[Construction and sequencing of full-length cDNA of peste des petits ruminants virus].
To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level.
Zhai
Jun-Jun
JJ
Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Gansu 730046, China.
Dou
Yong-Xi
YX
Zhang
Hai-Rui
HR
Mao
Li
L
Meng
Xue-Lian
XL
Luo
Xuo-Nong
XN
Cai
Xue-Peng
XP
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D016679:Genome, Viral; D008969:Molecular Sequence Data; D029021:Peste-des-Petits-Ruminants / Q000821:virology; D018107:Peste-des-petits-ruminants virus / Q000145:classification / Q000235:genetics*; D010802:Phylogeny; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary