吉林株犬新孢子虫SAG1基因片段的克隆及蛋白抗原性预测

Cloning of SAG1 Gene Fragment in Jilin Strain of Neospora caninum and Prediction of Its Protein Antigenicity

[目的]对犬新孢子虫SAG1基因进行克隆与序列分析。[方法]从细胞培养的吉林株虫体中提取犬新孢子虫DNA,根据已发表的犬新孢子虫SAG1基因的核苷酸序列,设计并合成1对特异性引物,采用PCR技术,扩增犬新孢子虫SAG1基因片段,并成功克隆到pMD18-Tsimple载体上,对经PCR、酶切鉴定为阳性的重组质粒进行序列分析。[结果]对犬新孢子虫SAG1基因进行克隆后,获得阳性重组质粒。克隆的SAG1基因片段长780 bp,编码260个氨基酸,与已发表的美国株犬新孢子虫SAG1基因核苷酸序列(AF132217)的同源性为99.0%,氨基酸序列同源性为98.8%。通过分子生物学软件对翻译水平进行预测,发现吉林株核苷酸序列并不影响氨基酸翻译与蛋白质的表达。[结论]该试验成功克隆重组质粒,为建立犬新孢子虫的高效表达系统奠定了基础。

[ Objective ] The purpose was to clone SAG1 gene of Neospora caninum and carry out sequence analysis. [ Method ] The N. caninum DNA was taken from insect body of Jilin strain that cultured in cell, a pair of specific primers were designed and synthesized according to the nucleotide sequence of N. caninum SAG1 gene that had published, PCR technology was adopted to amplify N. caninum SAGI gene fragment and successfully cloned into pMD18-T simple vector, the positive recombinant plasmid that was identified by PCR and restriction enzyme was conducted for the sequence analysis. [ Result ] After cloning of N. caninum SAG1 gene, the positive recombinant plasmid was obtained. The length of cloned SAG1 gene fragment was 780 bp and coded as 260 amino acids, and had the homology with the published SAG1 gene nucleotide sequence （ AF132217 ） of American strain of N. caninum, which was 99.0%, and the homology of amino acid sequence was 98.8%. The translation level was forecast through molecular biology software, and the nucleotide sequence of Jilin strain was found not to influence the amino acid translation and expression of protein. [ Conclusion ] The experiment successfully cloned the recombinant plasmid, which laid a foundation for the establishment of high efficient expression system for N. caninum.