CN106139138B - 一种细菌丝状血细胞凝集素Fha重组蛋白的应用 - Google Patents
一种细菌丝状血细胞凝集素Fha重组蛋白的应用 Download PDFInfo
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Abstract
本发明涉及分子疫苗学领域,具体的说是一种细菌丝状血细胞凝集素Fha重组蛋白的应用。细菌丝状血细胞凝集素Fha重组蛋白用于制备荧光假单胞菌的免疫疫苗。Fha重组蛋白的制备方法具体为以荧光假单胞菌TSS1为模板,采用F1/R1为引物进行PCR扩增,PCR产物与载体pET259连接,获得质粒pEtFha,转化大肠杆菌BL21(DE3)即可表达Fha重组蛋白。本发明的Fha重组蛋白作为疫苗能够有效保护大菱鲆抵御荧光假单胞菌侵染。
Description
技术领域
本发明涉及分子疫苗学领域,具体的说是一种细菌丝状血细胞凝集素Fha重组蛋白的应用。
背景技术
丝状血细胞凝集素(filamentous hemagglutinin,Fha)是细菌产生的一种粘附素,在细菌粘附宿主细胞表面的过程中发挥重要作用。Fha既可以在细菌外膜上表达,也可以分泌到细菌周围的环境中。在功能上,Fha可以介导细菌粘附宿主细胞,促进细菌生物膜形成和自凝集产生。另外,Fha还具有免疫调节作用。Fha也是一种重要的免疫原,在小鼠实验模型中对支气管败血波氏杆菌具有很好的免疫保护效应。
荧光假单胞菌是一种革兰氏阴性细菌,在自然界中广泛存在。荧光假单胞菌是重要的病原菌,不仅能够感染多种经济鱼类,对水产养殖业造成重大损失,还可以感染人类引发菌血症。目前尚无对于该菌的有效免疫防控方法。
发明内容
本发明目的在于提供一种细菌丝状血细胞凝集素Fha重组蛋白的应用。
为实现上述目的,本发明采用的技术方案为:
一种细菌丝状血细胞凝集素Fha重组蛋白的应用,Fha重组蛋白用于制备荧光假单胞菌的免疫疫苗。
所述Fha重组蛋白为以荧光假单胞菌TSS1为模板,采用F1/R1为引物PCR扩增,PCR产物与载体pET259连接,获得质粒pEtFha,所得质粒转化大肠杆菌BL21(DE3)即可表达重组Fha蛋白。
所述引物为F1:5’-GATATCATGCCGACTACTCCACACAG-3’;R1:5’-GATATCGAAGTCGACCTGATTGCGG-3’。
本发明具有如下优点:
本发明的细菌丝状血细胞凝集素Fha重组蛋白作为疫苗对荧光假单胞菌的侵染有显著免疫保护效应,并且无需商业化佐剂(如弗氏佐剂)。
附图说明
图1为本发明实施例提供的纯化的Fha重组蛋白(泳道2)。泳道1,分子量标准。
具体实施方式
下面结合实施例对本发明作进一步说明。实施例旨在对本发明进行举例描述,而非以任何形式对本发明进行限制。在本发明实施例中所涉及到的常规性实验方法均采用如下方法:
1.质粒提取、DNA(PCR)产物纯化皆使用“天根生化科技(北京)有限公司”的相应试剂盒。
2.质粒、DNA连接液转化进入大肠杆菌皆用Hanahan方法(Sambrook and Russell:Molecular Cloning:A Laboratory Mannual.Cold Spring Harbor Laboratory Press2001);
3.所有限制性内切酶和连接酶皆购自于“北京,纽英伦生物技术有限公司”。
实施例1
Fha重组蛋白rFha的制备
步骤1)rFha的表达载体pEtFha的构建:
荧光假单胞菌Fha的序列已被报道(GenBank accession number.WP_014719704.1)。该序列包含一个血凝反应活性域(haemagglutination activity domain),由氨基酸1-684组成。
pEtFha的构建如下:以荧光假单胞菌TSS1为模板,采用引物F1/R1扩增Fha的血凝反应活性域。PCR条件为:94℃ 60s预变性模板DNA,然后94℃ 40s,62℃ 60s,72℃ 60s,30个循环。PCR产物用天根的相应试剂盒纯化。将表达载体pET259(pET259构建过程参见HuYH,Zheng WW,Sun L.Identification and molecular analysis of a ferritin subunitfrom red drum(Sciaenops ocellatus).Fish Shellfish Immunol 2010;28:678-86)用限制性内切酶SwaI酶切后与上述纯化的PCR产物用T4DNA连接酶连接,连接液转化入大肠杆菌DH5α,在含卡那霉素(50ug/ml)的LB培养基上培养18-24小时,筛选转化子提取质粒,即为pEtFha。
所述菌株TSS1保存于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址,北京市朝阳区大屯路,保藏编号为:CGMCC No.2329,保藏日期:2008年1月9日,分类命名为荧光假单胞菌(Pseudomonas fluorescens);
所述LB组成成分按重量百分比计:1.0%蛋白胨,0.5%酵母粉,1.0%氯化钠,97.5%蒸馏水;
所述引物为F1:5’-GATATCATGCCGACTACTCCACACAG-3’;R1:5’-GATATCGAAGTCGACCTGATTGCGG-3’。
步骤2)rFha的表达和纯化:
rFha的表达和纯化如下:将上述步骤1)质粒pEtFha用常规方法转化大肠杆菌BL21(DE3)(购自于“天根生化科技有限公司”,北京),在含有卡那霉素(50ug/ml)的LB固体培养基上培养18-24小时,挑取转化子,将其命名为BL21/pEtFha。将BL21/pEtFha于含有卡那霉素(50ug/ml)的LB液体培养基中过夜培养;取1ml过夜后的培养液,加入100ml新鲜的含有卡那霉素(50ug/ml)的LB液体培养基中,于37℃下转速200rpm摇动培养至OD600为0.6,加入终浓度为1mM的IPTG,37℃继续以转速160rpm摇动培养4-5h,而后以5000g,4℃离心10min,收集菌液,加入5ml裂解液,在室温于摇床上缓慢摇动1-2小时,直至菌悬液变澄清为止。将菌液以10000g,4℃离心30min,回收上清。将上清中的蛋白用His Trap HP Columns(购于美国GE Healthcare公司)回收纯化,纯化的蛋白经SDS-PAGE电泳检测(8v/cm电压下电泳25-30min,随后15v/cm电压下电泳2-2.5h),测定其分子量大小,与预期的rFha分子量吻合(参见图1)。
所述裂解液含10mM NaH2PO4、10mM Tris和8M尿素,pH 8.0。
实施例2
rFha作为疫苗的免疫应用
步骤1)佐剂及疫苗混合液的制备。
佐剂对照液的制备:将5%(质量比)NaOH和5%(质量比)Al2(SO4)3以2:5体积比混合,将混合物以10,000g离心5分钟。将沉淀悬浮于PBS中至0.2mg/ml,即为佐剂。将PBS与佐剂等体积混合,即为佐剂对照液。
疫苗混合液制备:将上述实施例1纯化的rFha在PBS中稀释至200ug/ml,将稀释后的蛋白与佐剂对照液等体积混合,即为rFha疫苗混合液。
所述PBS组成成分按重量百分比计:0.8%NaCl,0.02%KCl,0.358%Na2HPO4.12H2O,0.024%NaH2PO4,余量为水。
步骤2)疫苗的免疫应用。将60条大菱鲆(每条重约14g)随机分为2组,每组35条。将这2组分别命名为A和B组。将A组的每条鱼腹腔注射100ul上述步骤1)的rFha疫苗混合液,将B组的每条鱼腹腔注射100ul上述步骤1)的佐剂对照液。
步骤3)荧光假单胞菌悬液的制备。在LB培养基中培养荧光假单胞菌TSS1至OD600为0.8,然后离心(5000g,4℃)10min。收集菌体,将其悬浮于PBS中至终浓度为1x108cfu/ml。
步骤4)疫苗免疫保护效应检测。在步骤2)免疫注射后的第30天,用上述步骤3)的荧光假单胞菌悬液腹腔注射步骤2)的2组鱼,每条鱼的注射量为50ul。在以后的20天中,每天观察并记录各组鱼的死亡情况。20天后,统计各组鱼的死亡率:A组,54%;B组,86%。利用下列公式计算相对免疫保护效率(RPS):
RPS=100x(1-免疫组鱼的总死亡百分比/对照组鱼的总死亡百分比)
根据此公式算出rFha的相对免疫保护效率为37%。因此,rFha作为疫苗能够有效保护大菱鲆抵御荧光假单胞菌侵染。
Claims (1)
1.一种细菌丝状血细胞凝集素Fha重组蛋白的应用,其特征在于:Fha重组蛋白用于制备荧光假单胞菌的免疫疫苗;
所述Fha重组蛋白为以荧光假单胞菌TSS1为模板,采用F1/R1为引物PCR扩增,PCR产物与载体pET259连接,获得质粒pEtFha,所得质粒转化大肠杆菌BL21(DE3) 即可表达重组Fha蛋白;
所述引物为F1:5’- GATATCATGCCGACTACTCCACACAG -3’;R1:5’-GATATCGAAGTCGACCTGATTGCGG -3’。
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CN105175529A (zh) * | 2015-07-16 | 2015-12-23 | 中国科学院海洋研究所 | 一种鱼类肿瘤坏死因子家族蛋白的重组蛋白及其应用 |
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