Ea3-1E与mChIL-15融合基因的构建及其真核表达质粒的免疫效果

Construction of fusion gene harbouring Ea 3-1E and mChIL-15 and immune efficacy of its eukaryotic expression plasmid in SPF chickens

应用重叠扩增PCR技术将鸡堆形艾美球虫子孢子表面抗原3-1E基因片段(Ea3-1E)与编码鸡白细胞介素15成熟蛋白基因片段(mChIL-15)进行串联连接,并在这2个基因片段之间引入4个柔性连接肽SPGS,获得融合基因Ea3-1E-linker-mChIL-15。以pcDNA3.1(+)为载体构建并鉴定真核表达质粒pcD-NA3.1-Ea3-1E-linker-mChIL-15。应用磷酸钙法将质粒体外转染293T细胞,通过间接免疫荧光技术和免疫组织化学技术对重组质粒的体外瞬时表达进行检测。将重组质粒pcDNA3.1-Ea3-1E-linker-mChIL-15、pcDNA3.1-Ea3-1E和pcDNA3.1分别于14和21日龄经腿部肌肉注射免疫SPF雏鸡,28日龄时,除非免疫非感染组外,各组感染5×104个堆形艾美球虫孢子化卵囊,观察真核表达质粒的免疫保护效果。结果显示,成功构建了融合基因3-1E-linker-mChIL-15,转染后第30小时可检测到融合基因编码蛋白在293T细胞中的瞬时表达。与质粒pcDNA3.1-Ea3-1E相比,质粒pcDNA3.1-Ea3-1E-linker-mChIL-15二次免疫后可提供明显的抗球虫免疫保护效果,提高相对增重率(96.48%),降低卵囊排出率(68.35%),降低十二指肠病变记分等。抗球虫指数为183.78。

The fusion gene,Ea3-1E-linker-mChIL-15,was constructed by co mbining 3-1E gene fragment encoding sporozoites surface antigen of Eime ria acervulina（Ea3-1E）and the gene fragment encoding m ature chicken interleukin 15（mChIL-15）with four flexible linker peptides SPGS by gene splicing overlap extension PCR technology.The eukaryotic expression plas mid pcDNA3.1-Ea3-1E-linker-mChIL-15 was constructed success fully. The recombinant plasmid was then transfected into 293T cells with Ca3（PO 4）2,and the transient expression of objective protein was detected by indirect immunofluorescence and immunohistochemistry.The recombinant plasmid pcDNA3.1-Ea3-1E-linker-mChIL-15,pcD NA3.1-Ea3-1E and pcDNA3.1 were used to immunize SPF chickens o f 14-or 21-day old by injection intra-muscularly in thigh muscle,respectivel y.Except for the control group,each group as challenged with 5×104 E.acervulina sporulated oocysts on 28 days of age,and the immune efficac y was observed.The results showed that the fusion gene Ea3-1E-mChIL-15 was successfully constructed,and the transient expression product of this fusion gene could be detected after transfection in 293T cell a t hour 30 post-transfection.The plasmid pcDNA3.1-Ea3-1E-linker-mChIL-15 could significantly provide immune protective efficacy against Eimeria challenge after two immunizations,compared to p lasmid pcDNA3.1-Ea3-1E,such as improving relative weight gain rate（96.48%）,reducing oocyst decrease ratio（68.35%）,decreasing average lesion sc ore in duodenum and so on.The anti-coccidial index was 183.78.