Location Of Fibroblast Growth Factor From Bombyx Mori Nuclear Polyhedrin Virus In Cells And Organs

Fibroblast growth factors were found in the vertebrates including human beings with multiple biological activities. Polyhedrin particles of Antheraea pernyi nuclear polyhedrin Virus (ApNPV) were purified from larva corpse of Anthraea pernyi. To understand more informations about the gene structures of the virus, the genomic DNA of ApNPV was extracted, and was digested by Hind III and Sal I , respectively. The fragments after digestion were inserted into vector. Three hundred positive clones were obtained and were sequenced. The result of homological analysis showed that an open reading frame is similar to the human Fibroblast Growth Factor gene. The fgf gene was found in the all of baculoviruses. Functions of the fgf gene in viral infection will be promoted. In order to find the function of the gene during the infection of baculovirus, the fgf gene of BmNPV was cloned, and prokaryotic expression plasmid, eukaryotic expression plasmid and baculoviruses expression plasmid were constructed, and the gene was expressed in these system respectively.The main conclusions are as followed:(1)The fgf gene from ApNPV was cloned into pUC18 plasmid. The coding region of this gene consists of 558bp nucleotides, and encodes a protein with 185 amino acid residues. This gene sequence has been submitted to the NCBI GenBank and the accession number is AY641527.(2)The fgf gene from BmNPV was cloned. The prokaryotic expression plasmid pET28a-fgf was constructed. The pET28a-fgf was transformed into BL21 and the gene was expressed in the formation of inclusion bodies induced by IPTG. The expression products with the weight of 27kDa fused with 6 X His ,were purified by Ni²⁺-NTA ion exchange resin.(3)The eukaryotic expression plasmid pCDN A3.1-gfp-fgf was constructed. The fgf gene products were located in the nuclei of the COS-7 cells. It showed that the protein was related to replication of DNA or affects trascription of genes.(4)The expression products of the fgf gene have different location in different cell lines. The protein was expressed in the cell membrane of TN5 and Sf9 cells, but it was expressed in the nuclei of BmN cells. Perhaps it is because that the fgf gene was cloned from BmNPV, and BmNPV only infected BmN cells.The gene is related to the infection of viruses, and to some specific cells as well.(5)Inject the 4th instar of silkworms with recombinant virus Bm-GFP-FGF, compared with silkworms with recombinant viruse Bm-GFP. Samples were obtained at variant time post injection, the protein expression was observed in the various tissues under a fluorescene microscope. The FGF fused with GFP was congregated in the blood, then in the trachea, the martensite, the middle part and rear end of silk gland. While in the silkworms injected with recombinant virus Bm-GFP, GFP was released in the late stage, and only at that time GFP was expressed in the trachea, the martensite, the middle part and rear end of silk gland. Hence, blood, trachea, martensite, middle part and rear end of silk gland are the targets of FGF. It played important role in the normal function of these organs.