Cloning,Expression And Analysis Of Bmlipase-1 Gene From Silkworm In Pichia Pastoris

Slikworm,Bombyx mori is an important economical insect.The sericulture industry is an important economical industry in China.But,silkworm diseases seriously threaten the development of the sericulture industry.Bombyx mori nucleopolyhedrosis caused by Bombyx mori nuclear polyhedrosis virus is the most serious silkworm virus disease which is highly contagious and can easily cause large-scale outbreaks,in the sericulture industry.Therefore,controlling the infection and spread of BmNPV and improving the resistance of silkworm to BmNPV have important significance for the development of sericulture.The lipase of Bombyx mori(Bmlipase-1)is a digestive enzyme specifically expressed in midgut tissues of silkeworm.Bmlipase-1 can inhibit virus in the initial stage of BmNPV infection.In other words,it has antiviral effect in the first line of immune defense in silkworm,but its antiviral mechanism is not clear yet.Cloning,expression and study of Bmlipase-1 is helpful to clarify its antiviral mechanism.In this study,the Bmlipase-1 gene was cloned and the codon of Bmlipase-1 was optimized.We try to expresse Bmlipase-1 in vitro using the Pichia pastoris eukaryotic expression system.And the expression results were analyzed and discussed.The main experimental methods and results of this study were as follows: 1.Cloning and bioinformatics analysis of Bmlipase-1We used midgut tissues of 5th instar silkworm as the experimental materials to extract its total RNA by TRIZOL method.We obtained the first strand cDNA by reverse transcription using Random hexamer primers.By designing primers and PCR amplification of Bmlipase-1,we obtained the Bmlipase-1 fragment,which was transformed into E.coli DH5? then.We successfully cloned the complete CDS sequence of Bmlipase-1,and performed bioinformatics analysis on it.The length of Bmlipase-1 CDS sequence is 885 bp,which encodes 294 amino acids.Its theoretical molecular weight is 31.74 kDa,theoretical isoelectric point is 9.33,instability index is 23.13,aliphatic index is 76.90,and grand average of hydropathicity(GRAVY)is-0.373.Bmlipase-1is a stable hydrophilic protein and its secondary structure is dominated by irregular curls.2.Construction of recombinant Pichia pastoris strain(GS115/pPICZ?A-Bmlipase-1)We linked cloned Bmlipase-1 gene to the shuttle plasmid vectors pPICZ?A and electrotransformed recombinant plasmid into P.pastoris GS115,thereby integrating the Bmlipase-1 gene into the P.pastoris genome.By screening methanol utilization phenotype,PCR amplification and Sequencing,we obtained the recombinant P.pastoris strain successfully.Lipase activity assay of transformants using tributyrin emulsified plate showed that the colonies of recombinant strains were slightly larger than the control strains,but the difference in the size of the transparent circles was not significant.We speculated that the appearance of transparent circles was due to the secretion of lipase from yeast itself,and Bmlipase-1 was not expressed,or it had a low expression level.3.Codon Optimization of Bmlipase-1 GeneWe performed codon optimization and gene synthesis on Bmlipase-1,named after opt-Bmlipase-1.We changed the codon usage bias in Pichia pastoris by upgrading the CAI from 0.68 to 0.97.GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA.The Stem-Loop structures,which impact ribosomal binding and stability of mRNA,were broken.4.Expression and expression analysis of Bmlipase-1 after codon optimizationWe linked opt-Bmlipase-1 to the shuttle plasmid vectors pPICZ?A and pPIC9 K respectively to construct P.pastoris expression vectors pPICZ?A-opt-Bmlipase-1 and pPIC9K-opt-Bmlipase-1,and electrotransformed recombinant plasmid into P.pastoris GS115.By screening methanol utilization phenotype,PCR amplification and sequencing,we obtained the positive transformants successfully.We performed methanol-induced shake flask fermentation experiments on recombinant P.pastoris strains and fermented to 144 hours.Then,we used the TCA method to concentrate the fermentation supernatant and performed SDS-PAGE.The result of electrophoresis showed that The fermentation supernatant of recombinant had many miscellaneous proteins than that of the control group.One of the SDS-PAGE results showed that there was a suspected target protein band at 144 h of fermentation.But other results of SDS-PAGE showed that there was no obvious target band.Fermentation supernatant of the control group had little or no protein,and the band was very light.Western Blotting and Ni-NTA affinity chromatography did not detect the expression of the protein of interest.There were many factors affecting the expression of foreign proteins in P.pastoris.Then,we analyzed and discussed the expression results from its own characteristics of gene and fermentation conditions and so on.