HSV-1 UL-12基因的克隆、表达及产物复性

Clone and Expression of HSV-1 UL-12 Gene and Product-renaturing

目的:克隆及表达单纯疱疹病毒Ⅰ型(HSV-1)UL12基因,对其表达产物HSV-1碱性核酸酶(AN)进行复性,为建立抗HSV-1药物的分子筛选模型奠定了基础,对于寻找抗病毒药物具有重要意义。方法:从HSV-1感染的Vero细胞中提取HSV-1基因组DNA,通过PCR克隆出UL12基因并对其测序;然后将HSV-1 UL12基因先克隆至pMD19-T载体,再亚克隆到pET32a表达载体上,并将重组载体转化到E.coli Rosetta菌中进行表达;最后用梯度盐酸胍对表达出的包涵体进行复性。结果:成功构建了pET32a-UL12重组载体,经序列比对,与GenBank上公布的HSV-1国际标准毒株17株的UL12基因序列的同源性达到了99.2%。在E.coli Rosetta菌中以包涵体的形式表达,经复性得到有活性的HSV-1碱性核酸酶(AN)。AN同时具有核酸外切酶和核酸内切酶的活性,与核酸作用60min时酶活性最高。结论:重组载体pET32a-UL12经表达、复性可获得有活性的HSV-1 AN。

Objective： Cloning of UL12 gene from herpes simplex virus type 1,was the foundation of establishing molecular models for HSV-1 drug screening.Method： The HSV-1 genome was extracted from Vero cells infected with HSV-1,plus,UL12 gene was amplified by PCR and then sequenced;HSV-1 UL12 was firstly cloned into pMD19-T vector and then subcloned into pET32a expression vector,and the recombinant vector was transformed into E.coli Rosetta bacteria to express;finally,the inclusion bodies expressed were renatured by a gradient of guanidine hydrochloride.Result： The HSV-1 UL12 gene has been successfully cloned and shares 99.2% of sequence alignment with the international standard HSV-1 stain 17.The HSV-1 alkaline nuclease expressed in inclusion bodies in E.coli Rosetta bacteria had both exonuclease and endonuclease activities after renaturation,which reached a highest activity when it acted on DNA for 60min.Conclusion： HSV-1 AN with biological activity was obtained after the recombinant vector pET32a-UL12 was expressed and renatured.