堆型艾美球虫表面抗原基因cSZ1在大肠埃希氏菌中的表达

根据已克隆的堆型艾美球虫 (Eimeriaacervulina)广东株子孢子表面抗原基因cSZ1的cDNA序列设计了特异性引物 ,用PCR方法扩增cSZ1的开放阅读框架 (ORF)后克隆至表达载体pET 32a( ) ,构建了重组表达质粒 pET 32a( ) cSZ1,并将其转化至大肠埃希氏菌BL2 1(DE3)。经IPTG诱导 ,获得了cSZ1重组抗原在大肠埃希氏菌中的高效表达 ,表达产物量可达菌体总蛋白的 9.3% ,融合蛋白的分子质量约为 4 0ku。重组菌诱导表达的产物经SDS PAGE后 ,用堆形艾美球虫感染鸡的超免疫血清进行免疫印迹分析 ,结果为阴性 ,提示cSZ1所编码的抗原可能主要是T细胞抗原表位

Expression of sporozoite surface antigenic gene cSZ1 of Eimeria acervulina Guangdong strain in Escherichia coli

The ORF of cSZ1 gene of Eimeria acervulina Guangdong strain was amplified by PCR using the specific primers designed according to the sequences of cSZ1(Gd) gene cloned in our laboratory, and its ligated products with vector pET-32a( ) were transformed into E.coli BL21(DE3) by the CaCl_(2) method. The transformants were identified by PCR amplification and endonuclease digestion. The sequences of positive clone were analyzed,and therecombinant protein was induced to beexpressedby 1 mmol/L IPTG in vitro. The molecular weight of cSZ1 recombinantprotein is about 40 ku, and the amount of recombinantprotein in total bacteria protein wasabout 9.3% . Western blotting result of the purified cSZ1recombinantprotein was negativewhen chicken hyperimmune serum of E. acervulinawas used as a probe.