Journal of Pure and Applied MicrobiologyVol. 9 No. 2

Cloning of 6.5 kb Segment of Cell Culture Adapted Classical Swine Fever Virus

Parveen Kumar, Pronab Dhar, Vikramaditya Upmanyu, Amit Kumar and Ashok Kumar Tiwari

Division of Biological Standardization, Indian Veterinary, Research Institute, Izatnagar -243122, India.

Received on 06 February 2015 and accepted on 10 March 2015

 

ABSTRACT

In this study, a 6.5 Kb fragment of cell culture adapted Lapinized Classical Swine Fever Virus (CSFV) was cloned into pTZ57R/T vector using the TA molecular cloning technique. Briefly, viral RNA was isolated using a modified RNA isolation protocol using Ribozol and RNeasy followed by cDNA synthesis using Clontech superscript RT. The cDNA obtained was used to synthesize 6.5 kb PCR products using (Long acting) LA-PCR. Gel purified PCR product was cloned with pTZ57R/T vector which was confirmed by RE digestion and partial sequencing. The same protocol can be utilized for cloning of similar sized amplified products in molecular biology laboratories.

Keywords : Classical swine fever, LA-PCR, TA Cloning.