VP3基因突变鸡传染性贫血病毒的构建

以含CAV标准毒CuX-1株VP3突变体的pTCAVM阳性质粒为基础,通过重叠PCR在基因组位点nt682、nt808、nt829处进行定点突变,构建pTCAVM1、pTCAVM2、pTCAVM3突变体;在此基础上,对上述3个位点进行组合定点突变,构建成功pTCAVMa、pTCAVMb、pTCAVMc突变体;此外,成功构建克隆出3个位点均发生突变的pTCAVMd突变体。所有的突变体都保证VP3定点突变位点的碱基变化,同时不引起VP2蛋白的氨基酸残基序列发生变化,保证了突变体和原始病毒抗原性的一致;将得到的突变体转染易感细胞MSB1,获得具有复制特性的病毒,将病毒接种1日龄的SPF雏鸡,发现病毒可以在鸡体内复制,并引起CAV感染的病理学变化。本研究成功获得了多株感染性克隆毒株,为研究CAV病毒致病性以及开发鸡贫血弱毒活疫苗奠定了坚实的基础。

Construction of VP3 mutation library of chicken anemia virus

Based on the positive plasmid which contains standard CuX CAV-1 with VP3 mutant,site-directed mutagenesis was introduced into the chicken anemia virus(CAV) genome at site nt682,nt808,nt829 by the method of overlap extension PCR,successfully constructed plasmids pTCAVM1,pTCAVM2 and pTCAVM3;with the combination of these three mutagenesis sites,and successfully constructed plasmids pTCAVMa,pTCAVMb and pTCAVMc,togetherwith successfully constructed plasmids pTCAVMd with mutagenesis at all three sites.Although VP3 gene over-lapped in VP2 gene,all VP3 mutants are guaranteed that the amino acid sequence of VP2 protein was not changed for the difference of ORF and degeneracy of codons,therefore the antigen characters of mutants and original virus are identical.By transfecting the mutants into susceptible cell MSB1to get a virus with copy characters,and then inoculated into 1-day-old SPF chicks,we found that the virus can replicate in chickens and cause histopathologic changes of CAV infection.This study successfully obtained multiple clones of infectious strains and lay a solid foundation for the study of the pathogenicity and development of live attenuated vaccines of Chicken infectious anemia(CIA).