绿荧光蛋白作标记研究伪狂犬病病毒在体内的增殖与分布

Besides its importance as an animal pathogen, Pseudorabies virus (PRV) has become more and more attractive as a model for studying the pathogenicity and the structure and function of genome at molecular level with the availability of an experimentally accessible natural virus-host system. Recently, Green fluorescent protein (GFP), a novel protein tag, was developed to study viral replication, location and function of a specific protein in live-cell level. In this project, the enhanced GFP (EGFP) will be inserted into the genomic DNA of three typical PRV, i.e., field strain (Ea), mutant strain Ea TK-gG-/LacZ+ and strain Bartha, to obtain three recombinant PRV expressing EGFP to study the replication and distribution of PRV in vivo. .After three years study, the following important results were obtained. (1) An EGFP expression cassette in which the EGFP gene was droved by the promoter of gG of PRV was obtained. This expression cassette can be used to study the function of the specific gene of PRV in the future. (2) Two universal transfer vectors, one locus on gG and another on gE and gI (gE/gI), were constructed, which lay foundation to develop polyvalent vaccines based on PRV. (3) Seven recombinant PRV expressing EGFP or its mutant were obtained, and the results of animal experiments showed that the virulence in vivo of TK-gG- was lower than that of strain Bartha, which indicated that the mutant strain TK-gG- was safe as vaccine and TK was an important factor relating to virulence. (4) Five genes which maybe involve in viral replication and latency were cloned and gC was verified to inhibit viral replication, while gI promote viral replication.The successful completion of this project not only lay foundation to study molecular pathogenicity of PRV, but also represent powerful tools to assess the safety of the existed gene-deleted vaccine and to develop a new generation of safe vaccine. In addition, many important techniques established in this project represent standard techniques for study the function of specific gene.

将绿荧光蛋白基因插入伪狂犬病菌病毒（PRV）的非必需基因gG和启动子下游，通过同源重榻浞直鹨隤RV鄂A野毒株、鄂A株TK.