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采用RT-PCR及RACE技术克隆朱砂叶螨Tetranychus cinnabarinus的热激蛋白90(HSP90)基因,并进行序列分析,得到一条长2595bp的cDNA序列,该序列开放阅读框(open reading frame,ORF)为2169bp,编码722个氨基酸,分子量约为83.45kDa,理论等电点为4.81,3′非编码区(untranslated region,UTR)为249bp,5′UTR为177bp。通过Antheprot分析发现5个HSP90家族的签名序列及胞质HSP90特征序列MEEVD。同源性分析表明,朱砂叶螨HSP90编码区核苷酸序列和其他已知的HSP90,尤其是节肢动物昆虫的HSP90,具有很高的相似性。将鉴定正确的原核重组表达质粒pET43a-TcHSP90,转化大肠杆菌Escherichia coli BL21(origami)进行原核表达,应用SDS-PAGE和Western blotting技术分离并检测融合蛋白,结果表明构建的原核表达质粒可以在宿主菌中稳定、正确表达。朱砂叶螨TcHSP90基因的克隆、原核表达,为进一步研究HSP90的性质和功能的研究提供有用的实验材料。
The heat shock protein 90 (HSP90) gene of Tetranychus cinnabarinus was cloned by RT-PCR and RACE and sequenced to obtain a 2595bp cDNA sequence with an open reading frame (ORF) of 2169bp , Encoding 722 amino acids with a molecular weight of about 83.45 kDa and a theoretical isoelectric point of 4.81. The 3 ’untranslated region (UTR) was 249 bp and the 5’UTR was 177 bp. The signature sequences of 5 HSP90 families and MEEVD of cytosolic HSP90 were found by Antheprot analysis. Homology analysis indicated that the nucleotide sequence of the HSP90 coding region of T. cinnabarinus had high similarity with other known HSP90, especially the arthropod insect HSP90. The prokaryotic recombinant plasmid pET43a-TcHSP90 was identified and transformed into Escherichia coli BL21 (origami) for prokaryotic expression. SDS-PAGE and Western blotting were used to isolate and test the fusion protein. The results showed that the prokaryotic expression plasmid could be expressed in host bacteria Stable and correctly expressed. Cloning and prokaryotic expression of TcHSP90 gene from Tetranychus cinnabarinus could provide useful experimental materials for further study on the properties and functions of HSP90.