[Construction and sequencing of full-length cDNA clone of swine vesicular disease virus strain HK'1/70].
By RACE, 2 overlapping cDNA fragments (3'PCR and 5'PCR fragments) covering the full genome of swine vesicular disease virus strain HK'1/70 were amplified from total RNA extracted from experimentally infected suckling mice. These fragments were cloned into pGEM-T Easy vector, respectively. 5'PCR fragment was digested by enzymes of Aat II and BssH II, and the Aat II-BssH II-digested 5'PCR fragment was obtained and cloned into the recombinant pGEM-T Easy vector containing 3'PCR fragment,the recombinant plasmid encoding full-length cDNA of SVDV HK'I/70 strain was then obtained and sequenced. The results showed that the complete genome of HK'1/70 was 7401 nucleotides (nts) long (excluding the poly (A) tract) which encodes a single polyprotein of 2185 amino acids, a 5'u ntranslating region (UTR) of 743 nts, a 3'UTR of 102 nts and a poly (A) tail at least 74 adenines. T' promoter was added at the 5'e nd of the full-length cDNA and an additional Pspl406I restriction site was added at the 3'e nd of poly (A) tail. The nucleotide and amino acid sequences were compared and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that HK'1 /70 belonged to the second antigenic group. SVD virus was antigenically closely related to Coxsackie B5 virus, and located on the branches of CB5 evolutionary tree. Successful construction of full-length cDNA clone of SVDV HK'1/70 strain lays foundation for rescuing SVDV effectively and enables further research of SVDV on molecular level.
Zheng
Hai-Xue
HX
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China. haixuezheng@163.com
Liu
Xiang-Tao
XT
Shang
You-Jun
YJ
Zhang
Miao-Tao
MT
Feng
Xia
X
Bai
Xing-Wen
XW
Wu
Jin-Yan
JY
Lv
Jian-Liang
JL
Sun
Shi-Qi
SQ
Yin
Shuang-Hui
SH
Guo
Jian-Hong
JH
Xie
Qin-Ge
QG
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D029822:Enterovirus B, Human / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013552:Swine
D018076:DNA, Complementary