弓形虫GRA1基因变异及真核表达载体的构建

目的：构建弓形虫致密颗粒抗原1（ GRA1）基因真核表达质粒。方法：根据GRA1基因已知序列，设计一对引物。用PCR技术从弓形虫RH株基因组DNA中扩增GRA1基因片段，插入pcDNA3质粒，转化大肠杆菌DH5α感受态细胞，经酶切及PCR鉴定、测序。结果：从弓形虫RH株基因组中扩增出775 bp的GRA1基因，测序显示该基因存在一135 bp的插入序列，使得所得PCR产物分子量大于预期值（628 bp）。该插入序列造成GRA1基因移码突变。结论：首次报道了变异的弓形虫RH株GRA1基因并构建了该变异基因的重组表达质粒。

Variation of GRA1 gene of Toxoplasma gondii and construction of its eukaryotic expressing plasmid

Objective To construt a recombinant eukaryotic expressing plasmid containing dense granules antigen (GRA1) gene of Toxoplasma gondii . Methods A pair of primers were designed for PCR according to the known sequence of GRA1 gene.The full fragment of GRA1 gene obtained by PCR amplification from genomic DNA of RH strain of Toxoplasma gondii was cloned into plasmid pcDNA3 orientatedly.The constructed recombinant plasmid pcDNA3 GRA1 was transferred into E.coli DH5α.The transformatants were screened and identified by restriction endonuclease digestion and PCR. The nucleotide sequences of the cloned genes were determined. Results A 775 bp GRA1 gene PCR product was obtained from genomic DNA of the RH strain of Toxoplasma gondii ,in which a 135 bp inserted sequence was found.Therefore the PCR product was longer than expected (628 bp).The frameshift mutation for GRA1 gene resulted from the insertion of exogenous sequence. Conclusion The inserted variation of GRA1 gene of the RH strain of Toxoplasma gondii is reported and its recombinant expressing plasmid is construted.