The Research Of Serpin-3from Bombyx Mori And The Interacting Proteins Of Bmserpin-3

Serine protease inhibitor gene families,involved in regulating physiological responsesof body.In order to study the serine protease inhibitors and the related factors worktogether to participate in the immune response,the serine protease inhibitorgene（serpin-3）of silkworm utilizing cDNA derived from fat body of silkworm as templatewas cloned and analyzed.The ORF of serpin-3was expressed by Prokaryotic expressionsystem, and the purified target protein used to study serpin-3could inhibit melanization inBombyx mori plasma. we performed pull-down assay and Mass spectrometry analysis toidentify two potential interacting proteins of Bmserpin-3from Bombyx mori blood.Theregulation of Bmserpin-3on these two proteins was explored by RNA interferenceassay.The main results are as follow:1Cloning and sequence analysising of serpin-3gene of Bombyx moriSerpin-3（The GenBank accession: NP₀01040318.1）was amplified by PCR utilizingcDNA derived from fat body of silkworm as template. The sequence analysis displayedthat the open reading frame of Bmserpin-3gene with a length of1311bp coding458amino acids; with a predicted signal peptide consisting of the first22residues. Thecalculated molecular mass and isoelectric point of the mature protein were49.33kD and5.28, respectively. Amino acid residues at the position of395-416were predicted as thereactive center loop, and the residue at position410of Lys was the predicted P1site.2Prokaryotic expression of serpin-3from Bombyx moriThe Serpin-5gene was connected to the prokaryotic expression vector pET-28a（+）,transferred to Escherichia coli BL21cells. Protein expression was induced by0.5mMIPTG. SDS-PAGE analysis indicated, a specific band at49kD was observed in theexperimental group, but not in the negative group,such as BL21（DE3）, BL21（DE3） withempty pET-28a（+）, BL21（DE3） not induced by IPTG.It was supposed that serpin-3genecould successfully express in E. coli,and purificated it.3Bmserpin-3protein regulating melanization reaction of Bombyx mori The amino acid sequence of Bmserpin-3was aligned with other previously identifiedinsect serpins that are involved in melanization reaction,such as Drosophilamelanogaster,Manduca sexta,Anopheles gambiae,the results showed that Bombyx moriserpin-3mature protein revealed the highest similarity with amino acid sequence of them.We measured protein concentration and PO activity in Bombyx mori larval hemolymphafter LPS treatment. The result showed protein concentration was increased compared tonative,and PO activity revealed an obvious increase after LPS injection, When theBmserpin-3was added to Bombyx mori larval hemolymph after LPS treatment, the POactivity revealed a decrease.Meanwhile,an obvious dose-dependent relationship betweenPO activity and Bmserpin-3was observed, Therefore, Bmserpin-3may be involved inmelanization reaction.4Identification of Interacting Proteins of Bombyx mori Serine Protease Inhibitor3（serpin-3）The prokaryotic expression recombinant protein after being induced by IPTG wassubsequently purified by using the dialysis purification technology. The purified proteinmolecular weight size consistent with the prediction by SDS analysis, and the purity oftarget protein is very high.We performed pull-down assay to identify two potentialinteracting proteins of Bmserpin-3from Bombyx mori blood using prokaryoticallyexpressed His-Bmserpin-3.They were identified as silkworm storage protein andsex-specific storage-protein2precursor by mass spectrometry by Mass spectrometryanalysis. Mainly involved in immunity, cell apoptosis,and so on.5The research of serpin-3of Bombyx mori target proteinThree siRNAs targeting different regions of Bmserpin-3mRNA were transfected intoBmN cells, screening for interference siRNAs effect is remarkable.Then the siRNA witheffective knockdown of the expression of Bmserpin-3were injected into day3fifth instarsilkworm larvae.Then The mRNA expression of sp2and sp3was detected by Q-PCR. Theresult showed the mRNA expression of sp2was significantly reduced when the expressionof Bmserpin-3was knocked down, while sp3’s mRNA expression was barely changed.These results indicate that serpin-3doesn’t affect Bmserpin-3at mRNA level,may regulateprotein expression of sp3at translation levels or after translation levels.This study we successfully cloned the cDNA of serpin-3,expressed and purified ofserpin-3.We used the expression of purified protein to identificated the interaction protein, the study found that Bmseripin-3to participate the melanization reaction.