原位杂交法检测hsp mRNA在猪组织细胞中的分布

Detection of hsp mRNA in the tissues of pigs by in situ hybridization

根据hsp70和hsp90 mRNA基因序列,设计并合成引物,将PCR扩增的基因片段克隆到pGEM-T载体,重组质粒经筛选、鉴定,利用体外转录系统,制作地高辛标记的RNA探针,建立检测hsps mRNA原位杂交方法。利用自建的原位杂交方法,对hsp70、hsp90 mRNA进行组织细胞定位研究。结果显示:hsp70 mRNA和hsp90 mRNA广泛分布于各组织细胞中,尤其在细胞核中相对集中,在肾小管上皮细胞胞浆中也呈强阳性着色。

According to the gene sequences of hsp70 and hsp90 mRNA, the primers were designed and synthesized. The fragments generated by RT-PCR were cloned into the pGEM-T vector and the recombinant plasmids were identified by PCR amplification. Digoxigenin （DIG） labeled RNA probes were prepared by transcription in vitro in order to establish ISH （in situ hybridization） method. The distributions of hsp70 mRNA and hsp90 mRNA in the tissues of pigs were examined by means of ISH method. Hsp70 mRNA and hsp90 mRNA were found in most of the detected tissues, preferentially in the nuclei and also in the cytoplasm of renal tubular epithelial cells.