猪去乙酰化酶SIRT2的克隆表达

Cloning and Expression of Sus Scrofa Sirtuin 2

目的:原核表达猪去乙酰化酶SIRT2(sirtuin 2,SIRT2)。方法:采用RT-PCR扩增包含SIRT2的完整编码序列;通过酶切连接将SIRT2完整编码序列克隆至表达载体pET28a(+)中,转入大肠杆菌Rosetta中进行诱导表达和亲和层析纯化;经免疫印迹Western-blotting分析初步评价SIRT2的抗原性和特异性。结果:重组原核表达质粒pET28a-SIRT2经测序证明构建正确,表达的重组蛋白相对分子质量约47 kDa,能被相关抗体所识别。结论:具有免疫原性的猪去乙酰化酶SIRT2在大肠杆菌中成功表达,可以此为基础进一步研究猪的SIRT2。

Objective; Express 8us scrofa sirtuin 2 （SIRT2） in prokaryotie system. Method： The fragment which included the SIRT2 com- plete coding sequence was obtained by RT - PCR and which was cloned into pET28a（ ＋ ） vector after enzyme digested. Then the recombi- nant vector was transformed into E, coli Rosetta to induce, express and purify, the antigenicity of recombinant SIRT2 was identified by Western -blotting. llesull ：The sequencing proved that recombinant plasmid pET28a -SIRT2 was constructed correctly. The expressed re- combinant protein with a relative molecular mass of about 47 kDa, was recognized by positive serum antibody. Conclusion ： SIRT2 was ex- pressed in E, coil and showed good specificity, which could as a base for the in - depth investigation of the role of SIRT2.