牙龈卟啉单胞菌血凝素2基因缺陷型突变株的构建

目的 构建牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)血凝素2(hemagglutinin-adhesin 2,HA-2)基因缺陷型突变株,为研究HA-2基因功能奠定基础.方法 PCR扩增PgHA-2基因两翼片段HA_u、HA_1,将抗性基因ermF-ermAM连接到两者之间构建打靶载体HA-ermF-ermAM.将HA-ermF-ermAM电转化PgATCC33277,基因同源重组整合进入PgATCC33277染色体,用选择性培养基筛选获得PgATCC33277HA-2基因缺陷型突变株.进行PgATCC33277HA-2基因缺陷型突变株与其野生株凝血功能试验,比较二者的凝血功能差异.结果 PCR、酶切和测序鉴定结果表明,打靶载体HA-ermF-ermAM成功构建.经PCR鉴定,打靶载体HA-ermF-ermAM通过同源重组整合进入PgATCC33277染色体,成功获得PgATCC33277HA-2基因缺陷型突变株.凝血实验结果表明,PsATCC33277HA-2基因缺陷型突变株与野生株相比凝血能力明显减弱.结论 成功获得PgATCC33277HA-2基因缺陷型突变株,为进一步研究HA-2的生物学性质奠定了基础.

Construction of the hemagglutinin-2-deficient mutant of Porphyromonas gingivalis

Objective To construct and identify the Porphyromonas gingivalis(Pg)ATCC33277hemagglutinin-2(HA-2)-deficient mutant. Methods The genomic DNA of Pg was isolated from PgATCC33277.The up/down stream genes of HA-2-HA_u,HA_1 were amplified by PCR,and inserted into pSY118 separately which contains a 2.1 kb antibiotic resistance ermF-ermAM cassette.The resultant 0recombinant plasmid-pSY118-HA was linearized as the gene targating fragment HA-ermF-ermAM and used in the electroporation of PgATCC33277.The Pg HA-2-deficient mutant was screened by allelic exchange.The test of aggregation of red blood cells was used to investigate the function change between PgHA2-deficient mutant and the wild type of PgATCC33277.Results The PgHA-2-deficient mutant was identified by PCR.The ability of Pg HA-2-deficient mutant to aggregate red blood cell Was significantly decreased compared with the wild type.Condusions HA-2-deficient mutant of Pg ATCC33277 was constructed successfully,which lays a foundation for further study of its biological function.