海豚链球菌C5a肽酶功能活性区域表达及其对斑点叉尾鱼回(Ictaluruspunctatus)的免疫保护作用

FRACTIONAL EXPRESSION OF FUNCTIONAL DOMAIN OF C5a PEPTIDASE OF STREPTOCOCCUS INIAE AND EVALUATION OF PROTECTIVE EFFICACY IN CHANNEL CATFISH(ICTALURUS PUNCTATUS)

本研究对海豚链球菌DGX07株C5a肽酶基因序列进行了克隆和生物信息学分析,表达了其功能活性区域,并以斑点叉尾(鱼回)(Ictalurus punctatus)作为受试动物,对该蛋白进行了免疫保护作用研究。结果显示,scpⅠ基因编码的氨基酸序列具有2个Peptidase_S8_S53超家族的保守结构域,1个PA超家族的保守结构域和1个Fn3-like超家族的保守结构域,推导其活性位点共有7个,位于前501个氨基酸;根据二级结构预测、疏水性分析、表面可及性、柔性分析和抗原表位分析,推断该氨基酸序列的B细胞表位区域主要集中在第67—1098个氨基酸之间。因此,本研究选取去掉信号肽的N段的前635个氨基酸(180—2085bp)进行表达。使用表达的重组蛋白pSCPⅠ免疫斑点叉尾,抗体效价结果显示:免疫后第四周抗体效价最高,第五周后抗体效价开始下降,相对免疫保护率为43.75%。以上结果表明重组pSCPⅠ蛋白具有较好的免疫保护作用,能够作为海豚链球菌亚单位疫苗的候选之一。

C5a peptidase gene of Streptococcus iniae strain DGX07 was cloned and sequenced. Bioinformatics analysis of C5a peptidase （scpⅠ） gene sequence demonstrated that the putative peptidase has two conserved domains that belongs to the Peptidase_S8_S53 superfamily, one to PA superfamily, and one to Fn3-likesuperfamily. The 7 putative active sits were within the former 501 amino acids from the N-terminal. Based on the secondary structure prediction, hydrophobicity analysis, surface accessibility, flexibility analysis, and epitope analysis indicated that the main B cell epitope regions were within 67-1098 amino acids from the N-terminal. The SCPⅠwas then expressed as partial SCPⅠ（180-2085bp）. Titer of serum of channel catfish （Ictalurus punctatus） challenged against pSCPⅠreached the peak at the fourth week. An immune protection rate of 56.3% was observed. These results suggest that the pSCPⅠhas considerable capacity of immune protection and can be listed as potential sub-unit vaccine candidate.