On The Function Of The ORF44 Gene Of Helicoverpa Armigera Nucleopolyhedrovirus: A Putative Functional Gene

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is a kind of large doublestranded cycle DNA virus, great progress has been made in its molecular and functional research. Open reading frame 44 (orf44) is a conserved gene, which function is unknown. This paper reported the functional anaysis of orf44.Bioinformatic anaysis shows that orf44 is 1137bp long, which encodes a putative protein, of 379 amino acides with a predicted molecular size of 42KDa. The protein was named Ha44. A late baculoviral transcription initation motif ATAAG was found 80nt upstream of the putative translational start site and an early baculoviral transcription initiation motif TATA was found 88nt upstream of the putative translational start site. The results of PSI-BLAST displayed that there were some homology sequences with Ha44, and all of them belong to group II NPV or Granulovirus(GV), but the functions of all the proteins were unknown, so the research of orf44 function is very important.With egfp as report gene, cloned egfp and or/44 into vector pIZ/V5-His, and transfected the recombinant plasmid to HzMA1 to get the fusion-expressing protein egfp-or/44. Fluorescence and laser scan confocal microscopy observation show that Ha44 was located in nucleus, it may act as an important functional protein in virus replication and proliferation.In order to know the function of or/44, we constructed the recombinant HaSNPV with or/44 deletion in E.coli BW25113. In this part, we designed a pair of primer, which contained the homology regions of orf44, and get the fragment P_hsp70-egfp-SV₄₀-Cm^r by PCR, which was used to recombination. The recombination system is pKD46, containingλphage recombinant system, and HaBacmid which is the bacterial artificial chromosome of HaSNPV. This recombinant will be used in the future researchs in the insect cells.Ha44 was express in E.coli BL21 with vector pGEX-KG, which moleculer weight is 68KDa. The fusing protein GST-Ha44 was purifed by SDS-PAGE, and used to generate antibodies in rabbit. Characterized through ELISA and Western-blot, the antibody of Ha44 was prepared for the future anayasis.