中文摘要：

为了鉴定猪链球菌2型（Streptococcus suis 2）噬菌体裂解酶（Ly ACCG/Ly ACC）催化域A5的关键氨基酸位点,本研究利用NCBI、Pfam和Uniport数据库,对催化域A5的氨基酸序列中的各位点的保守性进行了比对分析;以噬菌体SMP基因组为模板,PCR扩增lyacc基因,测序分析后构建原核表达质粒p SJ2-lyacc;以质粒p SJ2-lyacc为模板,通过PCR扩增的方法将催化域A5中3个高度保守的氨基酸位点（30位的天冬氨酸/D30、53位的苏氨酸/T53和95位的甘氨酸/G95）的密码子分别定点突变为丙氨酸（A）的密码子,经测序确证后将突变体质粒p SJ2-lyacc/D30A、p SJ2-lyacc/T53A和p SJ2-lyacc/G95A分别转化BL21（DE3）感受态细胞,同时将质粒p SJ2-lyacc作为对照平行转化,用IPTG于27℃诱导表达,菌体重悬后超声裂解,上清液过滤,获得的蛋白粗提液经SDS-PAGE分析,表明重组裂解酶Ly ACC及其突变体D30A、T53A和G95A的分子质量均约为30 ku;将重组裂解酶Ly ACC及其突变体D30A、T53A和G95A的蛋白粗提液用于平板裂解试验,结果显示,原核表达的重组Ly ACC的裂菌圈直径为2.2 cm;突变体G95A的裂菌圈直径仅为1.4 cm,裂菌活性部分降低;突变体D30A和T53A的裂菌圈消失,彻底失去裂菌活性。上述结果表明,D30和T53是Ly ACC的关键氨基酸位点。

英文摘要：

The purpose of this study lies in identification of the key amino acid sites in the domain A5 of lysin（LyACCG/LyiCC）encoded by phage infecting Streptococcus suis 2. Conservative levels of each amino acid site in the domain A5 was analyzed and calculated based on NCBI, Pfam and Uniport databases. The gene lyacc was amplified by PCR with the genome of SMP as template and confirmed by sequencing. The recombinant plasmid pSJ2-1yacc was then constructed. Using pSJ2-1yacc as template, three codons of the highly conserved amino acids located at 30 （isparagine, D）, 53 （Threonine, T） or 95 （Glycine, G） in the domain A5 were substituted with codons of alanine/i by PCR-based point mutation technology, respec- tively, and three mutated plasmids were named pSJ2-1yacc/D30A, pSJ2-1yacc/T53Aand pSJ2-1yacc/G95A.After verified by sequencing, three mutated plasmids and pSJ2-1yacc, respectively, were transformed into BL21（DE3） and were induced to express by IPTG at 27℃. The cells were resuspended in buffer and son- icated on ice. Cell debris were removed by centrifugation, and the supernatant were filtered to remove any remaining particles. SDS-PAGE analysis showed that molecular weight of.recombinated LyACC and mutatnts（D30A, T53A and G95A） were about 30 ku. Protein crude extracts of recombinated LyACC and mutatnts were used for lysis activity tests. The results showed that both mutants D30A and T53A completely lost their bacteriolytic activity, whereas G95A only decreased to a lysis diameter as 1.4 cm compared with LyACC as 2.2 cm. This study result confirmed that D30 and T53 were the key amino acid sites for the bacteriolytic activity of LyACC.