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目的探讨牙龈卟啉单胞菌W83PG0352基因突变株的构建和鉴定。方法本实验于2011年7—12月在中国医科大学口腔医学院中心实验室完成。建立PG0352突变质粒,在T载体上插入PG0352上游基因、红霉素抗性基因和PG0352下游基因,用电转化的方法将质粒转入牙龈卟啉单胞菌菌细胞中,用含有红霉素的TSB培养基筛选PG0352突变株,并通过PCR、RT-PCR和酶活性检测方法对突变株进行鉴定。结果在含有红霉素的TSB培养基中可见牙龈卟啉单胞菌的菌落,用PCR方法证实在这些菌落中PG0352基因被红霉素抗性基因所替代,且唾液酸酶活性丧失。结论通过电转化的方法可成功获得PG0352基因突变株,为研究PG0352基因的功能奠定基础。
Objective To investigate the construction and identification of the mutant strain of Porphyromonas gingivalis W83PG0352. Methods The experiment was performed at the Central Laboratory of Stomatology, China Medical University from July to December in 2011. The PG0352 mutant plasmid was constructed and the PG0352 upstream gene, erythromycin resistance gene and PG0352 downstream gene were inserted into the T vector. The plasmid was transformed into P. gingivalis cells by electrotransformation, The PG0352 mutant was screened by TSB medium and the mutants were identified by PCR, RT-PCR and enzyme activity assay. Results Colonies of P. gingivalis were visualized in TSB medium containing erythromycin. PCR was used to confirm that the PG0352 gene was replaced by the erythromycin resistance gene in these colonies, and the sialidase activity was lost. Conclusion The PG0352 gene mutant can be successfully obtained by electrotransformation, which lays the foundation for studying the function of PG0352 gene.