Abstract
Results of cloning X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate and analysis of their nucleotide sequences are presented. Obtained clones were added to the previously constructed clone library comprising clones of 8 genes of Krasnodar 06/12 isolate. Clones containing X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate will be used for recombinant protein obtaining and testing for their effect on in vitro virus reproduction and their role in the virus infectivity, level of clinical manifestations and virulence. Prokaryotic vector, pJET1.2/ blunt, was used. Thus, the clone library available at the FGBI “ARRIAH” Reference Laboratory for African swine fever was supplemented by pJET1.2-X69R, pJET1.2-A179L, pJET1.2-E248R, pJET1.2-I215L and pJET1.2-DP96R plasmid constructions containing 5 genes of ASF virus Krasnodar 07/17 isolate. Proportion of cloned virus genes was 3.01% of Krasnodar 07/17 isolate genome, hence, total amount of the clone library has reached 7.82%.
Highlights
Construction of representative DNA clone libraries is an efficient approach to the development of up-to-date diagnostic tools and recombinant vaccines
Cloning of African swine fever virus (ASFV) genes and analysis of their nucleotide sequences included several stages: 1. Primer design Optimal sites for primer design were defined basing on the publications and analysis of X69R, A179L, E248R, I215L and DP96R on the Georgia 2007/1 ASFV genome physical map (Fig. 1)
The selected oligonucleotide primers allowed for amplification of the ASFV full-size genes X69R, A179L, E248R, I215L and DP96R that code transmembrane proteins and proteins affecting the virus virulence and for determination of their nucleotide sequences for Krasnodar 07/17 isolate
Summary
Construction of representative DNA clone libraries is an efficient approach to the development of up-to-date diagnostic tools and recombinant vaccines. The gene clone library is represented by the selection of recombinant plasmids (or bacterial cell clones comprising them) bearing different genes of specific host. The first gene clone library was constructed by F. D. Kollnberger et al screened the library of expressing genes of African swine fever virus (ASFV) using polyclonal antisera (from pigs recovered from the infection with virulent ASFV) and identified 14 serological immunodeterminants including non-structural proteins (pB602L, pC44L, pCP312R, pE183L, pK145R and pK205R), structural proteins (pA104R, p10/pK78R, p30/pCP204L, p54/pE183L, p72/pB646L) and the following viral enzymes: ribonucleotide reductase (Fp334L, pF778R), DNA ligase (pNP419L) and thymidine kinase (pK169R) [9]
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