唑进行洗脱,收集洗脱峰;
1.2.8)用BufferA+20mM咪唑进行洗脱,收集洗脱峰;
1.2.9)用BufferA+40mM咪唑洗脱,收集洗脱峰;
1.2.10)用BufferA+100mM咪唑洗脱,收集洗脱峰;
1.2.11)用BufferA+150mM咪唑洗脱,收集洗脱峰;
1.2.12)取各洗脱峰样品100ul进行SDS-PAGE电泳;
1.2.13)结果发现,目标蛋白在40mM咪唑时被洗脱下来,纯度在90%以上,经bradford试剂盒进行蛋白质浓度测定后,调整浓度为0.2mg/mL,备用。至此制得鲍曼不动杆菌FhuPil融合蛋白。
1.3)抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体的制备
1.3.1)将步骤(1.2)所制鲍曼不动杆菌FhuPil融合蛋白与福氏完全佐剂混合,乳化后作为免疫原免疫雄性新西兰兔2只,每只兔子皮下注射总量为2ml,抗原总量为2mg/只。后每隔两周用FhuPil融合蛋白与福氏不完全佐剂形成的乳化液免疫一次,共免疫5次,抗原用量与初次免疫相同。五免后3-5天进行心脏大量取血,置37℃1小时,然后放冰箱4℃过夜,隔天取血清。
1.3.2)多克隆抗体效价的测定
以FhuPil融合蛋白为包被抗原,包被浓度为5μg/ml,每孔包被100μl,利用间接ELISA法检测血清抗体水平。实验组血清稀释倍数依次为:1:200、1:400、1:800、1:1600、1:3200、1:6400、1:12800、1:25600、1:51200、1:102400、1:204800;
ELISA板包被牛血清白蛋白作为阴性对照,用酶联检测仪测定OD450,满足P/N值>2.1为阳性。结果显示2只兔子的血清抗体效价均达到1:102400,说明免疫效果较好。
1.3.3)多克隆抗体的提取
用GE-HiTrap Protein A HP预装柱,按说明书纯化抗体,具体操作如下:
1.3.3.1)取5mL抗血清,加入0.5mL 1M Tris(pH8.0)调节到pH8.0,20,000g离心20min除去沉淀。
1.3.3.2)上柱后用10倍柱体积bufferA(100mM Tris-Cl,pH 8.0)洗涤后,再用10倍柱体积buffer B(10mM Tris-Cl,pH 8.0)洗涤。
1.3.3.3)用大约三倍柱体积的IgG洗脱缓冲液(100mM glycine,pH 3.0)洗脱IgG。(收集管内先预装0.1mL IgG-中和缓冲液(1M Tris-Cl,pH 8.0),每管装0.9mL洗脱液)
1.3.3.4)用50倍体积Tris(10mM Tris-Cl,pH 8.0)对洗脱液进行透析。
1.3.3.5)超滤浓缩后,调整浓度为5mg/ml,-70℃保存备用。至此制得抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体。
实施例2
抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体的制备:
1.1)鲍曼不动杆菌OmpPon融合基因的克隆
获取鲍曼不动杆菌表面蛋白OmpA及ponA(其NCBI蛋白质数据库中的accessionnumber分别为AJF83030及ADX05080)其胞外结构域中抗原表位最为丰富的肽段并找到其基因编码序列,优化其基因编码序列,并将此二序列用刚性连接肽(eaaakeaaak)的编码序列进行连接,形成融合基因。同时在该融合基因的5’引入酶切位点NdeI、3’端引入终止信号TAA和酶切位点BamHI后化学合成全基因序列,记为OmpPon。其基因全序列及编码的氨基酸序列如序列表所示。具体的说,OmpPon基因编码的蛋白质序列为鲍曼不动杆菌表面蛋白OmpA的31-173aa及表面蛋白ponA的406-572aa,两个蛋白序列中间以刚性连接肽(eaaakeaaak)进行连接。将此基因序列交由南京金斯瑞生物科技有限公司进行全基因化学合成,交货时该人工合成的基因片段连于载体pUC57上。将含有该段人工合成的DNA片段的载体pUC57用NdeI及BamHI进行双酶切后按常规方法回收目的片段,备用。同时采用NdeI及BamHI对载体pET-28a(+)进行双酶切,并按常规分子生物学方法将经双酶切后获得的OmpPon基因连入pET-28a(+)载体中,并转化大肠杆菌TOP10,构建pET-OmpPon表达载体。经酶切和序列测定证实表达载体构建无误。该载体表达重组OmpPon融合蛋白。
1.2)鲍曼不动杆菌OmpPon融合蛋白的表达与纯化
将上述鉴定正确的阳性克隆菌培养后提取质粒,按常规技术转入感受态E.coliBL21(DE3)中,转化完成后将菌液涂布于含50μg/mL卡那霉素的LB平板上,按常规方法筛选表达菌株。挑取pET-OmpPon转化的具有外源蛋白表达能力的单个菌落并接种入100mL LB培养基中,于37℃培养过夜。取出菌液后,按1:100接种于100mL含有50μg/mL卡那霉素的LB培养基中,于30℃培养至OD600=0.6时,加入1mol/L IPTG至终浓度为1mmol/L,于37℃摇菌培养,诱导融合蛋白表达。诱导6h后于8000r/min下离心10min收集菌体。将此菌体用50mLBufferA(50mM Na3PO4,0.5M NaCl;pH7.4)洗涤3次并用50mL上样缓冲液(50mM Na3PO4,0.5M NaCl;5mM咪唑,pH7.4)重悬后进行超声破碎,操作条件为:功率50W,工作时间2s,间隔时间3s,报警温度60℃,总时间30min。超声完成后,12000g离心15min分别收集沉淀和上清后进行电泳检测。发现重组OmpPon融合蛋白以部分溶解形式存在于菌体中(另一部分以包涵体形式存在)。薄层扫描显示,该重组蛋白占细菌总蛋白的30%以上。而将未经序列优化的野生型OmpPon基因按上述同样方式进行表达,未见表达产物,说明基因优化效果良好。将上述获得的超声破碎上清液用0.45μm的滤膜进行过滤后用His Trap affinity columns(GEhealthcare公司产品),按照说明书的方法进行重组OmpPon蛋白的纯化,具体方法如下:
1.2.1)连接层析***,该***包括进样管、蠕动泵(上海沪西分析仪器厂,型号DHL-A)、层析柱(GE healthcare公司产品,商品名His Trap affinity columns)及紫外检测器(上海沪西分析仪器厂,型号HD1),柱体积为2ml,预热紫外检测仪30min左右至读数稳定;
1.2.2)校对T%:调节光亮旋钮至显示100%;
1.2.3)旋转灵敏度至合适位置,一般用0.2A;
1.2.4)以上样缓冲液平衡层析***,至读数稳定后旋转“调零”至显示“000”;
1.2.5)上蛋白样品,流速控制在5ml/min以内,收集穿透液;
1.2.6)用上样缓冲液洗去未结合的蛋白,此过程中记录读数,直到读数不再变化为止,收集洗脱液;
1.2.7)用BufferA+10mM咪唑进行洗脱,收集洗脱峰;
1.2.8)用BufferA+20mM咪唑进行洗脱,收集洗脱峰;
1.2.9)用BufferA+40mM咪唑洗脱,收集洗脱峰;
1.2.10)用BufferA+100mM咪唑洗脱,收集洗脱峰;
1.2.11)用BufferA+150mM咪唑洗脱,收集洗脱峰;
1.2.12)取各洗脱峰样品100ul进行SDS-PAGE电泳;
1.2.13)结果发现,目标蛋白在40mM咪唑时被洗脱下来,纯度在90%以上,经bradford试剂盒进行蛋白质浓度测定后,调整浓度为0.2mg/mL,备用。至此制得鲍曼不动杆菌OmpPon融合蛋白。
1.3)抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体的制备
1.3.1)将步骤(1.2)所制鲍曼不动杆菌OmpPon融合蛋白与福氏完全佐剂混合,乳化后作为免疫原免疫雄性新西兰兔2只,每只兔子皮下注射总量为2ml,抗原总量为2mg/只。后每隔两周用OmpPon融合蛋白与福氏不完全佐剂形成的乳化液免疫一次,共免疫5次,抗原用量与初次免疫相同。五免后3-5天进行心脏大量取血,置37℃1小时,然后放冰箱4℃过夜,隔天取血清。
1.3.2)多克隆抗体效价的测定
以OmpPon融合蛋白为包被抗原,包被浓度为5μg/ml,每孔包被100μl,利用间接ELISA法检测血清抗体水平。实验组血清稀释倍数依次为:1:200、1:400、1:800、1:1600、1:3200、1:6400、1:12800、1:25600、1:51200、1:102400、1:204800;
ELISA板包被牛血清白蛋白作为阴性对照,用酶联检测仪测定OD450,满足P/N值>2.1为阳性。结果显示2只兔子的血清抗体效价均达到1:102400,说明免疫效果较好。
1.3.3)多克隆抗体的提取
用GE-HiTrap Protein A HP预装柱,按说明书纯化抗体,具体操作如下:
1.3.3.1)取5mL抗血清,加入0.5mL 1M Tris(pH8.0)调节到pH8.0,20,000g离心20min除去沉淀。
1.3.3.2)上柱后用10倍柱体积bufferA(100mM Tris-Cl,pH 8.0)洗涤后,再用10倍柱体积buffer B(10mM Tris-Cl,pH 8.0)洗涤。
1.3.3.3)用大约三倍柱体积的IgG洗脱缓冲液(100mM glycine,pH 3.0)洗脱IgG。(收集管内先预装0.1mL IgG-中和缓冲液(1M Tris-Cl,pH 8.0),每管装0.9mL洗脱液)
1.3.3.4)用50倍体积Tris(10mM Tris-Cl,pH 8.0)对洗脱液进行透析。
1.3.3.5)超滤浓缩后,调整浓度为5mg/ml,-70℃保存备用。至此制得抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体。
实施例3
高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒组成
包被抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体的酶标板、抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体、鲍曼不动杆菌阳性对照、阴性对照、洗涤液、酶标二抗、酶显色底物和终止液共同组成高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒。
(1)包被抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体的酶标板
用PBS缓冲液将抗鲍曼不动杆菌Fhue及Pilf蛋白多克隆抗体(实施例1制备所得)稀释至浓度为10μg/mL,按100μL/孔的量包被96孔EIA高效结合酶标板(型号:康宁costar2592),37℃包被2小时。取出后用250μL洗涤液洗板三次,甩干。用含有1%BSA的洗涤液作为封闭液,250μL/孔加入酶标板,37℃封闭1小时。取出后用250μL洗涤液洗板3次,每次一分钟,甩干,干燥后密封保存。
其中,PBS缓冲液配方:磷酸氢二钠1.4g,磷酸二氢钠0.2g,氯化钠8.5g,pH7.4加去离子水至1000mL;洗涤液:含0.01%Tween-20的PBS水溶液,pH为7.4;封闭液:含1%BSA的洗涤液水溶液,pH为7.4。
(2)抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体
将如实施例2所述抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体用上述封闭液配制成20μg/mL的溶液,按每个试剂盒5mL的量封装。
(3)酶标二抗
酶标二抗为辣根过氧化物酶标记的羊抗兔IgG,本实施例中其购自北京赛驰生物科技有限公司,产品编号030005-G,浓度为1mg/mL,使用时用PBS缓冲液稀释3000倍。按每个试剂盒0.1mL的量封装。
(4)酶显色底物
酶显色底物配置过程如下:
A液:(配置量1L)
1.称柠檬酸(含1分子结晶水,分子量210.14g)3.14g,醋酸钠(含3分子结晶水,分子量136.0)11.56g溶于970mL双蒸水,制成pH5.0的醋酸钠水溶液。
2.称非那西汀0.08g加入30mL双蒸水加热至100℃,溶解完全后加入第一步的溶液中。
3.再加入0.5g过氧化脲,混匀即可。
B液:(配置量1L)
往2L烧杯加入500mL甲醇,再加入1.27g 3,3,5,5-四甲基联苯胺TMB(SIGMA),60℃加热溶解后,再加入500mL丙三醇即可。
使用前将A、B液1:1混合,配制成酶显色底物。将A液及B液按每个试剂盒各5mL的量封装。
(5)阳性和阴性对照
阳性对照为甲醛灭活的鲍曼不动杆菌(ATCC19606),其制备方法如下:取巧克力液体培养基培养的鲍曼不动杆菌菌液,平板计数后离心收集菌体,用生理盐水重悬菌体并调整菌体浓度至1×109CFU/mL。取5mL菌液,加入分析纯甲醛至终浓度为1%,4℃灭活过夜。12000g离心10分钟后弃去上清液,沉淀加入2mL PBS缓冲液重悬后即为阳性对照。
阴性对照:阴性对照为甲醛灭活的大肠埃希菌(ATCC 25922),其制备方法如下:取LB液体培养基培养的大肠埃希菌菌体,平板计数后后离心收集菌体,用生理盐水重悬菌体并调整菌体浓度至1×109CFU/mL。取5mL菌液,加入分析纯甲醛至终浓度为1%,4℃灭活过夜。12000g离心10分钟后弃去上清液,沉淀加入2mL PBS缓冲液重悬后即为阴性对照。
(6)洗涤液
为PBST液,具体配置方法如(1)内所述。按每个试剂盒200mL的量封装。
(7)终止液
用双蒸水配制的1M HCL溶液。按每个试剂盒10mL的量封装。
实施例4
高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒的使用方法
1)待检样本的处理
按常规方法获得待检者的咽拭子,将其***装有500μL的洗涤液(PBST)的软质塑料管中,挤压塑料管壁,使拭子上的样品充分溶解。
2)加入对照和待检样本
取待检样本100μL加入相应的酶标孔,同时设阳性对照(100μL/孔)1孔,阴性对照(100μL/孔)3孔,37℃孵育1小时,后用250μL洗涤液洗板3次,甩干。
3)加入抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体
加入试剂盒中的抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体工作液,50μL/孔,37℃孵育1小时,后用250μL PBST洗涤液洗板3次,甩干。
4)加入酶标二抗
将实施例3所述试剂盒中的酶标二抗用PBS缓冲液做1:3000稀释,配制成工作液,按50μL/孔的量加入相应的酶标孔,37℃孵育1小时,后用250μL PBST洗涤液洗板3次,甩干。
5)加入酶显色底物
加入试剂盒中新鲜配制的酶显色底物,50μL/孔,显色15分钟。
6)加入终止液
每孔加入50μL终止液,终止反应。
7)测OD450nm值
酶标板置于酶标仪中测定OD450nm值。
8)结果判定
分别读取3孔阴性质控品及1孔阳性质控品样品的OD450nm值;3孔阴性质控品样品的OD450nm读数的平均值与3倍标准差之和即为CUT-OFF值;若上述人咽拭子样本的检测OD450nm值若大于CUT-OFF值即判断为此份临床咽拭子中鲍曼不动杆菌抗原为阳性,反之则判断为此份人咽拭子样本中鲍曼不动杆菌抗原为阴性;若阳性质控品样品的OD450nm值小于CUT-OFF值,则表明试剂盒失效。
实施例5
高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒的特异性和敏感性测定
1)特异性测定
为了验证本发明的高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒的特异性,按照实施例3及实施例4所述试剂盒组成和方法对浓度均为2×105CFU/mL的6株鲍曼不动杆菌菌株及17株非鲍曼不动杆菌标准菌株进行了检测,见表1。结果表明,本发明试剂盒对所有6株鲍曼不动杆菌菌株的检测结果均为阳性,而对其他17株呼吸道常见病原微生物的检测结果均为阴性。该试剂盒显示出良好的特异性。
表1
同时,对浓度为2×105CFU/mL的120株鲍曼不动杆菌临床分离株用本试剂盒进行检测,结果均显示阳性,显示出该试剂盒对临床分离的鲍曼不动杆菌的高度检测覆盖性。
2)敏感性测定
将鲍曼不动杆菌ATCC19606菌株接种于羊血巧克力培养基,35℃培养24小时后,生理盐水10倍梯度稀释,同时平板计数,得到菌体浓度为108-102CFU/mL的菌体溶液,然后将100μL菌液滴加于酶标板,按照实施例3及实施例4所述试剂盒组成和方法将进行检测。结果表明本发明试剂盒的检测敏感性为103CFU/mL。
序列表
<110> 湖北工业大学
<120> 一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒
<141> 2019-10-31
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1062
<212> DNA
<213> FhuPil的基因全序列(FhuPil)
<400> 1
catatgttcg atggcaacta cctggacccg gtagaaggta actctactga agttggtctc 60
aaatccgctt ggtttgacgg ccgtctgaac ggtaccctgg ctctgtacca catcaaacag 120
gacaacctgg cacaggaagc gggccaagtt acccgtaacg gtgttaaaga gacttactac 180
cgtgcagcga aaggcgctac atccgaaggt tttgaagttg aagtatcagg tcagatcact 240
ccagattgga acatcaccgc aggttactct caattttctg ctaaggacgc gaacgatgcg 300
gacgtaaaca ctcagcttcc gcgtaaaatg atccagagct tcactaccta taaactgcct 360
ggtaaactgg aaaacatcac agttggcggt ggcgtaaact ggcagtcttc tacctacgtg 420
aacgcaaaaa acccgaaaaa agtaatcgaa aaagttgagc agggtgacta cgctctggta 480
aacctgatgg cgcgttacca gatcactaag gacttttctg cacagcttaa cattaacaac 540
gttttcggtg gttctggtgg ttctggtggt tctggtggtt ctgcggttaa ggtacgcact 600
caactggcgg ctgaatatat ccgttcaggt gatctggact ccgcgaaacg ctccctggac 660
caggccctga gcgttgactc tcgtgacgcg acagcaaaca tgatgatggg catcctgctg 720
cagcaggagg gctctaaatc taacctggag aaagcggagc actacttcaa acgtgctatc 780
agttctgaac cggataacgc tcaggcgcgc aacaactatg gtacctatct gtaccagatg 840
gaacgttata acgacgcgat tgaacagttt cgtatcgcag gtgcgaccct gggttatgat 900
cagcgttatc aggcgctgga aaacctgggc cgcatctacc tgaagctggg taacatcgcc 960
agcgctgaaa aaactttcaa gcaggcactg ctggcgaacc gtgactccta catctctatg 1020
ctggagctgg ctgaaatctt ttacctgcag cagtaaggat cc 1062
<210> 2
<211> 350
<212> PRT
<213> FhuPil的蛋白序列(FhuPil)
<400> 2
Met Phe Asp Gly Asn Tyr Leu Asp Pro Val Glu Gly Asn Ser Thr Glu
1 5 10 15
Val Gly Leu Lys Ser Ala Trp Phe Asp Gly Arg Leu Asn Gly Thr Leu
20 25 30
Ala Leu Tyr His Ile Lys Gln Asp Asn Leu Ala Gln Glu Ala Gly Gln
35 40 45
Val Thr Arg Asn Gly Val Lys Glu Thr Tyr Tyr Arg Ala Ala Lys Gly
50 55 60
Ala Thr Ser Glu Gly Phe Glu Val Glu Val Ser Gly Gln Ile Thr Pro
65 70 75 80
Asp Trp Asn Ile Thr Ala Gly Tyr Ser Gln Phe Ser Ala Lys Asp Ala
85 90 95
Asn Asp Ala Asp Val Asn Thr Gln Leu Pro Arg Lys Met Ile Gln Ser
100 105 110
Phe Thr Thr Tyr Lys Leu Pro Gly Lys Leu Glu Asn Ile Thr Val Gly
115 120 125
Gly Gly Val Asn Trp Gln Ser Ser Thr Tyr Val Asn Ala Lys Asn Pro
130 135 140
Lys Lys Val Ile Glu Lys Val Glu Gln Gly Asp Tyr Ala Leu Val Asn
145 150 155 160
Leu Met Ala Arg Tyr Gln Ile Thr Lys Asp Phe Ser Ala Gln Leu Asn
165 170 175
Ile Asn Asn Val Phe Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly
180 185 190
Ser Ala Val Lys Val Arg Thr Gln Leu Ala Ala Glu Tyr Ile Arg Ser
195 200 205
Gly Asp Leu Asp Ser Ala Lys Arg Ser Leu Asp Gln Ala Leu Ser Val
210 215 220
Asp Ser Arg Asp Ala Thr Ala Asn Met Met Met Gly Ile Leu Leu Gln
225 230 235 240
Gln Glu Gly Ser Lys Ser Asn Leu Glu Lys Ala Glu His Tyr Phe Lys
245 250 255
Arg Ala Ile Ser Ser Glu Pro Asp Asn Ala Gln Ala Arg Asn Asn Tyr
260 265 270
Gly Thr Tyr Leu Tyr Gln Met Glu Arg Tyr Asn Asp Ala Ile Glu Gln
275 280 285
Phe Arg Ile Ala Gly Ala Thr Leu Gly Tyr Asp Gln Arg Tyr Gln Ala
290 295 300
Leu Glu Asn Leu Gly Arg Ile Tyr Leu Lys Leu Gly Asn Ile Ala Ser
305 310 315 320
Ala Glu Lys Thr Phe Lys Gln Ala Leu Leu Ala Asn Arg Asp Ser Tyr
325 330 335
Ile Ser Met Leu Glu Leu Ala Glu Ile Phe Tyr Leu Gln Gln
340 345 350
<210> 3
<211> 983
<212> DNA
<213> OmpPon的基因全序列(OmpPon)
<400> 3
catatgctgg gttacacctt ccaggatact cagcacaaca acggcggtaa agacggtgaa 60
ctgaccaacg gtccggaact gcaggacgac ctgttcgttg gtgctgcgct gggtatcgaa 120
ctgactccgt ggctgggttt cgaagccgag tataaccagg ttaagggtga cgtggatggc 180
ctggcagcgg gtgctgaata caaacagaaa cagatcaacg gtaacttcta cgttaccagc 240
gacctgatta ccaagaacta tgactctaaa atcaaaccgt atgttctgct gggtgcgggc 300
cactacaaat acgaaatccc ggacctttcc tatcacaacg acgaggaagg cactctgggt 360
aacgcgggtg ttggtgcttt ctggcgtctg aacgacgctc tgtctctgcg taccgaagct 420
cgtggtacct ataacgaagc tgctgctgct aaagaagctg ctgctgctaa aggctctatc 480
gaagctatcg taggtggtta caacttctac cagtccaagt ttaaccgtgc gctccagggc 540
tggcgccagc cgggctctac cattaaacct ttcctgtacg ctctggctct ggaacgtggc 600
atgaccccgt acagcatggt aaacgattct ccgatcacta ttggtaaatg gaccccaaaa 660
aattctgacg gccgttacct gggtatgatc ccgctgcgtc gcgctctgta cctgtcccgt 720
aacactgtat ccgttcgtct gctgcagact gttggcatcg aacgtacccg ccaactgttt 780
atggatttcg gtctgcagga agaccagatt ccacgtaact acactatcgc tctgggcact 840
ccgcaggtac tgccgatcca gatggctacc ggctacgcta ctttcgctaa tggcggctac 900
cgtgttcagc cacatttcat ccagcgtatc gaagacgcgt atggtaaagt aatttacgaa 960
gctaaaccgg aatataagga tcc 983
<210> 4
<211> 324
<212> PRT
<213> OmpPon的蛋白序列(OmpPon)
<400> 4
Met Leu Gly Tyr Thr Phe Gln Asp Thr Gln His Asn Asn Gly Gly Lys
1 5 10 15
Asp Gly Glu Leu Thr Asn Gly Pro Glu Leu Gln Asp Asp Leu Phe Val
20 25 30
Gly Ala Ala Leu Gly Ile Glu Leu Thr Pro Trp Leu Gly Phe Glu Ala
35 40 45
Glu Tyr Asn Gln Val Lys Gly Asp Val Asp Gly Leu Ala Ala Gly Ala
50 55 60
Glu Tyr Lys Gln Lys Gln Ile Asn Gly Asn Phe Tyr Val Thr Ser Asp
65 70 75 80
Leu Ile Thr Lys Asn Tyr Asp Ser Lys Ile Lys Pro Tyr Val Leu Leu
85 90 95
Gly Ala Gly His Tyr Lys Tyr Glu Ile Pro Asp Leu Ser Tyr His Asn
100 105 110
Asp Glu Glu Gly Thr Leu Gly Asn Ala Gly Val Gly Ala Phe Trp Arg
115 120 125
Leu Asn Asp Ala Leu Ser Leu Arg Thr Glu Ala Arg Gly Thr Tyr Asn
130 135 140
Glu Ala Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Gly Ser Ile Glu
145 150 155 160
Ala Ile Val Gly Gly Tyr Asn Phe Tyr Gln Ser Lys Phe Asn Arg Ala
165 170 175
Leu Gln Gly Trp Arg Gln Pro Gly Ser Thr Ile Lys Pro Phe Leu Tyr
180 185 190
Ala Leu Ala Leu Glu Arg Gly Met Thr Pro Tyr Ser Met Val Asn Asp
195 200 205
Ser Pro Ile Thr Ile Gly Lys Trp Thr Pro Lys Asn Ser Asp Gly Arg
210 215 220
Tyr Leu Gly Met Ile Pro Leu Arg Arg Ala Leu Tyr Leu Ser Arg Asn
225 230 235 240
Thr Val Ser Val Arg Leu Leu Gln Thr Val Gly Ile Glu Arg Thr Arg
245 250 255
Gln Leu Phe Met Asp Phe Gly Leu Gln Glu Asp Gln Ile Pro Arg Asn
260 265 270
Tyr Thr Ile Ala Leu Gly Thr Pro Gln Val Leu Pro Ile Gln Met Ala
275 280 285
Thr Gly Tyr Ala Thr Phe Ala Asn Gly Gly Tyr Arg Val Gln Pro His
290 295 300
Phe Ile Gln Arg Ile Glu Asp Ala Tyr Gly Lys Val Ile Tyr Glu Ala
305 310 315 320
Lys Pro Glu Tyr
Claims (8)
1.一种鲍曼不动杆菌表面蛋白Fhue及Pilf,其特征在于:所述鲍曼不动杆菌表面蛋白Fhue及Pilf的蛋白质序列是:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQ;
用于编码鲍曼不动杆菌表面蛋白Fhue及Pilf的蛋白质的基因全序列是:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCTTGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCGAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGTAAGGATCC。
2.用于制备如权利要求1所述的鲍曼不动杆菌表面蛋白Fhue及Pilf的方法,其特征在于:所述方法是:
分别获取鲍曼不动杆菌表面蛋白Fhue及表面蛋白Pilf胞外结构域中抗原表位最为丰富的肽段并找到该肽段基因编码序列,优化肽段基因编码序列,并将优化后的肽段基因编码序列用柔性连接肽的编码序列进行连接,形成融合基因;所述鲍曼不动杆菌表面蛋白Fhue及表面蛋白Pilf在NCBI蛋白质数据库中的accession number分别为KMV27515及AJF80497;所述柔性连接肽的序列是ggsggsggsggs;同时在该融合基因的5’引入酶切位点NdeI、3’端引入终止信号TAA和酶切位点BamHI后化学合成全基因序列,记为FhuPil;
所述FhuPil的基因全序列是:
CATATGTTCGATGGCAACTACCTGGACCCGGTAGAAGGTAACTCTACTGAAGTTGGTCTCAAATCCGCTTGGTTTGACGGCCGTCTGAACGGTACCCTGGCTCTGTACCACATCAAACAGGACAACCTGGCACAGGAAGCGGGCCAAGTTACCCGTAACGGTGTTAAAGAGACTTACTACCGTGCAGCGAAAGGCGCTACATCCGAAGGTTTTGAAGTTGAAGTATCAGGTCAGATCACTCCAGATTGGAACATCACCGCAGGTTACTCTCAATTTTCTGCTAAGGACGCGAACGATGCGGACGTAAACACTCAGCTTCCGCGTAAAATGATCCAGAGCTTCACTACCTATAAACTGCCTGGTAAACTGGAAAACATCACAGTTGGCGGTGGCGTAAACTGGCAGTCTTCTACCTACGTGAACGCAAAAAACCCGAAAAAAGTAATCGAAAAAGTTGAGCAGGGTGACTACGCTCTGGTAAACCTGATGGCGCGTTACCAGATCACTAAGGACTTTTCTGCACAGCTTAACATTAACAACGTTTTCGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTGCGGTTAAGGTACGCACTCAACTGGCGGCTGAATATATCCGTTCAGGTGATCTGGACTCCGCGAAACGCTCCCTGGACCAGGCCCTGAGCGTTGACTCTCGTGACGCGACAGCAAACATGATGATGGGCATCCTGCTGCAGCAGGAGGGCTCTAAATCTAACCTGGAGAAAGCGGAGCACTACTTCAAACGTGCTATCAGTTCTGAACCGGATAACGCTCAGGCGCGCAACAACTATGGTACCTATCTGTACCAGATGGAACGTTATAACGACGCGATTGAACAGTTTCGTATCGCAGGTGCGACCCTGGGTTATGATCAGCGTTATCAGGCGCTGGAAAACCTGGGCCGCATCTACCTGAAGCTGGGTAACATCGCCAGCGCTGAAAAAACTTTCAAGCAGGCACTGCTGGCGAACCGTGACTCCTACATCTCTATGCTGGAGCTGGCTGAAATCTTTTACCTGCAGCAGTAAGGATCC;
所述FhuPil基因编码的蛋白质序列是:
MFDGNYLDPVEGNSTEVGLKSAWFDGRLNGTLALYHIKQDNLAQEAGQVTRNGVKETYYRAAKGATSEGFEVEVSGQITPDWNITAGYSQFSAKDANDADVNTQLPRKMIQSFTTYKLPGKLENITVGGGVNWQSSTYVNAKNPKKVIEKVEQGDYALVNLMARYQITKDFSAQLNINNVFGGSGGSGGSGGSAVKVRTQLAAEYIRSGDLDSAKRSLDQALSVDSRDATANMMMGILLQQEGSKSNLEKAEHYFKRAISSEPDNAQARNNYGTYLYQMERYNDAIEQFRIAGATLGYDQRYQALENLGRIYLKLGNIASAEKTFKQALLANRDSYISMLELAEIFYLQQ;
所述FhuPil基因编码的蛋白质序列为鲍曼不动杆菌表面蛋白Fhue的509-688 aa及表面蛋白Pilf的28-184 aa;两段蛋白序列中间以柔性连接肽ggsggsggsggs进行连接;
将FhuPil的基因全序列按常规方法克隆入原核表达载体pET-28a(+),转入E.coliBL21(DE3)菌中,用IPTG诱导重组大肠杆菌表达,用Ni2+亲和层析法纯化重组FhuPil蛋白。
3.一种用于制备抗鲍曼不动杆菌表面蛋白Fhue及Pilf多克隆抗体的方法,其特征在于:所述方法是将如权利要求2所述的重组FhuPil蛋白作为免疫抗原,与弗氏佐剂混合后反复人工免疫健康的新西兰大白兔,抽血进行效价测定,分离高效价重组蛋白抗体并进行纯化,最终获得抗鲍曼不动杆菌表面蛋白Fhue及Pilf多克隆抗体。
4.一种鲍曼不动杆菌表面蛋白OmpA及ponA,其特征在于:所述鲍曼不动杆菌表面蛋白OmpA及ponA的蛋白质序列是:
MLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYNEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY;
用于编码鲍曼不动杆菌表面蛋白OmpA及ponA的蛋白质的基因全序列是:
CATATGCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC。
5.一种用于制备如权利要求4所述的鲍曼不动杆菌表面蛋白OmpA及ponA的方法,其特征在于:所述方法包括:
分别获取鲍曼不动杆菌表面蛋白OmpA及表面蛋白ponA胞外结构域中抗原表位最为丰富的肽段并找到该肽段基因编码序列,优化肽段基因编码序列,并将优化后的肽段基因编码序列用刚性连接肽的编码序列进行连接,形成融合基因;所述鲍曼不动杆菌表面蛋白OmpA及表面蛋白ponA在NCBI蛋白质数据库中的accession number分别为AJF83030及ADX05080;所述刚性连接肽的序列是eaaakeaaak;同时在该融合基因的5’引入酶切位点NdeI、3’端引入终止信号TAA和酶切位点BamHI后化学合成全基因序列,记为OmpPon;
所述OmpPon的基因全序列是:
CATATGCTGGGTTACACCTTCCAGGATACTCAGCACAACAACGGCGGTAAAGACGGTGAACTGACCAACGGTCCGGAACTGCAGGACGACCTGTTCGTTGGTGCTGCGCTGGGTATCGAACTGACTCCGTGGCTGGGTTTCGAAGCCGAGTATAACCAGGTTAAGGGTGACGTGGATGGCCTGGCAGCGGGTGCTGAATACAAACAGAAACAGATCAACGGTAACTTCTACGTTACCAGCGACCTGATTACCAAGAACTATGACTCTAAAATCAAACCGTATGTTCTGCTGGGTGCGGGCCACTACAAATACGAAATCCCGGACCTTTCCTATCACAACGACGAGGAAGGCACTCTGGGTAACGCGGGTGTTGGTGCTTTCTGGCGTCTGAACGACGCTCTGTCTCTGCGTACCGAAGCTCGTGGTACCTATAACGAAGCTGCTGCTGCTAAAGAAGCTGCTGCTGCTAAAGGCTCTATCGAAGCTATCGTAGGTGGTTACAACTTCTACCAGTCCAAGTTTAACCGTGCGCTCCAGGGCTGGCGCCAGCCGGGCTCTACCATTAAACCTTTCCTGTACGCTCTGGCTCTGGAACGTGGCATGACCCCGTACAGCATGGTAAACGATTCTCCGATCACTATTGGTAAATGGACCCCAAAAAATTCTGACGGCCGTTACCTGGGTATGATCCCGCTGCGTCGCGCTCTGTACCTGTCCCGTAACACTGTATCCGTTCGTCTGCTGCAGACTGTTGGCATCGAACGTACCCGCCAACTGTTTATGGATTTCGGTCTGCAGGAAGACCAGATTCCACGTAACTACACTATCGCTCTGGGCACTCCGCAGGTACTGCCGATCCAGATGGCTACCGGCTACGCTACTTTCGCTAATGGCGGCTACCGTGTTCAGCCACATTTCATCCAGCGTATCGAAGACGCGTATGGTAAAGTAATTTACGAAGCTAAACCGGAATATAAGGATCC;
所述OmpPon基因编码的蛋白质序列是:
MLGYTFQDTQHNNGGKDGELTNGPELQDDLFVGAALGIELTPWLGFEAEYNQVKGDVDGLAAGAEYKQKQINGNFYVTSDLITKNYDSKIKPYVLLGAGHYKYEIPDLSYHNDEEGTLGNAGVGAFWRLNDALSLRTEARGTYNEAAAAKEAAAAKGSIEAIVGGYNFYQSKFNRALQGWRQPGSTIKPFLYALALERGMTPYSMVNDSPITIGKWTPKNSDGRYLGMIPLRRALYLSRNTVSVRLLQTVGIERTRQLFMDFGLQEDQIPRNYTIALGTPQVLPIQMATGYATFANGGYRVQPHFIQRIEDAYGKVIYEAKPEY;
所述OmpPon基因编码的蛋白质序列为鲍曼不动杆菌表面蛋白OmpA的31-173 aa及表面蛋白ponA的406-572 aa;两段蛋白序列中间以刚性连接肽eaaakeaaak进行连接;
将OmpPon的基因全序列按常规方法克隆入原核表达载体pET-28a(+),转入E.coliBL21(DE3)菌中,用IPTG诱导重组大肠杆菌表达,用Ni2+亲和层析法纯化重组OmpPon蛋白。
6.一种用于制备抗鲍曼不动杆菌表面蛋白OmpA及ponA多克隆抗体的方法,其特征在于:所述方法是:
将根据权利要求5所述的重组OmpPon蛋白作为免疫抗原,与弗氏佐剂混合后反复人工免疫健康的新西兰大白兔,抽血进行效价测定,分离高效价重组蛋白抗体并进行纯化,最终获得抗鲍曼不动杆菌OmpA及ponA蛋白多克隆抗体,用封闭液稀释至终浓度为20µg/mL;所述封闭液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2 g/L,氯化钠 8.5 g/L,牛血清白蛋白10 g/L,所述封闭液的pH是7.4。
7.一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒,其特征在于:所述一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒包括包被如权利要求3制备得到的抗鲍曼不动杆菌表面蛋白Fhue及Pilf的多克隆抗体的酶标板、如权利要求6制备得到的抗鲍曼不动杆菌表面蛋白OmpA及ponA多克隆抗体、鲍曼不动杆菌阳性对照、阴性对照、洗涤液、酶标二抗、酶显色底物以及终止液;所述鲍曼不动杆菌阳性对照是灭活的鲍曼不动杆菌菌液;所述阴性对照是灭活的大肠杆菌菌液;所述洗涤液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2 g/L,氯化钠 8.5 g/L,Tween-20 0.5mL/L,所述洗涤液的pH=7.4;所述酶标二抗是辣根过氧化物酶标记的羊抗兔IgG;所述酶显色底物是TMB显色液;所述终止液是1M HCL溶液。
8.一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒的制备方法,其特征在于:所述一种高灵敏度的鲍曼不动杆菌抗原Elisa测定试剂盒包括包被如权利要求3制备得到的抗鲍曼不动杆菌表面蛋白Fhue及Pilf的多克隆抗体的酶标板、如权利要求6制备得到的抗鲍曼不动杆菌表面蛋白OmpA及ponA多克隆抗体、鲍曼不动杆菌阳性对照、阴性对照、洗涤液、酶标二抗、酶显色底物以及终止液;所述鲍曼不动杆菌阳性对照是灭活的鲍曼不动杆菌菌液;所述阴性对照是灭活的大肠杆菌菌液;所述洗涤液的组分及含量是:磷酸氢二钠1.4 g/L,磷酸二氢钠0.2 g/L,氯化钠 8.5 g/L,Tween-20 0.5 mL/L,所述洗涤液的pH=7.4;所述酶标二抗是辣根过氧化物酶标记的羊抗兔IgG;所述酶显色底物是TMB显色液;所述终止液是1M HCL溶液;
所述包被抗鲍曼不动杆菌表面蛋白Fhue及Pilf的多克隆抗体的酶标板的制备方法是:
1)制备抗鲍曼不动杆菌表面蛋白Fhue及Pilf多克隆抗体;
2)抗鲍曼不动杆菌表面蛋白Fhue及Pilf多克隆抗体的包被:
用PBS缓冲液将步骤1)制备得到的抗鲍曼不动杆菌表面蛋白Fhue及Pilf多克隆抗体稀释至浓度为10µg/mL,按100µL/孔的量包被96孔EIA高效结合酶标板,37℃ 2小时;取出后用250µL洗涤液洗板三次,甩干;用含有1% BSA的洗涤液作为封闭液,按250µL/孔的量加入酶标板,37℃封闭1小时;取出后用250µL洗涤液洗板3次,每次一分钟,甩干,干燥后密封保存;
其中,所述PBS缓冲液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2 g/L,氯化钠 8.5 g/L;所述PBS缓冲液的pH是7.4;
所述洗涤液的组分及含量是:磷酸氢二钠1.4g/L,磷酸二氢钠0.2 g/L,氯化钠 8.5 g/L,Tween-20 0.5mL/L,所述洗涤液的pH是7.4;
所述封闭液是含1% BSA的洗涤液的水溶液,所述封闭液的pH是7.4。
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