Cloning, Expression And Enzymatic Analysis Oflipasegene From Bombyx Mori Intestinal Bacterium, Bacillus Pumillus SW41

Bombyx mori is an important economical insect and plays a critical role in national economy. While thedisease ofsilkworm also affected the development of sericulture, especially Bombyx morinucleopolyhedrovirus(BmNPV),whichis the most harmful viral disease in sericulture. Among the long evolutionaryprocess, Bombyx mori have gradually formed some immune defense mechanisms of self-protection. Japanese scholars have found that the lipase from silkworm intestine shows strong antiviral activity against BmNPV, and then some domestic researchers cloned the lipase gene from silkworm intestine, they found the expression product also shows strong antiviral activity against BmNPV. Xiang yun-qing told us that when the silkworms were reared with mulberry leaves, the diversity of bacteria and dominant bacteria from the silkworm intestine was considerably richer compared with that of the larvae reared with tricuspid cudrania leaves. Xie hong-xia,Xue ying-wei, Wang xiao-qiang suggested that silkworm were reared with tricuspid cudrania leaves are sensitive toBombyx morinucleopolyhedrovirus(BmNPV), the activity of anti-BmNPV proteins and resistant enzyme ofsilkworm had declined in different degree. Feng Wei isolated and characterized nine lipase-producing bacteria from the silkworm intestinal dominant bacteria. Therefore, we suppose lipase-producing bacteria may play a role in resistance toBmNPV. In this study, we aimed at investigating the relationship between the lipase from silkworm intestinaldominant bacteria and BmNPV, expecting to provid novel ideas in forage of silkworm as well as its disease resistance and breeding.In this study, we used Bacillus pumilus SW41isolated from silkworm intestine as materials, the lipase gene of Bacillus pumilus SW41was cloned through molecular biology technologies and performed phylogenetic analysis ofthe sequence. Then we linked lipase gene to pCold plasmid and transfonnated them into Escherichia coliBL21(DE3) cells, finished prokaryotic expression and detected enzymatic activity. The experimental contents and results are as follows:1. Cloning of lipase gene and analysis of bioinformaticsGenomic DNA of Bacillus pumilus SW41was prepared, after designing primer and PCR amplification, we obtained648bp-long open reading frame of lipase gene, which can code215amino acids. We predicted the molecular weight of lipase protein was22.95kDa and the isoelectric point was9.92. Prediction of some online softwares showed that lipase protein has a signal peptide sequence of34amino acids in N-terminal, while neither transmembranedomain nor glycosylationsites was found. Therefore, our lipase was not glycoproteinbut secretory protein. Secondary structure of lipase indicated that it contains α/β folden structure and its domain belongs to α/β hydrolase family. Phylogenetic analysis implied that lipase of Bacillus pumilus SW41from silkworm intestine may have potential to resist BmNPV.2. Prokaryotic expression of lipase and Western blottingLinking full length fragment of lipase gene ORFto linearizated pCold I vector, obtained pCold Ⅰ-Lipase recombinant plasmid by characterizating of PCR and plasmid double digestion, transformated them into Escherichia coli BL21(DE3) cells, then proteinexpression was inducted for24h in condition of15℃and100rpm with1mM IPTG. SDS-PAGE found that lipase protein expressed in forms of solubility、insolubility and secretion, respectively. Western blotting determined the product was target protein.3. Detecting activity of recombinant lipaseActivity of recombinant lipase wasdetected by the means of Rhodamine B and NitrobenzeneFen. The result of Rhodamine B showed that colonies could grow in medium which covered with recombinant enzymatic solution while nothing existed in the control; besides, these colonies radiated orange fluorescence in UV light; those indicated that lipase existed in enzymatic solution and had some activity. We tested enzymatic activity as14.72U/mL of recombinant lipase in enzymatic solution by means of NitrobenzeneFen, this also suggested that the protein was provided with activity. The protein concentration ofenzymatic solution which contained recombinant lipase was0.09mg/mL viathe measurement of BCA method. It was no doubt that the determination of lipase activity canprovide foundation for follow studying such as function of lipase.