应用核酸原位分子杂交法检测弓形虫SAG1抗原基因在免疫小鼠体内的表达

DETECTION OF THE MRNA TRANSCRIPTION BY USING IN SITU HYBRIDIZATION IN THE MUSCLE OF MICE IMMUNIZED WITH RECOMBINANT PLASMID PCDNA3-SAG1 OF TOXOPLASMA GONDII

PCR扩增RH株弓形虫抗原基因SAG1编码序列 ,构建pcDNA3 -SAG1重组表达质粒。DNA免疫接种小鼠 ,3周后处死动物 ,取注射部位肌肉作冰冻切片 ,用原位分子杂交方法检测弓形虫 pcDNA3 -SAG1重组质粒在免疫小鼠肌肉内的表达。结果PCR扩增出SAG1编码区目的基因 ,片段大小为 10 2 0bp。测序、酶切分析表明构建的 pcDNA3 -SAG1重组表达质粒含有扩增的基因。利用原位分子杂交在免疫接种 pcDNA3-SAG1的小鼠骨骼肌中检测到紫蓝色的阳性杂交信号 ,提示局部肌肉有目的基因mRNA转录的发生。

The gene coding for the surface antigen SAG1 of Toxoplasma gondii was amplified by PCR. After the digestion of the PCR product and the vector pcDNA3 with HindIII and EcoRI,the target gene encoding SAG1 was successfully cloned into pcDNA3 vector. Frozen sections of skeletal muscle of mice inoculated with the recombinant plasmid pcDNA3 SAG1 and control pcDNA3 were in situ hybridized with dig labelled oligonucleotide probe.In situ hybridization results showed that much blue and purple granules, the positive hybridization signal, were detected in the cytoplasm in frozen sections of those mice immunized with pcDNA3-SAG1.These confirmed that mRNA transcription had been occurred in situ hybridization.