Expression Of S1 Glycoproteins From Infectious Bronchitis Virus Isolates Js/95/03 And Sd/97/01 In Insect Cells And Evaluation Of Their Immunogenicity

The entire SI genes from nephropathgenic strain JS/95/03 and respiratory strain SD/9710 I of infectious bronchitis virus were amplified by RT-PCR. The PCR products were cloned into the cloning vector pMDI8-T. Theii insertions and orientations in pMD 18-T were confirmed by PCR and restriction enzyme analysis. The resultant two recombinant plasmids designated as pMDJS95O3SI and pMDSD97OISI were used for Si gene sequencing. The nucleotide and deduced amino acid sequences of Si genes from two isolates and 10 reference strains in Genbank were compared in computer with DNAStar software. The results showed that JS/95/03 and SD/97101 genetically closely related to M4l and H120, respectively. iS195103 shared 99.2% nucleotide and 98.0% amino acid identity with M41, and SD/97/0l shared 97.5% nucleotide and 99.3% amino acid identity with HI 20, so it is speculated that they were possible variants of vaccine strains. Two isolates were different in pathogenicity, but they were genetically close. They shared 97.5% nucleotide and 95.3% amino acid identity. and were same in the number of amino acids, and the number and position of cysteine residues and potential glycosy!ation sites. Only 23 amino acid differences were found between SI glycoproteins of two isolates, and 15 of them occurred in the region of the first 130 amino acid residues. On the either side of position 126, 6 amino acid differences were detected and might be involved in pathogenicity. æ…¬he comparison of the predicted secondary structures of S I glycoproteins from two isolates revealed that only one or a few amino acid differences were responsible for different antigenicity and structure. æ…’o construct recombinant baculovirus expressing SI glycoprotein, pMDJS9SO3SI or pMDSD97OIS 1 was digested with BamH I and Sal I. The digested fragment containing SI gene of JS195103 or SDI97IOI was ligated into the baculovirus transposing vector pFASTBAC THa, and the recombinant transposing piasmid designated as pFASTJS95O3SI or pFASTSD97OISI was screened by restriction enzyme analysis and then transformed into Escherichia co/i DH1OBAC containing bacmid (a bacuiovirus shuttle vector) for transposition. The recombinant 106 bacmid named rBacmidJS95O3Sl or rBacmidSD97OlSl was verified for Si gene insertion by electrophosis and PCR. After transfection of recombinant bacmid DNAs into cells of the insect Spodoptera frugiperda (SP)), two recombinant baculoviruses rAcJS95O3S I and rAcSD97O ISI were obtained. The lysates of cells infected with recombinant baculoviruses were analyzed by SDS-PAGE and expression products of SI gene from JS/95/03 or SD/97/0 1 were detected by western blot and immunofluorescence assay (IFA). The results showed the recombinant baculovirus is fully capable of expressing the SI gene of JS/95/03 or SD/97/01. Maybe owing to the incomplete glycosylation in insect cells, the SI gene products had an Mr of only 61,000. In immunofluorescence and western blot tests, two expression products could reacted with polycolonal antibody against IBV M4 I strain, indicating they were possessed of the antigenic properties specific for native SI glycoproteins. The lysates of insect cells infected with rAcJS95O3S1 or rAcSD97Ol SI were used to immune two-week-old commercial ISA cockerels. The immunogenicity of recombinant SI (rS I) glycoproteins were evaluated based on the results from antibody responses detected by enzyme-linked immunosorbent assay (ELISA) after immunizations and the protection tested by...