【摘要】 目的不同分离株弓形虫α-Tubulin和β-Tubulin基因的PCR-RFLP分析及其杂交探针制备。方法PCR体外扩增α-Tubulin和β-Tubulin基因片段并进行RFLP分析;采用随机引物法用地高辛(DIG-11-dUTP)标记探针并同基因组DNA进行Southern杂交分析。结果从5株弓形虫分离株的基因组DNA中均扩增出α-Tubulin基因的818bp片段和β-Tubulin基因的959bp片段,RFLP分析弓形虫各分离株间酶切图谱未见差异。探针的特异性和敏感性较好。结论α-Tubulin和β-Tubulin基因在弓形虫中高度保守;α-Tubulin和β-Tubulin基因杂交探针可用于Southern杂交。更多还原

【Abstract】 Objective To analyze PCR RFLP of α Tubulin and β Tubulin genes among different geographical isolates of Toxoplasma gondii and prepare Digoxingenin labeled DNA probes for Southern blot analysis. Methods PCR amplification of a partial sequence of α Tubulin and β Tubulin genes followed by restriction enzyme digestion. Probes were prepared by Digoxingenin labeling using random priming method and were used in the Southern blot analysis. Results The 818bp fragment of α Tubulin gene and 959bp fragment of β Tubulin gene were successfully amplified from the genomic DNA of different isolates and showed no significant difference between these fragments in the restriction enzyme digestion analysis. Probes were hybridized to genomic DNA with its good specificity and sensitivity. Conclusion The fragment of α Tubulin and β Tubulin genes are highly conserved in different isolates of T.gondii and can be used as probes for Southern blot analysis.更多还原