鸡艾美耳球虫3-1E基因的克隆与表达

应用反转录 -聚合酶链式反应 (RT- PCR)技术 ,分别从柔嫩艾美耳球虫甘肃株 (E.tenella GS,Et GS)和堆型艾美耳球虫青海株 (E.acervulina QH,Ea QH)孢子化卵囊子孢子中提取的总 RNA扩增得到鸡球虫子孢子表面抗原 3- 1E基因 (Et GS3-1E和 Ea QH 3- 1E)。将 Et GS3- 1E与原核表达载体 p GEX- 6 P1连接 ,构建了的 p GEX- 3- 1E原核表达质粒 ,并获得融合蛋白的高效表达和纯化。序列分析表明 :Et GS3- 1E和 Ea QH 3- 1E的 ORF均为 5 13个碱基 ,共编码 171个氨基酸。Et GS3- 1E的蛋白分子量为 18.5 k D,Ea QH 3- 1E的蛋白分子量为 18.6 k D。ORF比较 ,Et GS3- 1E与文献报道的 Ea3- 1E比较 ,共有 2个核苷酸发生变异 ,核苷酸同源性为 98.8%,有 1个氨基酸发生变异 ,氨基酸同源性为 99.4 %;Ea QH 3- 1E与文献报道的 Ea3- 1E比较 ,共有 4个核苷酸发生变异 ,核苷酸同源性为 97.7%,有 3个氨基酸变异 ,氨基酸同源性为 98.3%;Et GS 3- 1E与 Ea QH 3- 1E比较 ,有 3个核苷酸发生变异 ,核苷酸同源性为 98.2 %,有 2个氨基酸发生变异 ,氨基酸同源性为 98.8%。获得了 Et GS 3- 1E融合蛋白的高效表达和纯化 ,表达率达 4 3.2 %。

Cloning and expression of the calmodulin-domain protein kinases gene (CDPK) from Eimeria tenella Gansu strain

The total RNA of Eimeria tenella Gansu(Et GS) strain and Eimeria. acervulina Qinghai(Ea QH) strain was extracted separately and used as the template for RT-PCR. The Et GS 3-1E gene and Ea QH 3-1E gene were cloned separately using specific primers. Sequence analysis suggested, the ORF 3-1E gene is consisted of 513 nucleotides encoding 171 amino acids. Compared Et GS 3-1E gene with the data of 3-1E gene in GenBank, there are two mutation nucleotides and the homology of nucleotides is 99.6%, there are only one mutation amino acid and the homology of amino acid is 99.4%. While compared Ea QH 3-1E gene with that of in GenBank, there are four mutation nucleotides and the homology of nucleotides is 99.2%, there are four mutation amino acid and the homology of amino acid is 98.3%. While compared Ea QH 3-1E gene with Et GS 3-1E gene, there are two mutation nucleotides and the homology of nucleotides is 99.6%, there are two mutation amino acid and the homology of amino acid is 98.8%. Et GS 3-1E gene was subcloned into high level expression vector pGEX-6P1 for construction expression plasmid pGEX-3-1E. After that, the recombinant plasmid was transformed into E.coli BL21. Then highly expression and purity fusion protein had been successfully gotten. The amount of fusion protein in total bacteria protein were 43.2%.