Cloning and expression of african horsesickness virus genome segment 10 in E-COLI
Abstract: The ten double stranded RNA genome segments of AHSV type 9 PI were extracted from Vero infected cells. The genome segment 10 (S10) underwent exponential amplification through applying Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR).
The primers used were designed so as to posses excess bases for restriction digestion with Xhol and Bam HI at the 5’ and 3’ termini respectively. Cloning of the amplified gene was carried out into the pET 15b expressing vector that was digested also with the same enzymes. Gel purification was applied before ligation using GeneClean protocol to to get rid of the excess bases that created from the restriction digestion. The pET 15 b system has its own translation signals and Histidine residues.
The clone was then transferred into E-coli competent cells ( The expression host) BL21 (DE3) containing a chromosomal copy of the T7RNA polymerase under the control of Lac UV5 promoter (Novagen). The expression was induced by the addition of Isopropyl ß-D thioglactopyranoside (IPTG). The protein extraction was done through lysing the bugs (bacterial cells) by mixing with non-ionic detergent. One step Rapid affinity purification with pET His. Tag system (Novagen) was manipulated in order to get NS3 in a semipure form. Further purification was achieved through passing the protein product in the recharged chelation resin. The presence and the right molecular weight of NS3 were assessed by running the product in PolyAcrylamide gel Electrophoresis (PAGE).
This study proved that NS3 could be obtained by expression of S10 in E-coli and the resultant protein could be successfully used as serogroup diagnostic antigen through coating plates for applying the indirect ELISA technique for the detection of AHS-NS3 polyclonal antibodies in serum of AHS infected horses particulary during the extremes.
Publication year 1995
Pages 102-108
Availability location معهد بحوث الامصال واللقاحات البيطرية
Availability number
Organization Name
City cairo
serial title the egyptian journal of immunology
ISSN 1110-4902
Author(s) from ARC
Agris Categories Animal diseases
Proposed Agrovoc African horsesickness virus;Horsesickness virus;
Publication Type Journal
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