中国百日咳鲍特菌血清型及其相关基因分析

Serotype and fimbriae-genotype analysis of Chinese Bordetella pertussis strains

目的了解我国1955-2005年临床分离百日咳鲍特菌及疫苗株的血清型及其相关基因特征。方法应用抗菌毛蛋白2（fim2）和3（fim3）单克隆抗体凝集反应分析我国不同时期分离的80株百日咳鲍特菌株和3株疫苗株的血清型，并与相应的多克隆抗体方法进行比较分析；同时应用PCR方法扩增这些菌株fim2和fim3基因，并对这些PCR产物进行DNA测序分析。结果研究表明，与多克隆抗体分析结果相比较，单克隆抗体玻片凝集方法和微量凝集反应除一株临床分离株的血清型分析结果不同，其余菌株的结果均一致。研究菌株的血清型分析结果显示17株临床分离菌株和CS与P3S10疫苗株的血清型为fim2＆3型、48株菌为fim2型、15株分离菌株与18530疫苗株为fim3型。在我国扩大免疫规划前分离菌株的血清型以fim2型和fim2＆3型为主，随着大范围的百日咳疫苗接种，l临床分离菌株中fim3型血清型细菌所占的比例逐渐增多。分析菌株中fim2和fim3的基因型以fim2-1（92．5％）和fim3-A（95．0％）亚型为主；18530疫苗株基因型为fim2-2和fim3-A，而其他两株疫苗株的基因型均为fim2—1和fim3-A。在2000年以后，分离出现含有fim3-B和fim3-D亚型菌株，这些结果表明随着我国大范围百日咳疫苗免疫接种，百日咳分离菌株表达的血清型及其基因序列也出现适应性变异。结论本研究证实了抗fim2和fim3单克隆抗体在百日咳血清型分析的特异性和实用性。对我国不同时期分离菌株和疫苗株的血清型分析，为我国百日咳疫苗株的检定、流行病学和细菌进化的研究奠定了基础。

Objective To understand serotype and fimbriae-genotype of tl. pertussis vaccine strains and isolates from different periods in China. Methods Serotype of eighty isolates and three vaccine strains were determined using anti-fim2 and fim3 monoclonal antibodies compared with polyclonal antisera. Fim2 and fim 3 genes were amplified by PCR and the amplified products were sequenced and analyzed . Results The serotype of three vaccine strains and all isolates but only one tested by the slide agglutination and microplate assay of anti-fim2 and fim3 monoclonal antibodies were the same in comparison with that of the slide agglutination of polyconal antisera. In this study, seventeen isolates and vaccine strains CS and P3S10 were fim2＆3 serotype, and forty-eight isolates were fim2 serotype while fifteen isolates and vaccine strain 18530 were fim3 serotype. The predominant serotypes were fim2 and fim2＆3 before Expanded Program on Immunization in 1978, while the fim3 became the most popular serotype after nation-wide pertussis vaccination in China. The fim2-1 and fim3-A genotype was the most common type, which was identified in 92.5% and 95.0% of the isolates, respectively. The genotype of vaccine strain 18530 was fim2-2 and fim3-A while other vaccine strains were fim2-1 and fim3-A. The isolates contained fim3-B and fim3-D subtypes were found since 2000. These data indicated that the serotype and fimbriae genotype of B. pertussis isolates have been changed for immune environment of national-wide pertussis vaccination in China. Conclusion The validity and specificity of anti-fim2 and fim3 monoclonal antibodies have been validated for serotyping of B. pertuzsis strains. The information of serotype and fimbirae genotype of B. pertussis vaccine strains and isolates from different time periods have been obtained. These data can facilitate the studies on quality control of vaccine strain, epidemiology and the evolution of B. pertussis in China.