猪磷酸二酯酶4B2的原核表达和活性鉴定

Prokaryotic Expression of Porcine Phosphodiesterase 4B2 (PDE4B2) and its Activity

使用RT-PCR方法扩增猪磷酸二酯酶4B2基因,将其克隆到表达载体,经PCR和测序鉴定的阳性重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导重组蛋白表达。结果显示,目的基因片段大小为1718 bp,与GenBank公布的猪PDE4B2基因序列同源性为99.7%。表达的重组蛋白以包涵体和可溶性蛋白两种形式存在。Western blotting检测结果表明,蛋白质大小约为66 ku,通过液相检测重组蛋白的cAMP-PDE活性为51.46%。制备的多克隆抗体有良好的特异性,为进一步检测天然蛋白PDE4B2和筛选PDE4B2的特异性抑制剂奠定了基础。

Porcine phosphodiesterase 4B2 gene was amplified by RT-PCR method and cloned into the prokaryotic expression vector.The positive recombinant plasmid,which was screened by PCR and sequencing,was transformed into E.coli strain BL21（DE3） and recombinant protein expression was induced with IPTG.The results showed that the target fragment was 1718 bp in length and has 99.7% homology with that of porcine counterparts deposited in GenBank.The protein was produced in inclusion bodies and soluble forms.The recombination PDE4B2 protein was 66 ku in molecular mass by Western blotting analysis.Activity of the cAMP-PDE was 51.46%,detected by high performance liquid chromatography（HPLC）.The polyclonal antibody raised against the recombinant PDE4B2 protein had a good specificity,which lays a foundation for nature PDE4B2 protein and inhibitor of PDE4.