犬瘟热病毒Lederle株H,N全基因序列分析及表达研究

采用RT-PCR方法自犬瘟热病毒Lederle株基因组RNA中扩增出H基因(1 911 bp)和N基因(1 707 bp)片段,序列测定和分析表明与Onderstepoort株、CDV3株等疫苗毒株的同源性在95%以上,而与野外分离株的同源性介于90%～95%;以获得的H,N全基因片段为模板,分别扩增H基因近3′端996 bp的基因片段以及N基因中间高度保守区562 bp的基因片段,经克隆测序后分别插入原核表达载体pET-32a中进行表达,结果显示H片段和N片段分别表达大小约为57 kD和37 kD的融合蛋白,Western blotting检测结果显示,表达蛋白均可与CDV抗血清发生阳性反应,提示原核表达产物具有与CDV抗血清结合的免疫反应性。

Sequence Analysis and Prokaryotic Expression of the H and N Gene of Canine Distemper Virus Lederle Strain

The haemagglutinin protein gene(H) and the nucleocapsid protein gene(N) were amplified from the genomic RNA of canine distemper virus Lederle strain by RT-PCR.The full length of the H fragment and the N fragment was 1 911 bp and 1 707 bp respectively.Sequence analysis showed that the H and N gene had an over 95% identity with vaccine strains such as Onderstepoort strain and CDV3 strain,while only 90%-95% with wild isolates.A 996 bp H fragment and a 562 bp N fragment were amplified and inserted into the prokaryotic expression vector pET-32a for induced expression.The size of the recombinant fusion proteins was 57 kD(H) and 37 kD(N) respectively.The recombinant fusion proteins showed positive reaction with anti-CDV canine serum by Western blotting.