伪狂犬病毒gI基因的克隆表达及其对病毒增殖的影响

Cloning, Expression of the gI Gene of Pseudorabies VirusEa Strain and Its Effection to Viral Replication

从伪狂犬病毒 (PRV)国内地方分离Ea株基因组DNA片段中克隆了完整的gI基因 ,序列分析结果表明 ,gI基因编码区全长 1 1 0 1bp ,可编码 3 6 6个氨基酸残基 ,二级结构预测具有典型I型膜蛋白特征。与GenBank中收录的国外Rice株的同源比较发现 ,Ea株gI在核苷酸和氨基酸水平上均存在多处突变 ,尤其是潜在胞浆区中连续两个碱基的缺失导致移码突变 ,致使gI基因的读码框架后移 ,从而导致Ea株gI较rice株长 1 6个氨基酸残基。将gI基因克隆到真核表达载体pcDNA3 1 +中的人巨细胞病毒早期启动子下游 ,构建的真核表达质粒转染PK 1 5细胞 ,间接免疫荧光检测证实gI获得正确表达。进一步测定天然缺失gI的PRV弱毒Bartha株在表达gI细胞系和空白载体转染的对照细胞系中的蚀斑形成单位 (pfu)和组织细胞培养半数感染量 (TCID50 ) ,结果显示 :Bartha株在表达gI细胞系中的pfu和TCID50 分别为对照细胞系的 1 6 4 %和 2 0 0 %。

The complete gI was cloned from the genomic DNA of Pseudorabies virus(PRV) Ea strain and the DNA sequence was determined by Sanger's sequencing technique. The nucleotide and deduced animo acids sequences indicated that the gI gene of PRV Ea strain is composed of 1101 base pairs and could encode 366 animo acids residues. The result of predicted II class structure showed that gI is a typical I class envelope protein. When compared with PRV Rice strain, there were multiple mutations in the gI gene of PRV Ea strain ,and the diversity of amino acid residues also existed, especially the deletion of two bases which results in the change of the reading frame and there were additional 16 animo acids residues in the potential cytoplasmic domains of Ea strain. The fragment containing the complete gI gene was further sub-cloned into the downstream of CMV promoter of eukaryotic expression vector pcDNA3.1+, resulting in a recombinant expression plasmid pSB209. pSB209 was transfected into PK-15 cell lines and the cells expressing gI were obtained in the selection of G418. Expressed protein was further detected by Indirect Immunofluorescence Assay(IFA). In order to explore the function of gI in the viral replication, the attenuated live vaccine strain, Bartha in which the gI is deleted, was chosen to determine the plaques forming units(pfu) and the tissue culture infectious dose(TCID 50) in PK-15 cell lines expressing the gI(PK gI). The data showed that the pfu and TCID 50 in PK+{gI} were 164% and 200% of those in control cell lines respectively. The above results indicated that the gI involve in and accelerate the viral replication.