Cloning and expression of Equine herpesvirus 1 glycoprotein D as a fusion protein in E. coli from a local isolate from El Zahraa Stud for Arabian Horses to be used as a diagnostic antigen for detecting the antibodies either in vaccinated or infected horse
Cloning and expression of Equine herpesvirus 1 glycoprotein D as a fusion protein in E. coli from a local isolate from El Zahraa Stud for Arabian Horses to be used as a diagnostic antigen for detecting the antibodies either in vaccinated or infected horse
Abd El-Hamid, M.I1, Seham, A.El-Zeedy1, El-Sanousi, A.A2, Reda, I.M2, Nehal, S. Saleh1, Abbas, A.M1
ABSTRACT
The current study focuses on The envelope glycoprotein D of EHV-1 (EHV-1gD) due to its essential role in virus infectivity and its function in entry of virus into cells and is considered as one of the most potent inducers of virus-neutralizing antibody among the spectrum of EHV-1 proteins .A wave of Abortions has been recorded in El Zahraa stud for Arabian Horses ,collected samples were either aborted fetal tissues either Lung and livers as well as Aborted placenta and nasal swaps from mares suffering from repeated Abortions, The current study used the gene synthesis technology introduced by Invitrogen to synthesis the glycoprotein D of the Kentucky Strain as reference strain to use it as appositive control for the envelope glycoprotein D of EHV-1 (EHV-1gD) cloned in pMA-T plasmid for optimizing the detection of the virus from field samples by PCR using 2 sets of primers one of them includes the whole length of the gene about 1209 bp while the nested one from the start ATG is 1000 bp ,seven out of nine samples have been reported positive by the 2 sets of primers, then the specific band at the expected size of the whole length of EHV-1 (EHV-1gD) gene of the local isolate was sent for sequence analysis, multiple alignment revealed single nucleotide substitution at the base pair number 121 from the start codon which give lead to single Amino Acid Substitution from CAG (Glutamine(Gln/Q)) which is considered as a polar Amino Acid to AAG (Lysine(Lys/K)) which is a basic Amino Acid, then the sequenced product of the local isolate was cloned Into PET 151 D topo plasmid with an N-terminal tag containing the V5 epitope and a 6xHis tag and transformed into Top 10 Ecoli cells just to maintain the stability and propagation of the cloned gene then the cloned plasmid isolated to be transformed again in Ecoli BL21 cell which include the T7 promotor for expressing the glycoprotein D, western blotting carried out on the induced culture at different expression times using the alkaline phosphatase labeled N-terminal anti histidine monoclonal antibodies revealed single bands at the expected size, the same samples have been reacted with serum collected from aborted and infected mares revealed band at the expected site using anti equine IgG labeled horse radish peroxidase, concluding that the expressed envelope glycoprotein D of EHV-1 (EHV-1gD) could be used as a good candidate for manufacturing of diagnostic Antigen for detection of the circulating antibody either in vaccinated or latent infected horses.
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