犬瘟热病毒小熊猫株H、F和N基因的克隆及表达

Cloning and Sequence Analysis of CDV H,F,N Gene

根据G enB ank中发表的犬瘟热病毒(CDV)的核苷酸序列,设计并合成了扩增CDV H、F和N基因的3对引物,经RT-PCR分别扩增获得了CDV小熊猫株(LP株)H、F和N基因,并对H、F及N基因进行了克隆和序列测定。序列分析表明,CDV LP株属于强毒谱系,与CDV流行株的亲缘关系近,H基因含有较多潜在的糖基化位点,F和N基因相对比较保守。将CDV LP株H、F和N基因克隆入真核表达载体pVAX 1的CM V启动子下游,构建了CDV基因疫苗表达载体pVAXLPH、pVAXLPF、pVAXLPN,体外转染BHK-21细胞,用间接EL ISA方法检测到目的蛋白的表达。用构建的3个表达质粒免疫小鼠,从小鼠血清中检测到了抗CDV抗体,初步证实用CDVH、F和N基因作为核酸疫苗免疫动物,可以激活机体的免疫应答。

The cDNAs of H, F and N genes of the CDVLP strain were amplified by RT-PCR, cloned and sequenced. Molecular and phylogenetic analyses of H gene revealed that the CDVLP strain genotype belongs to the lineage of the field isolate strains. The H gene has more potential glycosylation sites. The F and N genes are more conserved than the H gene. By molecular epidemiology assay, it implied that the CDVLP strain has higher immunogenicity than the vaccine strains. From a practical point of view, CDV DNA vaccine and recombinant vaccine based on canine adenovirus type-2 can be constructed with CDVLP H, F and N genes. The H, F and N genes of CDVLP strain were sub-cloned into the eukaryotic expression plasmid pVAX1, which is driven by the CMV promoter, named pVAXLPH, pVAXLPF and pVAXLPN, respectively. Purified plasmids DNA were transfected into BHK-21 cells by lipofectamine. The CDV H, F and N proteins were expressed in BHK-21, which do specifically react to the CDV antisera. The mice inoculated with these plasmids induced the immune response against CDV.