Sequence Analysis And Expression Of The Nucleoprotein Gene Of Avian Infectious Bronchitis Virus

Two pairs of primers for amplifying the nucleoprotein gene of avian infectious bronchitis virus (IBV) were designed. With the specific RT-PCR, the nucleoprotein(NP) genes of four IBV Chinese isolates, XL HD DB and DC, were amplified and cloned into the plasmid vector pUC19 at SinaI site. The generated four recombinant plasmid designated as pUC 1 9-XJ-N pUC 19-DC- N pUC 1 9-HD-N. pUC 1 9-DB-N were identifided harboring NP gene by PCR restriction endonuclease digestion and sequencing. By CLUSTAL analysis software, the NP genes and deduced amino acid sequence of four Chinese isolates were compared with those published ten IBV strains. The result indicated that the four Chinese isolates can be classified to the same genotype with N 1/62 strain and VICS swain. DC strain was more divergent than the other three isolates. The homology of the nucleotide of the NP gene between these 14 strains is 86%-98%, and the homology of the amino acid sequence is 88%-97%. The complete coding region of NP gene of DB strain was then reaniplified from pUC19-DB-N by PCR., and subcloned into baculovirus transfer vector pBlueBacHis B. The recombinant baculovirus transfer vector containing NP gene in correct orientation was designated as rBac-DB-N. The transfer vector was used to cotransfect insect cell Sf9 with linearized baculovirus DNA mediated with CellFectin reagent. The recombinant baculoviruses were generated and by three cycle of plaque cloning with the chromogenic substracte X-gal in agarose overlay. 2Kb fragment was amplified from the recombinant baculovirus DNA with primers Npalb, which confirmed that the nucleoprotein gene was integrated into baculovirus genome. The expression of NP in Sf9 cell was determined by Western blot with polyclonal antibodies against IBV. The infected cells were collected at different time post-infection, analyzed by SDS-PAGE, the result showed that the expressed product had a molecular mass of approximately 56KDa, the expression level of the recombinant proteins was up to 19.4% of the total cell protein.