牙龈卟啉菌基因文库的建立

CONSTRUCTION OF GENOMIC DNA LIBRARY FOR PORPHYROMONAS GINGIVALIS

目的 通过构建牙龈卟啉菌 47A- 1的基因文库 ,为进一步深入研究牙龈卟菌的基因结构奠定基础。方法 选择 Sau3AI对牙龈卟啉菌 47A- 1染色体 DNA作酶切 ,获得的 DNA片段与 Bam HI酶解的质粒 p U C18作体外连接并转化大肠杆菌 JM10 9,在含有 IPTG、X- gal和氨苄青霉素的 L B琼脂平板上 ,用蓝、白筛选法选出含重组质粒的大肠杆菌扩增并分析。结果 得到的重组质粒经酶切、电泳分析 ,证实包含牙龈卟啉菌 47A- 1的插入片段。结论 牙龈卟啉菌 47A-

The purpose of this study is to construct genomic DNA library for Porphyromonas gingivalis. Methods:Total cellular DNA from P. gingivalis 47A 1 was extracted with hexadecyltrimethyl ammonium bromide(CTAB) and phenol:chloroform:isoamyl a alcohol lcohol. Purified bacterial DNA was digested with Sau3AI, yielding fragments with cohesive ends complementary to the pUC18 cohesive ends, which was digested with restriction enzyme BamHI. The digested bacterial DNA fragments was ligated to the linear pUC18 and used to transform competent Escherichia coli JM109.Transformants containing insert DNA were selected on Luria Bertani(LB) plates containing ampicillin, 5 bromo 4 chloro 3 indoy1 3 galactoside(X gal) and isoprogy1 β D thiogalactoyranoside(IPTG).Results:Recombinant clones produced white colonies. 10 white colonies from the plate were randomly selected and analyzed. The recombinant plasmids were purified and digested with EcoRI and HindIII.The presence of inserts were detected by restriction mapping.Conclusion:Construction of genomic DNA library for Porphyromonas gingivalis 47A 1 succeeded.