Molecular Cloning of the thdF Gene from Streptococcus Agalactiae

• Main Author: 邵逸昕
• Other Authors: 邵卓凡
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/45663417275579734324

碩士 === 國立臺灣大學 === 微生物學研究所 === 86 === 　　In the genus of streptococcus, Streptococcus agalactiae (group B streptococcus, GBS) is the only microorganism expressing hippuricase activity that can degrade hippuric acid into glycine and benzoic acid. In the attempt to isolate hippuricase gene (hipO) of GBS, a pair of primers were designed from the hipO gene nucleotide sequence of Camphylobacter jejuni. Using GBS genomic DNA as template, a 2.0 Kb DNA fragment was obtained by PCR. Then, the PCR product was labeled with digoxigenin-dUTP and used as a probe to screen GBS genomic library constructed with Lambda DASH Ⅱvector. 　　A clone positively reacted with the probe was isolated and purified its DNA. The phage DNA fragment positively reacted with the probe was isolated and subcloned to pUC18 and sequenced. The nucleotide of the inserted DNA fragment was verified containing a partial thiophene degradation gene (thdF)fragment. Comparing amino acid sequence of thdF with that of other bacteria, the GBS gene had high homology with the gene of Escherichia coli, Bacillus subtilis, Borrelia burgdorferi, Haemophilus influenzae, Pseudomonas putida and Mycoplasm gentialium. By PCR study, thdF gene seems to be existed in some but not all GBS strains. Using the partial thdF gene containing recombinant plasmid to transform a thdF mutant strain E.coli NAR 967 for expression assay, none of transfornants was obtained recovering wild strain phenotype. Many questions are discussed in detail.