中文摘要：

目的在毕赤酵母中表达HSV-1复制的关键酶--碱性脱氧核糖核酸酶（alkaline deoxyribonuclease，AN），检测其核酸外切酶活性，从而进一步研究AN的抑制剂。方法以GenBank发表的HSV-1的17株UL12基因为模板，在不改变ULl2氨基酸序列的情况下选择毕赤酵母偏好密码子，设计合成新的基因UL12．2，定向克隆进毕赤酵母表达载体pPIC9K中，经酶切和测序鉴定证明重组载体构建成功。线性化的重组载体pPIC9K—UL12．2，电转化至毕赤酵母菌GS115，用组氨酸缺陷平板选择His＋转化子，并进行表型测定，筛选出高抗性转化子。采取甲醇诱导表达，对诱导表达96h的上清进行离子交换层析，SDS．PAGE检测经纯化的AN，并对AN进行核酸外切酶活性检测。结果重组质粒pPIC9K-UL12—2在GS115中成功分泌表达，表达产物纯化后经SDS-PAGE检测可见目的蛋白条带，目的蛋白显示出核酸外切酶活性。结论毕赤酵母成功表达了具有核酸外切酶活性的碱性核酸酶AN，为抗HSV-1新药的研发奠定了基础。

英文摘要：

Objective Alkaline deoxyribonuclease （AN） is required for efficient Herpes Simplex Virus（HSV-1） replication. We prepared active AN and identified exonuclease activity for further exploration of the inhibitors of AN. Methods The UL12 gene from 17 strains of HSV-1 published in GenBank was used as a template .Without changing its amino acid sequence, UL12-2 gene was synthesized by using the preferential condons of P. pastoris, and inserted into vector pPIC9K. Both enzyme digestion and sequencing proved that recombinant plasmid pPIC9K- UL12-2 was constructed correctly. The linearized recombinant vector pPIC9K-ULI2-2 was transformed into P. pastoris GS 115 by electroporation. HIS＋ transformant was screened according to histidine defect flat. High resistance transformants were screened and confirmed Mut phenotype for expression under induction of methanol. After 96h induction, the supernatant was carried out on ion exchange chromatography. The purified AN protein was identified by SDS-PAGE and determined for exonuclease activity. Results Recombinant plasmid pPIC9K-UL12-2 was expressed successfully in GS 115. Purified products are identified target protein fragment by SDS-PAGE and objective protein shows exonuclease activity. Conclusion The alkaline nuclease with a certain exonuclease activity was expressed in P. pastoris GS 115, which pioneers new thinking to develop antiHSV-1 new drugs.