Molecular genetic studies on pathogenicity of genus Bacteroides.

1. 将一个编码脆弱拟杆菌YCH46神经氨酸酶的基因克隆到cosmid载体pHC79上。神经氨酸酶基因nanH从cosmid亚克隆，位于1.8千碱基对的XhoI-KpnI限制性内切核酸酶片段中。对插入物进行DNA测序，发现一个编码296个氨基酸的开放阅读框。预测的蛋白质序列与已知的细菌神经氨酸酶序列没有明显的相似性。将含有nanH基因的5.4 kb BamHI-SaII片段克隆到大肠杆菌-拟杆菌穿梭载体pVAL-1中。质粒pKK105由大肠杆菌HB101与均匀拟杆菌BU1001的结合质粒R751介导。发现携带pKK105的菌株BU1001粗细胞提取物的神经氨酸酶活性比携带lame质粒的菌株HB101高1600倍。利用穿梭质粒pVAL-1，采用高压电穿孔法转化白螺旋体菌株5482。利用优化后的方法，每杯pVAL-1质粒dna的转化效率高达3xlO^5个转化子。构建了携带致死基因pnd的质粒载体pUCST5，用于重组质粒的正选择。

1. A gene encoding neuraminidase of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The neuraminidase gene nanH was subcloned from the cosmid and was located within a 1.8-kilobase-pair XhoI-KpnI restriction endbnuclease fragment. DNA sequencing of the insert revealed an open reading frame which encodes 296 amino acids. The predicted protein sequence had no marked similarity to known bacterial neuraminidase sequences. A 5.4-kb BamHI-SaII fragment containing the nanH gene was cloned into the Escherichia coli-Bacteroides shuttle vector pVAL-1. The resulting plasmid, pKK105, was mobilized from E. coli strain HB101 to Bacteroides uniformis strain BU1001 by the conjugative plasmid R751.The neuraminidase activity in crude cell extracts from strain BU1001 carrying pKK105 was found to be 1600-fold greater than that found in strain HB101 carrying the lame plasmid.2. High voltage electroporation was applied to transformation of B. thetaiotaomicron strain 5482 using the shuttle plasmid pVAL-1. With the optimized procedure, B. thetaiotaomicorn was transformed with an efficiency up to 3xlO^5 transformants per mug pVAL-1 plasmid DNA.3. A plasmid vector pUCST5 carrying a lethal gene pnd was constructed for the use of positive-selection of recombinant plasmids.