猪圆环病毒2型湖南分离株HNLYYA1全基因组的多引物滚环扩增和序列分析

Multiply-primed rolling-circle amplification and sequence analysis of PCV2 genome of Hunan strain HNLYYA1

首先采用常规PCR法对7份湖南省某地区猪病料进行猪圆环病毒2型(PCV2)的检测,然后对1份PCV2阳性病料的总DNA进行多引物滚环扩增(MPRCA)反应,最后利用限制性内切酶(Eco RⅠ和NcoⅠ)酶切MPRCA产物。结果显示,酶切后的MPRCA产物条带与PCV2基因组或片段的大小一致。将回收的目的条带与Psp72载体连接,转化DH5α菌,最后对提取的重组质粒进行测序和序列比对。获得的PCV2分离株HNLYYA1提交于Gen Bank(登录号KJ867555),其全基因组大小为1 767 bp,基因型为PCV2b-1C。该株PCV2 Cap蛋白与其他毒株进行比对,发现其具有独特的氨基酸突变,NLS区34位组氨酸(H)突变为酪氨酸(Y)、LOOP GH区169位精氨酸(R)突变为甘氨酸(G)、LOOP HI区206位赖氨酸(K)突变为异亮氨酸(I)。本研究揭示了湖南PCV2分离株HNLYYA1的全基因组特征,并为环状病毒的全基因组分离研究提供新的技术途径。

PCR was used for detection of PCV2 DNA in seven samples of diseased pigs in a certain area of Hunan Province at first,then Multiply-primed rolling-circle amplification（MPRCA） reactions were used for detecion of total DNA of positive PCV2 samples,and MPRCA products were digested with restriction enzyme（Eco RⅠand NcoⅠ）.The results showed that the size of the digested MPRCA products was the same with PCV2 genomes or fragments.The MPRCA products were cloned into Psp72 vector,then recombinant plasmid was transformed into bacteria DH5α.Finally,the extracted recombinant plasmid was sequenced and aligned with other sequences.The whole genome was submitted to Gen Bank with the accession number of KJ867555.Sequence alignment and phylogenetic analysis revealed that the genome sequence of PCV2 strain HNLYYA1 consisted of 1 767 nucleotides,and its genotype was PCV2b-1C.There was an unique amino acid mutation of PCV2 cap protein when it was aligned with sequences of other strains.The 34 th histidine into tyrosine in NLS,the 169 th arginine was mutated into glycine in LOOP GH,and the 206 th lysine was mutated into isoleucine in LOOP HI.The study reveals the characteristics of complete genome of PCV2 HNLYYA1,and provides a new technical approach for the studies of completegenomes of circovirus.