猪圆环病毒1型和2型四川分离株全基因克隆分析

参照Genbank收录猪圆环病毒(porcine circovirus,PCV)1、2型序列,设计两对特异性引物,分别扩增猪圆环病毒1型和2型四川分离株(PCV1-YA1株和PCV2-SC株)全基因组,获得了预期目标大小一致扩增产物,分别将其克隆于PMD18-T载体,分别命名为PMD18-T-PCV1-YA与PMD18-T-PCV2-SC。经酶切鉴定和序列测定证实,成功克隆了PCV1-YA1株1759bp和PCV2-SC株1767 bp的全基因组序列。所得序列与GenBank中的国内外其它PCV1和PCV2毒株进行比较分析,其同源性分别达到98.8%~99.9%与93.6%~98.6%;而PCV1-YA1株和PCV2-SC株之间的同源性则仅为69.2%,进一步分析两个毒株的主要阅读框架,毒株间ORF1核苷酸序列及推导的氨基酸同源性均为85.3%,而ORF2的核苷酸序列及推导的氨基酸同源性分别为66%和65.2%。推导显示两毒株之间ORF1编码产物疏水性区域分布有相似之处,而ORF2编码产物的跨膜区存在差异。

Cloning and Sequencing of Complete Genome of Porcine Circovirus Type 1 (PCV1) and Porcine Circovirus Type 2 (PCV2)

One pair of primers was designed based on the Genbank published sequence of Porcine Circovirus type 1(PCV1) to amplify the complete PCV1 genome of YA1 strain,resulting a 1759bp PCR product.Then cloned the PCR product into pMD18-T vector to obtain the recombinant vector PMD18-T-PCV1-YA.Enzyme and sequencing analysis shows that the complete sequence of PCV1 was successfully cloned.Sequenced similarity analysis showed that PCV1-YA1 strain shared 98.8%～99.9% nucleotide sequence identity with the other six strains in GenBank.Complete PCV2(1767bp) sequence of SC strain was cloned into PMD18-T-PCV2-SC successfully using the same method,and sequenced similarity analysis showed that PCV2-SC strain shared 93.6%～98.6% nucleotide sequence identity with other strains in GenBank.The nucleotide sequence identity between PCV1-YA1 and PCV2-SC is only 69.2%.The two major open-reading frame analysis shows that the nucleotide sequence identity of ORF1 and ORF2 between PCV1-YA1 and PCV2-SC are 85.3% and 66%,and their deduction amino acids identity are 85.3% and 65.2% respectively.The hydrophobic prediction revealed that PCV1 ORF1 is similar to PCV2 ORF1,and the transmembrane prediction revealed ORF2 are difference between the two strains.