马传染性贫血病毒强、弱毒株dUTPase结构与酶活性比较

为研究尿嘧啶脱氧核糖核苷三磷酸酶(dUTPase)在马传染性贫血病毒(equine infectous anemia virus,EIAV)致弱过程中的作用,探索dUTPase结构与功能的关系,分别对EIAV强、弱毒株dUTPase的编码基因进行了结构分析,并在大肠杆菌中进行了表达.经镍-次氮基三乙酸(Ni-NTA)金属亲合层析方法对表达产物纯化后,用3H标记底物的方法测定了重组强、弱毒株dUTPase的活性.证明所表达的两种重组dUTPase均具有水解dUTP的功能,但重组弱毒株dUTPase的活性显著高于重组强毒株dUTPase的活性.结果提示,由于EIAV疫苗株在驴白细胞上连续传代培养,使病毒dUTPase的活性增强和复制能力提高,而决定酶活性改变的分子基础是dUTPase编码基因中的两个氨基酸发生了突变.此结果对其它慢病毒病的免疫预防具有重要参考价值.

Studies on Structure and Function of dUTPase of EIAV--Structural Analysis, Expression of Genes Encoding dUTPase from Virulent and Vaccine EIAV Strains and Characterization of Recombinant Proteins

In order to investigate the effects of dUTPase on attenuation of EIAV strain during passages in the host cell and to demonstrate its structural and functional relationship, the genes encoding dUTPase of virulent and vaccine EIAV strains were analyzed and expressed in E. coli. The recombinant proteins were purified with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography and identified by means of isotopic assay (tritium-labeled substrate). It was shown that the two recombinant dUTPase had the activity of hydrolyzing dUTP, but there were some differences in activity between two recombinant dUTPase. The results suggest that the activity of EIAV dUTPase is increased and the replication ability is enhanced during its series passages in donkey leukocyte. The molecular base of activity alteration of the enzyme is that the two amino acids have mutated in the gene structure of dUTPase of the vaccine strain. This study provides important value for immunoprophylaxis of the other lentivirus infections.