摘要
目的 将弓形虫 14 3 3(Toxo14 3 3)信号转导蛋白基因亚克隆入表达载体pBK CMV ,用于寻找弓形虫新的诊断和候选疫苗分子以及寄生虫 宿主相互作用等研究。方法 根据Toxo14 3 3核苷酸序列设计并合成一对引物 ,以弓形虫速殖子mRNA为模板 ,RT PCR法扩增Toxo14 3 3DNA片段 ,通过TA连接将其克隆入 pGEM T载体、经测序证实序列完全正确。再将该目的基因亚克隆入表达载体 pBK CMV中 ,转化大肠杆菌XL1 blue,提取质粒 ,经双酶切法和以该质粒为模板PCR法证实。结果 Toxo14 3 3信号蛋白基因ORF含 798bp ,编码 2 6 5个氨基酸。 结论 获得表达质粒pBK CMV/Toxo14 3 3阳性重组体 ,为原核和真核基因表达奠定了基础。
Objective 14-3-3 signal transduction protein gene of Toxoplasma gondii(Toxo14-3-3) was subcloned into eukaryotic expression vector pBK-CMV for characterization of a new candidate vaccine and diagnostic molecules and for studies of the interaction between parasite and host cells. Methods A pair of primers were designed and synthesized based on the DNA sequence cloned from the enteroepithelial stage of Toxoplasma gondii. The DNA fragment encoding Toxo14-3-3 was amplified by RT-PCR from RNA of RH strain tachyzoites. The purified PCR products were ligated to T-vector pGEM-T. The inserted DNA fragment was confirmed by DNA sequencing. Then the target gene was subcloned into eukaryotic expression vector pBK-CMV. E.coli XL1-blue strain was transformed and the transformants were screened with LB Kanamycin media. The recombinant vector pBK-CMV /Toxo14-3-3 was identified by restriction endonuclease digestion and PCR. Results A open reading frame(ORF) of 798 bp was verified,which codes 265 amino acids. Conclusion A positive recombinant clone pBK-CMV /Toxo14-3-3 was obtained which lays a foundation for further expression recombimant.
出处
《安徽医科大学学报》
CAS
2001年第1期12-14,共3页
Acta Universitatis Medicinalis Anhui
基金
安徽医科大学青年科研基金资助项目! (编号 5 2 2 0 45 )
