title: Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.
abstract: To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.
authors.affiliation: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Aman
authors.initials: A
authors.lastname: Kamboj
authors.affiliation: Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
authors.forename: Mohini
authors.initials: M
authors.lastname: Saini
authors.affiliation: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India.
authors.forename: Lekshmi S
authors.initials: LS
authors.lastname: Rajan
authors.affiliation: Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
authors.forename: Chhabi Lal
authors.initials: CL
authors.lastname: Patel
authors.affiliation: Division of Biological Products, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India.
authors.forename: V K
authors.initials: VK
authors.lastname: Chaturvedi
authors.affiliation: Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India. Electronic address: praveen.indian@gmail.com.
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Gupta
mesh_terms: D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D006692:Classical Swine Fever Virus / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D007194:India; D013552:Swine; D014162:Transfection
chemical_list: D018076:DNA, Complementary
keywords: Classical swine fever virus; Full-length cDNA clone; Overlap extension PCR; in vitro recombination cloning