牙龈卟啉单胞菌血凝素2基因缺陷型突变株的构建

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

目的构建牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）血凝素2（hemagglutinin—adhesin2，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HAu、HAl，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，PgATCC33277HA-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。

Objective To construct and identify the Porphyromonas gingivalis （Pg）ATCC33277 hemagglutinin-2 （HA-2）-deficient mutant. Methods The genomic DNA of Pg was isolated from PgATCC33277. The up/down stream genes of HA-2-HAu, HAl were amplified by PCR, and inserted into pSYll8 separately which contains a 2. 1 kb antibiotic resistance ermF-ermAM cassette. The resultant recombinant 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. Conclusions HA-2-deficient mutant of Pg ATCC33277 was constructed successfully, which lays a foundation for further study of its biological function.