一株减毒百日咳鲍特菌的构建及生物学特性分析

Development and biological properties of an attenuated Bordetella pertussis strain

百日咳是传染性强、感染率高的急性呼吸道传染病,主要感染婴幼儿,是婴儿死亡的主要原因之一。百日咳鲍特菌(Bordetella pertussis)是引起百日咳的最主要病原菌。近年来世界各地多次出现百日咳暴发,迫切需研制更加有效的新型百日咳疫苗。本研究构建了一株减毒百日咳活疫苗BPTM1,利用同源重组方法敲除编码百日咳鲍特菌主要毒力因子百日咳毒素(pertussis toxin,PTX)和皮肤坏死毒素(dermonecrotic toxin,DNT)的基因,并用大肠埃希菌的同源基因置换了负责气管细胞毒素(tracheal cytotoxin,TCT)转运的基因amp G。通过聚合酶链反应验证毒素及相关基因的敲除和置换,蛋白免疫印迹法检测表明PTX的S1亚基未表达。体外生长曲线和体内定植曲线均表明,相比于野生型百日咳鲍特菌BPMM,减毒BPTM1的生长和定植能力未受影响,其所致肺部病理效应减轻,而所诱导的百日咳鲍特菌特异性Ig G、Ig G1、Ig G2a抗体保持高水平。本研究表明,减毒百日咳鲍特菌BPTM1有可能成为百日咳候选疫苗。

Whooping cough is a highly contagious and acute respiratory disease which mainly affects children.Bordetella pertussis is responsible for a large part of these cases.Despite widespread vaccination,re-emergence of pertussis has been reported both in developed and developing countries.It is necessary to develop newvaccine candidates with high effectiveness.In this study,we constructed a live attenuated strain named BPTM1,in which two genes encoding major toxins PTX and DNT were removed,and amp G gene responsible for the transportation of toxin TCT was replaced by a homologous gene from Escherichia coli by homologous recombination.The decreased expression of PTX was supported by Western bolting.The knock-out or replacement of the three genes was confirmed at DNA level by polymerase chain reaction（PCR）.The attenuated strain,BPTM1,showed similar growth curves in vitro and colonization curves in vivo as the parent strain BPM M.Although BPTM1 had a significant reduction in lung pathology than BPM M,it induced the same levels of Ig G,Ig G1,Ig G2 a as BPM M in the host.These results suggest that attenuated Bordetella pertussis BPTM1 may become a candidate vaccine for pertussis.