摘要
目的克隆并鉴定弓形虫PRU株表面抗原SAG2C基因序列和cDNA序列,对比不同毒力弓形虫株(PRU、RH、ME49)中SAG2C基因序列,进行生物信息学分析。方法根据SAG2C基因已知序列设计合成一对引物,应用PCR技术从Prugniaud(PRU)株弓形虫基因组DNA和总RNA中扩增SAG2C基因,克隆入pMD19-T载体并进行序列测定。应用DNAMAN软件、NCBI网站(http://www.ncbi.nlm.nih.gov/)分析3种虫株之间SAG2C基因的同源性;利用生物信息学网站ExPASy(http://us.expasy.org/)对获得的基因及推导出的氨基酸序列进行生物信息学分析。结果PCR扩增得到弓形虫PRU株SAG2C基因及其全长cDNA序列,酶切及PCR鉴定获得了正确的重组质粒,测序结果表明,获得SAG2C基因序列1225bp,全长cDNA序列1095bp,编码364个氨基酸。同源性分析显示,弓形虫Prugniaud株和RH株SAG2C基因同源性为97.14%;Prugniaud株与ME49株cDNA序列同源性为96.89%;编码氨基酸序列同源性为92%,N端为信号肽,C端疏水序列预测它为糖基磷脂酰肌醇固着蛋白,存在13个潜在抗原表位及多个保守功能区域。结论成功克隆了弓形虫缓殖子期特异抗原SAG2C基因序列及其全长cDNA序列,序列分析显示其为糖基磷脂酰肌醇固着蛋白。
Objective To clone, identify and analyze the SAG2C cDNA of Toxoplasma gondii PRU strain. Methods The genomic DNA and cDNA sequences of SAG2C gene were PCR amplified from Toxoplasma gondii PRU strain genomic DNA and RNA, respectively. The PCR fragments were cloned into the pMD19-T vector. Positive clones were screened and identified by endonuclease digestion and PCR. The sequence of insert was further determined by sequencing. The homology of SAG2C among T. gondii PRU, RH and ME49 strains was analyzed by the software. Results The 1 225 base pair of genomic DNA and the 1 095 base pair of cDNA fragments were PCR amplified and subcloned into pMD19-T vector. The sequencing result showed that they are the SAG2C gene and full-length cDNA of SAG2C gene. The deduced amino acid sequence of PRU strain SAG2C gene showed a 92% homology with that of ME49 strain. The N-terminal of SAG2C is a signal peptide, and the C-terminal is a hydrophobic region which suggesting it is a GPI-anchored surface protein. There are 13 potential antigenic epitopes and s set of conserved domains in SAG2C. Conclusion The coding sequence for a bradyzoite-specific antigen, SAG2C, was successfully cloned and identified from T. gondii PRU strain.
出处
《热带医学杂志》
CAS
2008年第3期193-197,200,共6页
Journal of Tropical Medicine
基金
国家自然科学基金(No.30671837)
