The Function Of BmNPV Orf98a And Orf99 Gene And Confirmation Of P74 Overlapping SORF

Baculovirus is a kind of arthropod virus with highly specific host requirements.With the deepening of scientific research, great progress is made in the study of baculovirus upon the expression of foreign gene, gene therapy and new insecticide. At present the most thorough study of baculovirus is Ac MNPV and Bm NPV. Through gene deletion studies, the basic function of many of the genes in the baculovirus has proven,but there are still parts of genes with unknown functions, especially some s ORF coding protein has a molecular weight of less than 100 amino acid residues. This study selected Bm Orf98 a, Bm Orf99 two genes from Bm NPV, of which the function is unknown, as the research object, using red recombination system to knockout Bm Orf98 a Bm Orf99 respectively. By comparing the differences of knockout recombinant virus and wild-type virus in the morphogenesis, replication and proliferation, the function of Bm Orf98 a and Bm Orf99 was defined, and the contents of molecular biology of Bm NPV were enriched.In addition, we have a new forecast to a s ORF and it overlaps with the P74 Bm NPV. Through experiments, we prove that s ORF can code protein,providing a new member for the Bm NPV gene family.1. Function of Bm Orf98 a geneThe Bm Orf98 a gene is located between the 94474-94647 nt in genomic Bm NPV,the total length of it’s ORF is 174 bp,coding 57 amino acids,weighing 6702 Da. Results of homology search show that Bm NPV, Ac MNPV and Bombyx mandarina NPV in the whole genome sequences of baculovirus signing in Gen Bank have Homologous gene encoding Bm Orf98 a. The consistency of Bm Orf98 a in different Bm NPV is 98-100%.The homology between Bm NPV and Ac MNPV reached 91%.The results of the phase analysis show that Bm Orf98 a is a late gene.Through Red restructuring system, we construct the knockout virus Bm NPV-Bm Orf98 a KO-polh-GFP,the repair virus Bm NPV-Bm Orf98 a RE-polh-GFP, wild type virus Bm NPVBm Orf98 a WT-polh-GFP and we transfect the DNA of Bm NPV- Bm Orf98 a KO-polh-GFP and Bm Orf98 a WT-polh-GFP to Bm N cells. The transfection shows the knockout Bm Orf99 can still proliferate. After virus DNA transfected from 24 to 72 hours, the culture supernatant of knockout virus transfected cells is slightly higher than control virus Bm NPV-Bm Orf98 a WT-polh-GFP at the BV level. After transfected for 72 hours, a little differences exist in terms of TCID50 of the former between them.Transfection of 6 to 72 hours, we can find Bm Orf99 knockout has no appreciable effect on the assemble of virus and the formation of polyhedron through transmission electron microscope. After knockout virus is infected for 72 hours, the expression of Bm Orf99,which locates in another chain of the Viral DNA and has partial overlap with the 3’end of Bm Orf98 a, increases by 2.61 compared with the control virus. All these above show that Bm Orf98 a isn’t necessary for replication. Lack of Bm Orf99 shows no obvious effect on the structure of virus and the level of BV. Besides, Bm Orf98 may raise the expression level Bm Orf99.2. Study on the function of Bm Orf99 geneThe Bm Orf99 gene is located between the 94540-94725 nt in genomic Bm NPV, the total length of it’s ORF is 186 bp,coding 61 amino acids,weighing7100 Da. Results of homology search show that there are only 8 kinds of viruses contine Bm Orf99 homologous gene. Analysis of homology comparison shows Bm NPV Orf99 has high homology with protein of Bombyx mandarina NPV, Rachiplusia ou MNPV, Autographa californica NPV and has a little homology with protein of other baculovirus.The results of evolutionary analysis based on the amino acid sequence show that Bm Orf99 has the closest genetic relationship with Ac122 protein. Indecating that Bm Orf99 isn’t the functional conservative gene of Nuclear multi angle virus and isn’t indispensable.The results of the phase analysis show that Bm Orf98 a is an advanced gene.Through Red restructuring system, we construct the knockout virus Bm NPV-Bm Orf98 a KO-polh-GFP, the repair virus Bm NPV-Bm Orf98 a RE-polh-GFP,wild type virus Bm NPV-Bm Orf98 a WT-polh-GFP and we transfect the DNA of Bm NPV-Bm Orf98 a KO-polh-GFP and Bm Orf98 a WT-polh-GFP to Bm N cells. Thetransfection shows the knockout Bm Orf99 can still proliferate. Bm Orf99 has no appreciable effect on the assemble of virus and the formation of polyhedron.After transfected for 72 hours, TCID50 of the former is slightly higher than that of the l atter. Transfection of 6 to 72 hours,knockout virus transfected cells is slightly higher than control virus at the level of virus DNA. After knockout virus is infected for 72 hours, the expression of Bm Orf98 a increases by 2.21 compared with the control virus.All these above shows that Bm Orf99 is the early gene of virus and isn’t necessary for replication. Lack of Bm Orf99 can encourage replication and increase the level of BV without affecting the structure of virus. Besides, Bm Orf99 may work through the mediation of other genes of virus.3. Confirmation of overlapping s ORF with P74 geneIt is predicted that there is a s ORF encoding 37 amino acid residues in the P74 Bm NPV gene. The DNA sequence was cloned into the prokaryotic expression vector p Easy 、DE3、 and is induced expression by IPTG. The results of SDS-PAGE and blotting Western shows Bm Orf P74 s ORF was expressed. We prepare anti Bm P74 s ORF polyclonal antibody using recombinant protein immune mice. Cellular immune fluorescence results show Bmp74 s ORF is a small peptide coding gene and it’s expression product is mainly located in the nucleus of Bm N cells. The results of transcription analysis showed that s ORF Bmp74 was the early gene.