关键词: 淋病奈瑟球菌, 转化, 细菌, 质粒, 头孢曲松, 抗药性, 细菌, penA基因, 最小抑菌浓度

Abstract: 【Abstract】 Objective To evaluate the effect of different experimental conditions on the in vitro transformation efficiency for Neisseria gonorrhoeae. Methods The penA genes were amplified using genomic DNA of the gonococcal strain D as a template. The overlap extension PCR method was used to prepare the drug-resistant penAA501V gene, and the drug-resistant penAH041 gene was purchased and served as a positive control. Plasmids containing the penA/penAA501V/penAH041 genes were prepared and transformed into DH5α competent cells of Escherichia coli, and then were cloned in the blue-white screening medium; plasmids were extracted by using the MiniBEST plasmid purification kit from Japanese TaKaRa company and the TIANpure mini plasmid kit from Beijing Tiangen Biochemical Technology Co., Ltd separately. The penA plasmids, penAA501V plasmids, penAH041 plasmids, penAA501V genes, and penAH041 genes were used as substrates and transformed into gonococcal recipient strains A43 and A49 by using the direct incubation method, and the transformation rates （CFU/μg） and minimum inhibitory concentrations （MICs） of ceftriaxone （CRO） were measured. The penAA501V and penAH041 genes were also used as substrates and transformed into gonococcal recipient strains A43 and A49 by using the liposome method, and the transformation efficiency and CRO MICs were determined. Results Using genomic DNA of the gonococcal strain D as the template, the 1 749-bp penA and penAA501V genes were successfully obtained. After extraction of plasmids and electrophoretic analysis, the plasmids containing the gonococcal penA, penAA501V, and penAH041 genes extracted with TaKaRa kits mainly showed bands at the position of 2 500 bp, with relatively high migration rates, while the plasmids extracted with Tiangen kits mainly produced bands at the position of 5 000 bp, with relatively low migration rates. After transformation by using the direct incubation method, the MIC of CRO was 0.001 μg/ml for the recipient strain A43 containing the wild-type penA gene, which increased to 0.002, 0.004, and 0.004 μg/ml after transformation with the penA plasmids, penAA501V plasmids, and penAA501V genes respectively, while the MICs both rose to 0.512 μg/ml for strains A43 transformed with the positive control penAH041 plasmid and penAH041 gene; after incubation with various substrates, the MICs were all 0.016 μg/ml for the strain A49, which were the same as the MIC for the blank control strain （incubated without any substrates）. After transformation by using the liposome-based method, the MIC of CRO was 0.002 μg/ml for the strain A43 containing the wild-type penA gene, and 0.016 μg/ml for the corresponding strain A49; after transformation with penAA501V genes, the MIC rose to 0.004 μg/ml for the strain A43, but remained 0.016 μg/ml for the strain A49; the MICs both rose to 0.250 μg/ml for strains A43 and A49 after transformation with positive control penAH041 genes, which were 125 times and 15.6 times that for the blank control strain, respectively. Conclusions The approach with plasmids as substrates and using the direct incubation method exhibited better transformation efficiency for the gonococcal strain A43. Different plasmid extraction methods may affect transformation efficiency, and the liposome-based method could be used for the strain A49 whose transformation efficiency was low when using the direct incubation method.

Key words: Neisseria gonorrhoeae, Transformation, bacterial, Plasmids, Ceftriaxone, Drug resistance, bacterial, penA gene, Minimum inhibitory concentration