柔嫩艾美耳球虫YZ株SO7基因的克隆与序列分析

Clone and sequence analysis of gene SO7 of Eimeria tenella YZ strain

对柔嫩艾美耳球虫(E im eria tenella)YZ株折光体SO 7基因进行克隆,并对其核苷酸及氨基酸进行序列分析。以纯化的发育7 h的E.tenella YZ株卵囊孢子提取的总RNA为模板,设计特异性引物,应用两步法RT-PCR技术扩增出SO 7基因特异性片段。将扩增出的片段插入pUCm-T载体,随机选择经蓝白斑筛选、PCR及酶切鉴定为阳性的两个克隆分别进行测序分析,并对结果进行验证。结果表明:两个阳性克隆所含插入片段的DNA序列完全一致,含全长为651个核苷酸的开放阅读框,富含AGC和CTG重复序列,编码区核苷酸与LS18株和B J株的同源性都为99.5%,与HB株同源性为99.4%,与GD株同源性为99.7%。其推测的氨基酸序列与LS18株和GD株的同源性都为99.1%,与B J和HB株同源性为98.6%。通过对其蛋白抗原性、表面可能性、亲水性、柔性区、二级结构等参数预测表明,YZ株核苷酸的变异未引起其蛋白主要特性和B细胞表位的变化。该结果为研制E.tenella的基因工程疫苗奠定了基础。

SO7 gene of Eirneria tenella YZ strain isolated from Yangzhou city was amplified by reverse transcription polymerase chain reaction （RT-PCR） from total RNA of sporulating ooeysts at 7 hours, and cloned into pUCm-T vector. Two positive clones was identificated by blue/white selection, restriction enzyme digestion, and PCR method. Nucleotide sequence of the two inserted fragments was determined by dideotide chain-termination method, results indicated that SO7 gene of E. tenella YZ strain include 651 bp opening read frame which encoded for a polypeptide of 216 amino acids. The ORF sequences of the gene showed 99.5% homology compared with LS18 strain and BJ strain, but 99. 4% with HB strain, and 99. 7%0 with GD strain. As its deduced amino acid ~quence concerned, it showed 99.1% homology compared with LS18 strain and GD strain, and 98.6% with BJ strain and HB strain. Analysis of secondary structure, flexible regions, antigenic index, and surface probability of SO7 protein of YZ strain indicated that they coincided with other isolated strains, and may he a suitable gene for studying vaccine against Eimeria infection.