Cloning And Sequence Analysis And Expression Of Serpin-6 Gene In Bombyx Mori

In this study the head,midgut,fat body,silk glands,testis,ovarian and haemolymph were collected from Day-3 fifth instar silkworm(Bombyx mori,"Daizo") larvae.The gene of Serpin in Bombyx mori(Serpin-6) was cloned by PCR using fat body cDNAs as templates. The Semi-quantitative analysis was carried out by using cDNA of head,midgut,fat body,silk glands,testis,ovarian and haemolymph as templates.For another semi-quantitative analysis,fat body and haemolymph were collected from Day-3 fifth instar larvae injected with LPS at different periods.E.coli prokaryotic expression system was used for expression study of serpin-6 gene.The main results are as follows:1.Expected Serpin sequence of Bombyx mori including full-length ORF was obtainded by gene splicing,following BLAST aligning the Serpin conserved sequence and Bombyx mori EST database.According to the sequence,proper primer was designed. Afterwards,taking it as primer and mRNA extracted from fat body of Day-3 fifth instar "Daizo" larvae as templates,Serpin-6 gene of Bombyx mori(The GenBank accession numbers:EU159447) was cloned by RT-PCR.According to sequence analysis,the 1 242 bp coding region of Serpin-6 encoding 413 amino acid residues results in theoretical molecular weight of 46.47 kDa,isoelectric point of 5.48 and without signal peptide sequence this gene molecular weight is about 44.62 kDa.Comparied to other insects,the Serpin-6 gene shows highest amino acid identity with Serpin-6 of Manduca sexta(77.67%),and 39.51%identity with Serpin-9 of Anopheles gambiae,only 22.37% identity with Serpin-5 of Bombyx mori.It reveals that Serpin-6 of Bombyx mori and Serpin-6 of Manduca sexta may belong to orthologous gene and play a same or similar roleThe result of alignment between this gene and the silkworm genome shows that the gene consists of eight exon and seven intron.Analyzing the Serpin-6 gene of Bombyx mori by Signal P3.0 Server Progam predicts that the 1st to 17th amino acid residues of N-terminal is the signal peptide sequence.2.According to the ORF sequence of Serpin-6 gene of Bombyx mori,appropriate primers were designed.Afterwards,semi-quantitative RT-PCR was carried out using cDNA of head,midgut,fat body,silk glands,testis,ovarian,haemolymph of contract Day-3 fifth instar larvae("Daizo"),fat body and haemolymph of larvae injected with LPS at different periods respectively as templates.The expected size of PCR products was about 349 bp.After 1.2%Agarose gel electrophoresis,semi-quantitative calculation was processed using actin3 gene of Bombyx mori as reference.The result ofsemi-quantitative analysis about contrat silkworm showed that Serpin-6 gene was highly expressed in head,testis,ovarian,while lowly expressed in the midgut,fat body,silk glands and haemolymph.For larvae injected with LPS,the results showed that this gene was highly induced and highly expressed in the fat body and haemolymph,and the highest expression was at 6h after injection.After 6h post-infection,expression level decreaced with time,to be the lowest at 24h post-infection in this study.It is inferred that this Serpin gene may play an important role in immunity of Bombyx mori.Further research shoude be done to confirm that if it have some other function due to its different expression level in different tissue.3.The Serpin-6 gene without signal peptide was constructed to the prokaryotic expression vector pET-28a(+) and then transformed into E.coli BL21.And lmmol/L IPTG was used to induce the target protein to express.SDS-PAGE analysis indicated that the Serpin-6 gene was expressed in the form of fusion protein.There was a specific band with relative molecular weight of 45 kDa on PAGE profiles for the BL21 with Serpin-6 induced by IPTG,comparied to the contract BL21,BL21 with empty pET-28a(+) or BL21 with Serpin-6 not induced by IPTG,which indicated that Serpin-6 gene was expressed in E. coli.Western blot analysis showed a obvious band on the expected position(45 kDa),in accordance with the molecular weight of the fusion Protein,the specific band not detected for the other three group,which further confirmed that the Serpin-6 gene without signal peptide was successfully expressed.No specific band for Serpin-6 with signal peptide in the same condition inferred that signal peptide sequence may affect the expression of Serpin-6 in prokaryotic expression system.The successful cloning,semi-quantitative analysis of tissue expression,differential expression afte LPS infection and prokaryotic expressing research of Serpin-6 gene of Bombyx mori has further analyticed the relationship between Serpin and immunity of Bombyx mori.It also provides reference for studing immune defense of Bombyx mori and other Lepidoptera insects.