Molecular cloning and restriction endonuclease analysis of 0.4 kb Hin dIII'O' fragment of bovine herpesvirus 1 DNA.

ABSTRACT

Bovine herpesvirus 1 DNA has been isolated by SDS lysis of the virus purified from potassium tartrate (10-50%) density gradient centrifugation. The quality and quantity of viral DNA was checked by UV spectrophotometry and ethidium bromide stained agarose gel electrophoresis. The 0.4 kb Hin dIII'O' fragment of BHV-1 DNA was selectively cloned into Hin dIII cut pUC9 plasmid DNA (2.665 kb). Recombinants were screened by white/blue colonies as well as Hin dIII restriction enzyme analysis. On restriction endonuclease analysis of recombinant plasmid DNA (p-BH-0) with several restriction enzymes, viz., Sau 3A, Hin fI, Rsa I, Sal I, Dra I, Bgl I, Bgl II, Sma I, Hpa I, Stu I, Mlu I, Xho I, Kpn I, Hae III, Eco RI, Bam HI, Pst I, Pal I, revealed insert viral DNA having sites for Hin fI, Hae III, Rsa I, Sma I, only. Further, the partial restriction map of the recombinant plasmid DNA was constructed using above enzymes.