Hemagglutinin (H) gene of canine distemper virus cloned in pTargeT mammalian expression vector induces neutralizing antibody response in dogs

Hemagglutinin (H) gene of canine distemper virus (CDV) was amplified by RT-PCR and cloned in pTargeT™ mammalian expression vector for subsequent expression and DNA vaccine studies. The RNA isolated from commercially available canine distemper vaccine was successfully amplified in RT-PCR assay using specific primers designed for amplification of complete ‘H’ gene of CDV. The recombinant pTargeT.cdv.h was constructed by cloning RT-PCR amplified CDV of ‘H’ gene in mammalian expression vector pTargeT™ in right orientation which was confirmed by restriction enzyme digestion. The E. coli DH5α competent cells transformed with this recombinant plasmid were grown in LB broth at 37°C in an orbital shaker overnight and the plasmid DNA was extracted and purified using ‘HISPEED Plasmid Maxi kit’ (QIAGEN, Germany). One hundred microgram of purified recombinant pTargeT.cdv.h was injected intramuscularly in thigh muscle of two months old dogs, while in control dogs pTargeT vector (without insert) was injected. Two dogs were kept as un-immunized healthy control. The dogs were bled 21 days post injection and sera were assessed for CDV antibody in serum neutralization assay which indicated that 1:40 dilution of pTargeT.cdv.h vaccinated dog sera neutralized 100 TCID₅₀ virus completely while vector DNA immunized and un-immunized healthy dog sera did not show neutralization of CDV indicating that the pTargeT.cdv.h construct is a potential candidate for CDV vaccine.