| Silkworm pebrine caused huge economic losses to China’s sericulture every year. Its pathogen is Nosema bombycis, which belongs to microsporidia and kill silkworm larva by systemic infection. Studies on molecular pathogenesis of N. bombycis are important to provide more solutions to prevent pebrine. Serpin is a protein superfamily with serpin domain. Many serpins exist in blood as proteases inhibitor. However some of serpins don’t have the inhibiting function, play roles as storage protein, energy transporters, etc. Serpins have played important roles in the fields of medicine and immunology. N. bombycis encodes4479genes, including a cluster of serpin genes. Analyses of comparative genomics showed that serpins only exist in Nosema genus. There are19serpin genes predicted in N. bombycis genome, their roles in infection and proliferation processes are still unknown. Here, we first obtained the protein of rNbSerpin6, rNbSerpinl3and r△NbSerpinl3(without the signal peptide) through the baculovirus expression system; meanwhile, we verified the inhibitory activity of r(△)NbSerpinl3to trypsin in vitro.1. Domestication of Sf21cells and construction of recombinant bacmidSf21cells are semi-adherent cells, used SFM900Ⅱ, serum-free medium to culture Sf21cells. Optimization of culture conditions, we got the optimal cultural condition as28℃,120r/min and4×106cells per milliliter. Inserted NbSerpin6and NbSerpinl3into the donor vector pFastBacHTb respectively and got recombinant plasmids pFastBacHTb-NbSerpins. Then transformed pFastBacHTb-NbSerpins into E. coli DH10competent cells by the shutter vector and recombined the target genes to the bacmid of AcNPV by transposase expressed by transformed gene. Finally, we got recombinant bacmid with reliability higher than99%by blue-white spots and three antibiotics screening. Mass cultured recombinant strains to prepared the recombinant bacmid with transfection level.2.Baculovirus expression and protein purification of NbSerpin6and (△)NbSerpinl3Rcombinant bacmid of NbSerpins with transfection level transfected Sf21cells with1μg per well in six-well plates. After96h post-transfection, we collected P1generation virus. Then infected Sf21cells with P1generation virus in the25square centimetres cell culture bottles and collected P2generation virus after72h post-infection. Detected P2generation virus titer in six-well plates and infected P2generation virus to Sf21cells in250mL bottles for6multiplicity of infection (MOI) Three days after infection, we collected P3generation virus supernatant and cells precipitation, then inoculated P3generation virus to the Sf21cells in500mL bottles for protein expression. Collecting cells precipitation after60h post-infection, NbSerpin13and NbSerpin6were purified with His-affinity chromatography. SDS-PAGE and Western bloting analysis confirmed that we had obtained recombinant proteins by baculovirus expression system.3.Analysis of inhibitory activity of NbSerpinsInhibitory activity of NbSerpinl3, NbSerpin6was analyzed in system as follow: purified protein of NbSerpins as inhibitors, commercial trypsin and protease K as substrates, casein as protease’s substrate, PMSF as positive control and no inhibitor as negative control, we detected inhibition efficiency respectively. The results show that (△)NbSerpinl3have significant inhibition activity for trypsin, however there is no inhibitory activity for protease K. The inhibitory activity ratio for trypsin of NbSerpinl3to PMSF is0.41and△NbSerpinl3to PMSF is0.51. NbSerpin6has no inhibiting activity to both trypsin and protease K. |
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