单增李斯特菌胶体金免疫层析试纸条的研制
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摘要
目的以单增李斯特菌(LM)内化素A蛋白(InlA)单克隆抗体为基础,研制其胶体金免疫层析检测试纸条。方法采用DNAStar软件对LM inlA全长基因编码蛋白进行抗原表位分析,截取部分inlA基因片段构建原核表达质粒,诱导表达和纯化获得重组蛋白。以该蛋白免疫BALB/c小鼠,筛选高效分泌抗InlA单克隆抗体的杂交瘤细胞株,制备单克隆抗体;以双抗体夹心的原理研制胶体金免疫层析检测试纸条,并对其特异性、敏感性、稳定性进行评价。结果筛选到2株高效分泌抗InlA单克隆抗体的杂交瘤细胞株,抗体属于IgG1亚类,小鼠腹水抗体效价为1∶64 000;研制的试纸条可与LM发生阳性反应,而与非LM李斯特菌、链球菌、鼠伤寒沙门菌、大肠杆菌O157∶H7等食源性致病菌均不发生阳性反应;LM纯培养物敏感性为2.4×105cfu/ml,模拟猪肉样品敏感性为4.0×106cfu/ml;4℃保存期可达16周以上。结论研制的胶体金免疫层析试纸条具有快速、特异、敏感等优点,可以用于样品中LM的快速检测。
Objective To develop a colloidal gold strip for detection of Listeria monocytogenenes( LM) based on the monoclonal antibody against LM internalin A( InlA) protein. Methods After analyzing the antigenic epitopes of LM inlA full-length gene encoded protein using DNAStar software,the target inlA gene fragment was selected to construct the prokaryotic expression plasmid,and the recombinant InlA protein was prepared by inducible expression and used to immunize the BALB / c mice. The specific monoclonal antibody against LM InlA protein was prepared. Based on the principle of doubleantibody sandwich method,the colloidal gold strip was developed,and its specificity,sensitivity,and stability were evaluated. Results Two hybridoma cell lines were identified to specifically secret anti-InlA monoclonal antibodies,and the antibody subclasses were IgG1 subtype. The antibody titers of acites were 1∶ 64 000. The colloidal gold strip showed positive reaction with the LM strains,but showed negative reactions with Listeria other than LM,as well as other food-borne bacteria such as Streptococcus,Salmonella typhimurium and EHEC O157∶ H7. The detection limits for LM pure cultures and analog samples were 2. 4 × 105and 4. 0 × 106cfu / ml,respectively. The strip could be stored at 4 ℃ for more than 16 weeks. Conclusion The colloidal gold strip could be used to detect LM in food sample rapidly,sensitively and accurately.
Objective To develop a colloidal gold strip for detection of Listeria monocytogenenes( LM) based on the monoclonal antibody against LM internalin A( InlA) protein. Methods After analyzing the antigenic epitopes of LM inlA full-length gene encoded protein using DNAStar software,the target inlA gene fragment was selected to construct the prokaryotic expression plasmid,and the recombinant InlA protein was prepared by inducible expression and used to immunize the BALB / c mice. The specific monoclonal antibody against LM InlA protein was prepared. Based on the principle of doubleantibody sandwich method,the colloidal gold strip was developed,and its specificity,sensitivity,and stability were evaluated. Results Two hybridoma cell lines were identified to specifically secret anti-InlA monoclonal antibodies,and the antibody subclasses were IgG1 subtype. The antibody titers of acites were 1∶ 64 000. The colloidal gold strip showed positive reaction with the LM strains,but showed negative reactions with Listeria other than LM,as well as other food-borne bacteria such as Streptococcus,Salmonella typhimurium and EHEC O157∶ H7. The detection limits for LM pure cultures and analog samples were 2. 4 × 105and 4. 0 × 106cfu / ml,respectively. The strip could be stored at 4 ℃ for more than 16 weeks. Conclusion The colloidal gold strip could be used to detect LM in food sample rapidly,sensitively and accurately.
引文
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[5]崔焕忠,张辉,王兴龙.单增李斯特菌单克隆抗体的制备及胶体金试纸的研制[J].中国兽医科学,2010,40(1):45-50.
[6]段霞.单核细胞增生李斯特氏菌胶体金免疫层析方法的建立[D].南昌:南昌大学,2011.
[7]李峰.应用胶体金免疫层析技术检测单核细胞增生李斯特菌[D].长春:吉林大学,2012.
[8]罗立新,缪珑,潘力,等.快速检测产单核李斯特菌免疫胶体金层析法的研究[J].中国卫生检验杂志,2006,16(2):154-156.
[9]罗雁非,贾芙蓉,丁旭,等.胶体金标记单克隆抗体测定单增李斯特菌试剂盒制备[J].中国实验诊断学,2009,13(9):1231-1234.
[10]谢士嘉,王静,王振国.利用胶体金免疫层析技术快速定量检测单增李斯特菌方法的建立[J].中国国境卫生检疫杂志,2010,33(2):126-129.
[11]熊国华,于丽,曹际娟,等.单增李斯特菌免疫胶体金试纸条快速检测[J].中国公共卫生,2008,24(2):248-249.
[12]喻翠翠,孟宪荣,栗绍文,等.猪结合珠蛋白克隆、原核表达及单克隆抗体制备[J].中国兽医学报,2010,30(9):1245-1247.
[13]彭珍,魏华,万翠香,等.单核增生李斯特菌Internalin A的克隆表达及单克隆抗体制备[J].中国微生态学杂志,2009,21(12):1073-1076.
[14]Cossart P.Illuminating the landscape of host-pathogen interactions with the bacterium Listeria monocytogenes[J].PNAS,2011,108(49):19484-19491.
[2]Vazquez-Boland J A,Kuhn M,Berche P.Listeria pathogenesis and molecular virulence determinants[J].Clin Microbiol Rev,2001,14(3):584-640.
[3]Yucel N,Citak S,Onder M.Prevalence and antibiotic resistance of Listeria species in meat products in Ankara,Turkey[J].Food Microbiology,2005,22:241-245.
[4]中华人民共和国卫生部.GB 4789.30—2010食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验[S].北京:中国标准出版社,2010.
[5]崔焕忠,张辉,王兴龙.单增李斯特菌单克隆抗体的制备及胶体金试纸的研制[J].中国兽医科学,2010,40(1):45-50.
[6]段霞.单核细胞增生李斯特氏菌胶体金免疫层析方法的建立[D].南昌:南昌大学,2011.
[7]李峰.应用胶体金免疫层析技术检测单核细胞增生李斯特菌[D].长春:吉林大学,2012.
[8]罗立新,缪珑,潘力,等.快速检测产单核李斯特菌免疫胶体金层析法的研究[J].中国卫生检验杂志,2006,16(2):154-156.
[9]罗雁非,贾芙蓉,丁旭,等.胶体金标记单克隆抗体测定单增李斯特菌试剂盒制备[J].中国实验诊断学,2009,13(9):1231-1234.
[10]谢士嘉,王静,王振国.利用胶体金免疫层析技术快速定量检测单增李斯特菌方法的建立[J].中国国境卫生检疫杂志,2010,33(2):126-129.
[11]熊国华,于丽,曹际娟,等.单增李斯特菌免疫胶体金试纸条快速检测[J].中国公共卫生,2008,24(2):248-249.
[12]喻翠翠,孟宪荣,栗绍文,等.猪结合珠蛋白克隆、原核表达及单克隆抗体制备[J].中国兽医学报,2010,30(9):1245-1247.
[13]彭珍,魏华,万翠香,等.单核增生李斯特菌Internalin A的克隆表达及单克隆抗体制备[J].中国微生态学杂志,2009,21(12):1073-1076.
[14]Cossart P.Illuminating the landscape of host-pathogen interactions with the bacterium Listeria monocytogenes[J].PNAS,2011,108(49):19484-19491.