罗非鱼源无乳链球菌(Streptococcus agalactiae)Sip基因的克隆、鉴定及分子特性分析

CHARACTERIZATION OF THE Sip GENE IN THE STRAIN STREPTOCOCCUS AGALACTIAE ISOLATED FROM TILAPIA

利用设计的特异性引物,扩增出了分离自患病罗非鱼无乳链球菌强毒株Sip基因,并将其克隆到pMD19-T载体上,之后对重组质粒进行了PCR和双酶切(BamHⅠ+HindⅢ)鉴定,并利用多种生物信息学软件对Sip基因进行分子特性分析。结果显示,罗非鱼源无乳链球菌Sip编码氨基酸序列具有极高保守性,与人源、哺乳动物源无乳链球菌亲缘性达100%,存在1个由25个氨基酸组成的信号肽,具有1个与免疫调节功能相关的LysM结构域,具有与蛋白翻译后修饰功能相关的磷酸化位点33个和N-糖基化位点2个,编码多肽链中疏水区大于亲水区,是一种膜外蛋白。密码子偏爱性分析表明,罗非鱼源无乳链球菌Sip基因密码子使用频率差异较大,密码子偏爱性与真核生物较为接近。

The Sip gene of a virulent strain of Streptococcus agalactiae isolated from Tilapia was amplified using the PCR method with specific primers and then cloned into pMD19-T vector. The presence of the recombinant plasmid was confirmed by the PCR method combined with the restriction enzyme digestion method （BamHⅠ and HindⅢ）. The results showed that the amino acid sequence derived from the Sip of S. agalactiae in Tilapia was highly conserved and had a 100% of homology among strains isolated from humans and other mammals. The polypeptide contained a signal peptide consisting of 25 amino acids and a LysM functional domain which was related to immunoregulation. The polypeptide had a number of important sites related to post-translational modification, including 33 phosphorylation sites and 2 N-glycosylation sites. The hydrophobic regions of the Sip were larger than the hydrophilic regions and the Sip was predicted to be located outside of the cell membrane. An analysis of codon bias demonstrated that the codon usage frequency of Sip in the strain S. agalactiae was distinctly different and it resembles more closely to those of the eukaryotes.