Studies On The Molecular Cloning, Expression And Characterization Of 3 Protein Genes From Insect

With the development of the study on the life sciences,such as molecular biology and genetic engineering technology,it has become possible to make the research of the insects in medical use in a molecular level.In this study,we propose the molecular cloning,expression and characterization of 3 insect protein genes,implementing 2 aspects as follows.1.Molecular Cloning,expression and Chzracterization of AgCRP gene from the mulberry longicorn beetle,Apriona gemariThe gene AgCRP,which was screened from the ESTs of the larvae of mulberry longicorn beetle,was amplified by RT-PCR.The result of sequencing showed that the full length cDNA of AgCRP was 207bp encoding a 69 amino acid polypeptide.The indexing of NCBI database and the alignment of other amino acid sequences results showed that the mutual peptide of AgCRP has a amino acid sequence identity of 62%with the anti-bacterial gene AlCRP from Acalolepta luxuriosa,and has a CSαβ(cysteine stabilizedαβ)motif of "C…CXXXC…C…CXC" consensus sequence,which is similar to other insect defensins.AgCRP gene has been submitted to GenBank as a first reported sequence(Accession number:AY771360).Northern blot analysis revealed AgCRP exhibited fatbody-specific expression and was up-regulated by wounding,bacterial or fungal challenge.Thus,it is belong to the insect defensin family.The restriction fragments of AgCRP by EcoRⅠand XhoⅠwas inserted into the same multiple cloning sites of the pBacl to construct a transfer vector pBac-CRR Then,through the cell transfection agent Lipofectin,the pBac-CRP was cotransfected into Sf-9 insect cell with bAcGOZA bacularvirus DNA.The cell showed the infection morphology obviously,which suggested the construction of recombinant virus is successful.Meanwhile,it is the first time to express AgCRP in bacularvirus-insect cell expression system.2.Molecular Cloning,expression and Characterization of BiVP and BiLP gene from the bumblebee,Bombus ignitusThe gene BiVP and BiLP geng,which were screened from the ESTs of the bumblebee, were amplified by RACE-PCR and RT-PCR.The result of sequencing showed that the full length cDNA of BiVP and BiLP were 1083bp and 954bp encoding the polypeptides of 360 and 317 amino acid,respectively.The indexing of NCBI database and the alignment of other amino acid sequences results showed that the BiVP gene belong to the serine protease family for containing a conserved catalytic triad activity site formed by His,Asp and Ser conserved residues,and the BiLP gene was thought to be a lipase for having a lipase protein characteristic motif of "GXSXG" consensus sequence,respectively.Both gene are first reported sequence.The BiLP gene has been submitted to GenBank(Accession number: EU443786).Northern blot analysis reveal both BiVP and BiLP genes exhibited fatbody specific expression.The restriction fragments of BiVP and BiLP by XhoⅠand BamHⅠwere inserted into the same multiple cloning sites of the pBac1 to construct a transfer vector pBac1-BiVP and pBac1-BiLP,respectively.Then,through the cell transfection agent Lipofectin,the pBac1-BiVP and pBac1-BiLP were cotransfected into Sf-9 insect cell with bAcGOZA bacularvirus DNA.The cell showed the infection morphology obviously,which suggested the construction of recombinant virus is successful.Meanwhile,it is the first time to express BiVP and BiLP in bacularvirus-insect cell expression system.The recombinant BiVP was purified by fast protein liquid chromatography with about 51 times purification degree and 57.8%yield.During the determination of enzyme activity,the recombinant BiVP is found to has a optimal pH value of pH3.0,a stable enzyme activity over a wide range from pH2.0-pH5.0,a optimal temperature of 45℃,and a stable enzyme activity for at least 10min at 45℃.It is the first time to report the determination of recombinant BiVP enzyme activity.