Exoression And Purification Of Bombyx Mori Sensory Neuron Membrane Protein Gene BmSNMP By Fusion With Baculoviral Polyhedrin

In the process of responding to chemical signals from the environment, the olfaction of insects plays a very important role on the behaviors including the choosing of habitat, foraging, information transmission and seeking for mate, oviposition site, host and quarry, etc. Sensory neuron membrane protein （SNMP） is one of the important proteins involved in the odor recognization of insects, and belongs to CD36 gene family in human gene, which is the only gene of CD36 family found in neurons. For a long time, the olfaction of Bombyx mori, specially the imago, is the good model for studies on chemical communiscation of insects.A new gene was screened from the cDNA library of silkworm pupae in our laboratory. We analyzed the sequence by bioinformatic tools and found that it contained an open reading frame （ORP） of 1569 bp encoding 522 amino acids with a predicted molecular mass of 56.76 kD and the isoelectric point was 8.58. The predicted protein contains a conserved domain of CD36, and it has high homology with SNMP protein of many other species, so we named it BmSNMP（Bombyx mori SNMP） gene and its accession number in Genbank is NM₀01043721. The ORP of BmSNMP gene was amplified by PCR and constructed the prokaryotic expression vector pET-32a（+）-polh-tev-BmSNMP.The recombinant plasmid was transformed into E.coli BL21（DE3）. After induced with IPTG, the recombinant fusion protein was high level expressed. We used differential centrifugation, differential pH buffer and Ion-exchange chromatography to purified fusion protein Polh-tev-BmSNMP based on the character of polyhedrin. The fusion protein Polh-tev-BmSNMP was digested by TEV protease. We constructed the baculovirus transfer vector pFastBac KTb-polh-BmSNMP, and the recombinant transfer plasmid was transformed into the Bacmid DNA by homologous recombination successfully （the recombinant Bacmid was named Bacmid-polh-tev-BmSNMP）. We infected BmN cells with Bacmid-polh-tev-BmSNMP by liposome and got the recombinant virus of vBmpolh-BmSNMP. The recombinant virus was amplified and infected the normal BmN cells. The expressed recombinant protein was identified by Western blotting. By the same method, we constructured the recombinant virus vBmpolh-BmSNMP-EGFP. After infected the normal BmN cells, we found the green fluorescence distributed around the membrane of cell showing that the recombinant BmSNMP could be expressed in eukaryotic cells as a membrane protein.The transcription level of mRNA in different tissues of fifth instar larva was also analyzed by the real-time fluorescent quantificational PCR, and found that the transcription of BmSNMP Gene gradually decreased from fat body to epidermis. All the results above will provide us important evidences for further studies on the function of BmSNMP. What’s more, RNA interference was applied to silence the target mRNA followed by detecting the cell condition with flow cytometer. The results suggest the interfered cells were decreased in the S-phase of the cell cycle. Meanwhile, the gene BmSNMP fusion with polh seemed likely to regulation of the cell cycle.