| In the redox cycling process, aerobic organisms often produce large amounts ofReactive Oxygen Species (ROS, including:O2._,.OH and hydrogen peroxide, etc.) ROSare normal byproducts of oxygen metabolism, plays a role of messenger in cellsignaling and maintaining the body’s normal metabolism according to some papers.However, under extreme conditions, such as exposure to UV and heat, a large amount ofROS will dramatically increase. Excessive level of ROS may cause finally damage tothe macromolecules of living body, such as the DNA molecular structural damage andlipid peroxidation. Irreversible damage to active structure of enzyme and a range ofother oxidative damage lead to various physiological diseases of organisms. It has beenreportedseveral critical enzymes that played impotant roles in the equilibrium ofmetabolism and organism’s survival. The superoxide dismutase (SOD), as an crucialantioxidant enzyme existed in almost all aerobic organisms is the only one that candirectly participate in the metabolism of ROS superoxide class of enzymes.After being infected by NPV virus, ROS levels of silkworms (Bombyx mori)significantly increased. By screening silkworm immune microarray data, we also foundthat the expression levels of Bmsod3genes significantly increased after being inducedby B. mori NPV and several other major pathogens. We assumed that BmSOD3proteinmight be associated with NPV resistance. High level of ROS, such as superoxide anion,might cause damage to tissue cells. As a first line of defending antioxidant enzymes,SOD protein may play a pivotal role in the antioxidant effect. Based on this, we havefirst cloned and expressed silkworm Bmsod3gene in prokaryote BL21strains, theresults obtained are as follows.1) BmSOD3, as an extracellular SOD protein has a high protein sequencesimilarity with other insects’ SOD3protein, such as65%of similarity with Anophelesgambiae. Bmsod3has only one locus on nscaff2828of chromosome8. The length of theentire CDS of Bmsod3gene is only606bp. BmSOD3protein, as a extracellular SODprotein, the probability of having a signal peptide is0.843. The predicted splice site ofsignal peptide is located between the21th amino acids (glycine) and the22th aminoacid (glutamic). The predicted theoretical BmSOD3protein isoelectric point is5.87.The theoretical mass of BmSOD3protein is18.96kD, and the total mass is about21kDplus with histidine tag. We have also calculated the active centre of reaction for the catalytic dismutation of the protein BmSOD3is located in65th,80th,100th,140thsite ofthe protein sequence, Cu2+binding centre located in65th,80th,140thsite of the proteinsequence, and Zn2+binding centre located in80th,90th,100thsite of the protein sequence.Interestingly, Cu2+, Zn2+binding sites are all where catalytically active sites are.2) We have analyzed gene expression of Bmsod3in tissue via RT-PCR method. Wefound that the expressive abundance of Bmsod3in the midgut and malpighian tubuleswere completely consistent with the data expreesed in genechip. After the NPVinduction, the abundance of Bmsod3gene expressed in treated group had significantlyincreased compared with the control.3) The Bmsod3gene has been cloned into the Trans-T simple vector, and then itwas transferred into expression vector. We used the mature expression system consistedof pET28(a) and BL21(DE3). His-tag in the pET28(a) vector will be beneficial forpurification of protein. During purification, when the concentration of imidazole in theelution buffer reached to300mM, the protein started to be eluted and could be elutedwell in concentration of500mM imidazole. The purified target protein could further berenatured or used to prepare for the preparation of polyclonal antibodies.4) The form of BmSOD3protein expressed in BL21is inclusion body. We purifiedit by using nickel affinity chromatography and his-tag expressed with target protein. Werefolded inclusion bodies by dialysis method then analyzed its activity. The resultsshowed that refolded protein has a low SOD activity. |
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