罗非鱼无乳链球菌强毒株基因组表达文库的构建及鉴定

Construction and identification of genomic expression library of Streptococcus agalactiae virulent strain Hod-1 isolated from Oreochromis niloticus

为了筛选罗非鱼（Oreochromis niloticus）无乳链球菌（Streptococcus agalactiae）在鱼体内特异诱导表达基因，该研究构建了罗非鱼无乳链球菌强毒株Hod-1的基因组表达文库。采用Sau3AI对无乳链球菌菌株Hod-1基因组DNA进行不完全酶切，回收大小为0．5—2．0kb的DNA片段，并与经BamHI单酶切且去磷酸化的pET-28a／b／c表达载体连接，转化表达宿主菌BL21（DE3）。结果显示，所构建的无乳链球菌菌株Hod．1基因组表达文库含1．024×10^5个克隆，远大于覆盖无乳链球菌全基因组所需的最小库容量（45579个）。PCR检测结果显示重组子中外源片段的插入率为94％，插入片段大小为0．5—2．0kb，且分布均匀，测序结果显示插入序列与GenBank中无乳链球菌基因组序列同源性高达99％以上。结果表明该研究成功构建了罗非鱼无乳链球菌的基因组表达文库，该文库可用于无乳链球菌在罗非鱼体内诱导表达基因的筛选。

To screen the specific genes of Streptococcus agalactiae induced in Oreochromis niloticus, the genomic expression library of the virulent strain Hod-1 was constructed. The Sau3A I was used to digest genomic DNA of S. agalactiae strain Hod-1 incompletely. The DNA fragments ranging from 0.5 kb to 2 kb were recycled and ligated into pET-28 a/b/c expression vectors which had been diges- ted by BamH I and dephosphorylated by SAP before using. The recombinant vectors were transformed into BL21 （DE3）. The results show that the genomic expression library of S. agalactiae contained 1. 024 ×10^5 clones, considerably more than the minimum number of clones （45 579 clones） covering the whole genomic DNA of S. agalactiae. Results of PCR detection show that the rate of fragment insertion was 94% and different sizes of inserted fragments ranged from 0. 5 kb to 2.0 kb. The results of sequencing show that the homology between inserted sequences and the genomic DNA sequnce of S. agalactiae was over 99%. In conclusion, the genomic expression library of S. agalactiae strain Hod-1 was constructed successfully, which can be used for screening genes of S. agalactiae induced expression in 0. niloticus,