Cloning of F gene of Newcastle disease virus HeB02 isolate and the study of its DNA vaccine / 生物工程学报

ABSTRACT

In order to amplify F gene of NDV HeB02 strain, one pair of primers was designed according to the GenBank sequence, and a 1.66 kb F gene fragment was obtained by RT-PCR. Sequence analysis indicated that the homologies of the nucleotide sequence of HeB02 strain to those of F48 E9, La Sota and Clone30 strains were 88.1%, 84.9% and 83.8% respectively. The expression plasmid pSV-F was constructed by inserting the F gene into the pVAX1 vector, and transfected into the cultured COS 7 cell line via liposomes. The specific 5.9 kD protein was detected by SDS-PAGE and the immunogenicity of the expressed F protein was confirmed by Western blot, ELISA and neutralization test. 3 week-old SPF chickens were subcutaneously immunized twice at week 0 and 3 with 50 microg DNA of plasmid pSV-F by electroporration. 5 weeks later, all chickenss were challenged with 100 x EID50 of NDV HeB02 strain, 1 week post challenge all chickenss were sampled by larynx swabbing to isolate virus and the HI level of NDV was measured. The results indicated that the virus isolation was negtive in all vaccinated chickenss and positive in all control chickens. The HI titres reached to 8.3log2 +/- 1.30 and 7.2log2 +/- 1.23 induced by NDV vaccine and positive cells (pSV-F), respectivily, the HI titres induced by Control cells (pVAX1) was not detected. Furthermore, the HI titres reached to 9.8log2 +/- 1.55 and 8.9log2 +/- 1.77 in vaccinated group with NDV vaccine and positive cells (pSV-F), respectivily, were sinificantly higher than that of the control cells (pVAX1) immunized group( HI titers was 3.0 log2 +/- 1.40, P < 0.01) after challenge. These results showed that the plasmid pSV-F could be as a candidate of DNA vaccine to provide protective immune response against NDV infection.