CLONING AND SEQUENCING OF GENOME SEGMENT 10 OF AFRICAN HORSESICKNESS VIRUS TYPE 9

Document Type : Research article

Authors

Abstract

African horsesickness virus, Type 9 Plum Island isolate, (AHSV, T9 PI) was p < /strong>ropagated on VERO cells till reaching 70% CPE. The double stranded (ds) RNA genome was extracted using the Phenol Chloroform Method,
 
CLONING AND SEQUENCING OF GENOME OF AHS VIRUS
(FAVALORO et al, 1980), CDNA of S10 was obtained by applying the Reverse Transcryptase (RT) reaction. The exponential amplification of S10 cDNA from the dsRNA genome was achieved through performing the Polymerase Chain Reaction (PCR) using 2 primers designed in accordance with the published sequence of the cognate gene. Ligation of S10 to the PCR TM11 vector as well as Transformation of the "One Shot" competent cells (NV, F) with Such a p < strong>lasmid was carried out following the instructions of the TA cloning Manual (Invitrogen). The assertiveness of the proper ligation and transformation was assessed by applying the minilysate preparation protocol and checked up in 1.2% agarose gel electrophoresis for the right size of S10 gene ( ~700 bp) For obtaining an adequate amount of plasmid BNA needed for sequence analysis of S10, PEG plasmid preparation protocol was performed. The nucleotide sequence of S10 was determined by using version 2.0-DNA Sequencing Kit based on the strategy of the Dideoxy Termination Reaction (SANGER et al 1977). This study proved that there is a high degree of homology in the nucleotide Sequence of AHSV-T9 PI, S10 and the published sequence of the cognate gene so that it could be of diagnostic value. It is also a preliminary step for further investigations for the role of, s10 gene in AHS virus replication as well as its pathogenesis. This work was accomplished in Plum Island laboratories (USA) and the associated authors shared in.

Keywords

Files

Share

How to cite

Statistics