Preparation And Tissues Expression Analysis Of Polyclonal Antibody Of Serine Protease Inhibitor5from Bombyx Mori

In this study, the serine protease inhibitor gene（serpin-5）of silkworm (Bombyxmori,"Daizo") utilizing cDNA derived from fat body of silkworm as template wascloned and analyzed. Prokaryotic expression system was used to get recombinant targetprotein, and the purified target protein was used as antigen to immunize KM mouse forthe sake of producing polyclonal antibody against serpin-5. The midgut, silk glands, fatbody, tuba Malpighii, testis and ovarian from the silkworm on the3rdof the5thinstarwere analyzed in tissue expression at the protein level. The BmN cells and silkworm onthe3rdday of the5thinstar were interfered using RNAi method, and the level of genetranscription was determined by quantitative analysis. The main results are showed asfollows:1.Serpin-5was amplified by PCR utilizing cDNA derived from fat body ofsilkworm on the5th instar as template. The sequence analysis displayed that theserpin-5gene with a length of1140bp coding379amino acids; the molecular weight ofthe predicted protein was42.6kD; and the isoelectric point was6.02. The targetSerpin-5(The GenBank accession: AY566165) was its open reading frame (ORF)without signal peptide sequence. It was supposed that the amino acid residues from1to16was signal peptide sequence after analyzing serpin-5by Signal P3.0Server.2.The Serpin-5gene without signal peptide was connected to the prokaryoticexpression vector pET-28a (+) and then transformed into E. coli BL21(DE3). And1mmol/L IPTG was used to induce the target protein to express. SDS-PAGE analysisindicated after recombinant transforming bacteria of serpin-5induced by IPTG. And thefive treated groups were: BL21(DE3), BL21(DE3) with empty pET-28a(+), BL21(DE3) not induced by IPTG, pET28a-serpin-5induced by IPTG, and supernatant aftersonication of pET28a-serpin-5induced by IPTG. The results showed that there was aspecific band with relative molecular weight of42.6kDa on PAGE profiles forpET28a-serpin-5induced by IPTG and supernatant after sonication of pET28a-serpin-5 induced by IPTG, in accordance with the molecular weight of serpin-5fusion protein. Itwas supposed that serpin-5gene could successfully express in E. coli and mostlyexisted in the form of inclusion bodies.3.The prokaryotic expression recombinant protein after being induced by IPTGwas subsequently purified by Ni column. The purified protein was employed as antigento immunize KM mouse. The titer and specificity of the acquired antibody wasvalidated by ELISA and Western blot, and its titer was1:24000.4.The results showed that serpin-5gene was highly expressed in testis and ovarian,which may be related to silkworm reproductive function. But,the expression in themidgut was lower, and lowest in the silk glands, tuba Malpighii and fat body.5.The designed siRNA fragments in BmN cells were filtered in order to get validfragments which had remarkable interference effect. The gene transcription level oftestis, ovarian, silk glands, fat body and blood at24h,36h,48h was determined byquantitative analysis after transfecting valid fragments to silkworm on the3th of the5thinstar. Compared to the control group,the results showed that transcriptional level ofmRNA in experimental group was no obvious changes testis, ovarian and fat body;however, reduced three times in blood and skin and about9times in tuba Malpighii.What’s more, the activity of phenol oxidase continued to rise as time went by, it rose1.5times at48h. As stated above, we speculated that the serpin-5may existed inhibitionrelationship with the phenol oxidase and may be involved in the innate immuneresponse of silkworm.This study we successfully cloned the cDNA of serpin-5, constructed a recombina-nt expression vector, carried on prokaryotic expressing research and the preparation ofpolyclonal antibody. Besides, the expression speculated of serpin-5protein in testis,ovarian, midgut, fat body, silk glands and tuba Malpighii were tested by polyclonalantibody, and the initial results we acquired was expected to be beneficial to thefurniture research of the function of serpin-5protein.