Helicoverpa Armigera Single Particles Embedded Nuclear Polyhedrosis Virus Of P40, Eng Cloning And Chi Genes In E. Coli And Insect Cell Expression

The genomic DNA library of plaque-purified Cl clone of Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) was constructed. Two ORFs encoding p40 and chitinase, respectively, were identified and analysed. Furthermore, expression of the chitinase gene both in E. coli and insect cell was also conducted in this paper.HaSNPV C1 clone was propagated by infecting third instar //. armigera larvae with the viral polyhedra. Genomic DNA was extracted from purified polyhedra. Digestion of genomic DNA with 7 restriction endoriucleases( BamH I, EcoR I, EcoR V,Hind, Pst I, Xba I and Xho I) resulted in 11,19, 22, 13, 6, 16 and 5 fragments (longer than 0.8kb), respectively. Length of these fragments was calculated by comparing with A DNA/Hind III marker, and the genomic DNA length was predicted about 130 kb. Based on the restriction analysis, the genomic library was constructed by clonning the fragments produced by digestion of the genomic DNA with Hind III,Xba I, Xho I,HindIII+Xba I, HindIII+PstI, HindIII+XhoI.The HaSNPV HindIII-H , J fragments clones encoding a Helicoverpa zea nucleopolyhedrovirus(HzNPV) p40 like gene were screened by restriction analysis and terminal sequencing methods. The HaSNPV p40 gene exhibits 98% identity with HzSNPV p40 gene at the nucleotide sequence and has a conserved baculovirus late transcription strart motif ATAAG. The HaSNPV p40 is potential to encode a protein of predicted size 36. 58 kD that has about 55% identity with BmNPV/p40 and AcMNPV gp41. Comparing HaSNPV p40 residue 28-277 region which have a hydrophilic domain with SeMNPV, LdMNPV, AcMNPV, BmNPV and OpMNPV homologue,they have above 60% similarity. Among 7 predicted protein,3 cycteines are conserved.A phylogenetic tree of 7 baculovirus p40 gene was drawn by using Genetyx programe .The result showed that the evolutionary rate of p40 genes is in some degree different to that of polyhedrin genes.The HaSNPV HindIII-E fragment (10.6 kb) was cloned and sequenced. 9 ORFs, which are initiated with a methionine codon and encode a polypeptide of at least 50 amino acids,including the ORF coding the chitinase, were identified. The chitinase gene encoded a predicted 65.5 kD peptide which had 91%, 66% and 55%identity with HzSNPV chitinase, AcMNPV chitinase and Serratia marcescens chiA, respectively. The putative catalytic domains of HaSNPV chitinase and Serratia marcescens chih share 78% identity and 86% similarity. A signal peptide and endoplasmic reticulum(ER) location motif were identified by comparing with HzNPV chitinase gene.The complete chitinase gene (chi) and that deficient putative signal peptide encoding region (chis-} were amplified from HaSNPV Hindlll-E fragment by using PCR method. These two target genes were inserted into the expression vector pET28a, under the control of phage T7 promoter and with a His tag in their N terminal, respectively. Then the recombinant vectors were transformed into ?coli (DE3) for expression. An expressed band of about 60 kD was identified by SOS-poly acrylamide gel electrophoresis and further confirmed by Western blot.chiS- gene was inserted into donor plasmid pFASTBACHTa under the control of Ph promoter and flanked by the left and right ends of Tn7. The target gene was transposed to the target Bacmid in ?coli (1)1110), by Tn7 transposition function. Recombinant Bacmid was screened by intra-allelic complementation and antibiotic resistance methods and then transfected into Tn-5Bl~4 cell mediated by Lipofectin . An expressed protein band of 60 kD was determined by SDS-PAGE.