小鼠Hsp84真核表达质粒的构建及鉴定

Construction and verification of eukaryotic expression vector for mouse Hsp84

目的:构建小鼠Hsp84基因真核表达质粒。方法:采用逆转录、热降落PCR方法,扩增BALb/c小鼠Hsp84基因片段,将其连入pMD18-T载体,筛选阳性克隆、测序验证。然后以重组T载体为模板,PCR扩增目的序列,插入真核表达质粒pcD-NA3.1中,转化大肠杆菌DH5α,通过琼脂糖电泳、双酶切挑选和验证阳性重组克隆。结果:RT-PCR自小鼠脑组织中扩增出目的基因片段,克隆入pMD18-T载体后,测序结果证明为BALb/c小鼠Hsp84基因.插入目的基因的真核表达载体的酶切谱与设计一致。结论:成功构建了小鼠Hsp84编码框全长的真核细胞表达质粒。

Objective： To construct the eukaryotic expression vector of mouse Hsp84.Methods：Amplify the Hsp84 gene by reverese transcription and touchdown PCR ,and then the PCR products was ligated into pMD18-T vector. The positive was selected and verified by agorose electrophoresis and sequencing.The positive clone was used as template and further amplified with the primers containing endodigestion sites and protective hases in both terminals.The PCR products were then inserted into eukariotic expression vector pcDNA3. 1.DH-5 α was transformed with ligated plasmids .Check for the presence of positive clones by means of agorose electrophoresis and en- dodigestion. Results ：The sequence inserted into the recombined positive T vectors was corresponding to the BALb/c mouse Hsp84 gene provided in Gene bank. The presence of positive eukary, otic expression vector was verified by double endodigestion. Conclusions：The eukaryotic expression plasmid for mouse Hsp84 was successfully constructed.