堆型艾美耳球虫子孢子表面抗原基因cSZ1的克隆及序列分析

Cloning and Sequencing of Sporozoite Surface Antigen Gene cSZ1 of Eimeria acervulina Guangdong Strain

根据已报道的堆型艾美耳球虫(Eimeriaacervulina)子孢子表面抗原基因cSZ1的cDNA序列设计特异性引物,以孢子化12h的E.acervulina广东株卵囊的总RNA为模板,用RT-PCR方法成功克隆了其子孢子表面抗原基因cSZ1(cSZ1(Gd))的cDNA。所克隆cSZ1(Gd)的cDNA全长940bp,其中第3～512位是其阅读框架,共编码170个氨基酸,两端为非编码区。与已报道的cSZ1基因的cDNA序列相比,cSZ1(Gd)有4个位置的核苷酸发生了变异,即原序列中第294、443、569、586位的G、G、G、A在cSZ1(Gd)分别为A、A、A、G,二者核苷酸序列的同源性为99.57%(936/940),其中阅读框架的核苷酸同源性为99.61%(508/510)。比较根据核苷酸序列推导的氨基酸序列,第294位的变异导致了所编码氨基酸由缬氨酸(V)变为异亮氨酸(I),而第443位的变异则为无义突变,氨基酸序列的同源性为99.41%(169/170)。

The sporozoite surface antigen gene cSZ1 of E. acervulina Guangdong strain (cSZ1(Gd)) was amplified by RT-PCR using the specific primers that designed according to the published sequences of cSZ1 gene. Purified PCR products that ligated with vector pGEM-T easy were transformed into E. coli DH5α, and the transformants were identified by the methods of PCR amplification and digestion of endonuclease. The length of cSZ1(Gd) cDNA was 940 bp. Its ORF encoded 170 amino acids and was located in the 3～512 nucleotide region. There was 99.57%(936/940) homology in nucleotide sequence between cSZ1(Gd) and the original cSZ1.2 of the 4 nucleotide mutations were within ORF, in which the mutation located in 294^(th) bp resulted in the change of the corresponding amino acid in predicated amino acid sequence, but another mutation located in 443^(th) bp was a silent mutation. As a result, the deduced amino acid sequence of cSZ1(Gd) was 99.41%(169/170) homology with the cSZ1 reported.