假性狂犬病毒隸屬於阿爾伐泡疹病毒亞科的一員。泡疹病毒DNA結合蛋白質（DNA-binding protein; DBP）除了為病毒複製所必須，也可能參與病毒基因體的重組。為了探討假性狂犬病毒DBP的特性，我們進行假性狂犬病毒DBP基因的選殖、定序、表現及特性分析。由實驗室之前對於假性狂犬病毒cDNA定位的實驗結果顯示DBP基因位於病毒基因體0.17 map unit的位置；由定序結果推測假性狂犬病毒DBP基因轉譯區長3531核?酸，可轉譯出1177個氨基酸、約125,000道爾頓的蛋白質，其氨基酸序列和單純泡疹病毒一型的氨基酸序列具有58.5%的相似性。將此段基因的完整轉譯序列架接於表現載體pET-28c(+)，在大腸桿菌BL21(DE3)pLysS細胞中大量表現。在經refresh至OD600為0.6、IPTG濃度為0.1 mM的誘導狀態下，可以大量表現出分子量約125,000道爾頓的重組DBP，和預期的重組蛋白質分子量相符。此大量表現的重組蛋白質，在其N端帶有來自表現載體的六個histidine標記，故可以金屬親合性色層分析法，純化假性狂犬病毒DBP。進一步以重組假性狂犬病毒DBP探討其特性，首先以DNA電泳移動分析偵測DBP-DNA複合物形成的情形，結果證實DBP具有結合DNA的能力。不論是單股DNA或雙股DNA都可當做假性狂犬病毒DBP的受質。以競爭性結合分析DBP對鹼基的偏好性，發現假性狂犬病毒DBP對鹼基偏好性依次為G、C、T、A。在本實驗中，也證實了假性狂犬病毒DBP具有使互補的單股DNA再黏合為雙股DNA的功能；而參與重組的蛋白質，例如噬菌體T4 gp32蛋白質及噬菌體λ的β蛋白質也具有這項功能，因此假性狂犬病毒DBP是否參與病毒基因體的重組，仍需進一步探討。; Pseudorabies virus (PRV) is a member of Alphaherpesvirinae. Herpesvirus DNA-binding protein (DBP) is essential for viral DNA synthesis. Several researches also suggested that DBP might play a role in the process of recombination. To study the biochemical properties of PRV DBP, we cloned and sequenced the PRV DBP gene. PRV DBP gene has an open reading frame of 3531 nucleotides, capable of coding a 1177-amino-acid polypeptide of 125-kDa, and the amino acid sequences share a 58.5% similarity with herpes simplex virus 1. In order to characterize the PRV DBP, this protein was expressed in Escherichia coli with the use of a pET expression vector to produce an N-terminal fusion with six histidine residues. The bacteria were induced with 0.1 mM IPTG when the OD600 reached to 0.6 and a 125-kDa recombinant protein was observed in SDS-PAGE. The recombinant protein was further purified by nickel-affinity chromatography to homogeneity. The recombinant DBP exhibited a DNA-binding property by electrophoretic mobility shift assay, and it bound to both single-stranded and double—stranded DNA. The base preference of DBP was evaluated by competitive binding assay and showed that the binding preference was listed in the order G, C, T, A. Moreover, PRV DBP could promote the renaturation of complementary single strands of DNA, indicated that PRV DBP possessed a similar function as bacteriaphage T4 gp32 protein and λ phage β protein, two proteins involved in DNA recombination. Thus, the PRV DBP involved in viral DNA recombination was suggested in this study and the role of PRV DBP in the process of DNA recombination that occurred during PRV infection will be further elucidated.
