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目的:利用pGenesil-1质粒构建针对热休克蛋白HSP70-2的短发夹RNA(shRNA)表达载体。方法:设计针对HSP70-2表达shRNA结构的互补DNA序列,经退火成双链,分别克隆至带有U6启动子的质粒载体pGenesil-1中,构建重组质粒,转化大肠杆菌DH5α菌株,扩增,提取质粒,酶切鉴定后测序分析。利用质粒转染大鼠精原细胞,用RT-PCR和Western blotting法检测转染后HSP70-2 mRNA和蛋白表达变化。结果:成功构建靶向HSP70-2的3个shRNA重组质粒载体pGenesil-1-HSP70-21、pGenesil-1-HSP70-22和pGenesil-1-HSP70-23。酶切鉴定和测序分析重组质粒,shRNA编码序列与设计的片段完全一致,经酶切凝胶电泳证实载体构建成功。质粒筛选实验提示pGenesil-1-HSP70-23质粒对精原细胞HSP70-2基因的抑制作用最强,而pGenesil-1-HSP70-21的抑制作用最弱。结论:成功构建表达靶向HSP70-2的shRNA重组质粒载体。
OBJECTIVE: To construct a short hairpin RNA (shRNA) expression vector targeting heat shock protein HSP70-2 by using pGenesil-1 plasmid. Methods: The complementary DNA sequence targeting shRNA expression of HSP70-2 was designed and annealed into double-stranded DNA and cloned into plasmid pGenesil-1 with U6 promoter respectively to construct a recombinant plasmid. The recombinant plasmid was transformed into E. coli DH5α, amplified, Plasmids were extracted and identified by restriction analysis. The plasmid was transfected into rat spermatogonia, and the expression of HSP70-2 mRNA and protein was detected by RT-PCR and Western blotting. Results: Three shRNA recombinant plasmids pGenesil-1-HSP70-21, pGenesil-1-HSP70-22 and pGenesil-1-HSP70-23 were successfully constructed. Enzyme digestion and sequencing analysis of the recombinant plasmid, shRNA coding sequence and the design of the fragment exactly the same, confirmed by restriction endonuclease gel electrophoresis vector was successfully constructed. Plasmid screening experiments showed that pGenesil-1-HSP70-23 plasmid had the strongest inhibitory effect on spermatogonia HSP70-2 gene and pGenesil-1-HSP70-21 had the weakest inhibitory effect. Conclusion: shRNA recombinant plasmid vector expressing HSP70-2 was successfully constructed.