柔嫩艾美耳球虫长春株SAG2基因的克隆与序列分析

Cloning and Sequence Analysis of SAG2 Gene in Eimeria tenella Changchun Strain

根据已发表的柔嫩艾美耳球虫(Houghton株)基因序列,设计一对特异性引物,通过RT-PCR扩增柔嫩艾美耳球虫长春株SAG2基因,将扩增的片段连入pMD18T载体,筛选阳性克隆进行测序和序列分析。结果表明,成功克隆了柔嫩艾美耳球虫长春株SAG2基因,测得ORF全长813bp;与GenBank中收录的Houghton株柔嫩艾美耳球虫的SAG2基因序列相比,有4个碱基发生变异,两者的核苷酸序列同源性为99.50%;柔嫩艾美耳球虫长春株SAG2基因的推导氨基酸序列同源性为99.63%,其中,A185G变异导致了编码氨基酸由谷氨酸变为甘氨酸(E62G),A247T变异导致了编码氨基酸由蛋氨酸变为亮氨酸(M83L),而其他核苷酸的突变为无义突变;SAG2抗原的N端和C端疏水性较强,并且N端前23个氨基酸为跨膜信号肽区,而中间有许多亲水性区域,且显示很强的抗原性。柔嫩艾美耳球虫长春株与Houghton株的SAG2基因编码蛋白相似性很高,具有很强的抗原性,可以作为球虫疫苗的候选抗原来进一步研究。

According to the SAG2 gene sequence of E.tenellapublished in GenBank,apair of specific primers was designed.Then,the SAG2 gene in E.tenella Changchun strain was amplified by RT-PCR and ligated into pMD18T vector,the positive clone was sequenced.The results showed that the ORF of E.tenellaof Changchun strain SAG2 gene is 813bp,compared with the published SAG2 gene（Houghton strain）sequence,four nucleotide mutation sites appeared in the SAG2 gene of E.tenella Changchun strain.There is 99.50% homology in nucleotide sequence,and 99.63% homology in its encoding region sequence.Among these mutation sites,A185G mutation led to substitution of glutamic acid by glycine,and A247T mutation led to substitution of methionine by leucine.There is strong hydrophobic at N-terminal and C-terminal of SAG2,and there is 23 amino acid transmembrane signal peptide at N-terminal,however,there are a lot of hydrophilic zones in the middle portion,and showed strong antigenicity.These suggest that there is high homology between Changchun strain and Houghton strain of E.tenella SAG2 gene,the protein encoded by SAG2 gene in E.tenella Changchun strain had strong antigenicity,it can be used as a candidate antigen for vaccine development.