Based On The Baculovirus Expression System To Express Exogenous Genetic Research

SummaryThe insect baculovirus expression system is a useful tool for the efficient production of eukaryotic proteins that require correct folding and posttranslational modification such as signal peptide processing, phosphorylation and glycosylation. The baculovirus expression system has been proved to be very efficient for heterologous protein production than that obtained in naturally producing mammalian cells. Superoxide dismutase (SOD) is a metalloenzyme, which catalyzes the conversion of the superoxide radicals into molecular O2 and H2O2 and thus forms a crucial part of the cellular antioxidant defense mechanism. The amount of SOD present in cellular and extracellular environment is crucial for the prevention of disease linked to oxidative stress. We used the silkworm, Bombyx mori, larvae as a bio-reactor and expressed the Mn-SOD by the recombinant bacmid baculoviruses expression system. The fifth instar silkworm larvae expressed SOD 96 h post-infection with recombinant virus (rBacmid/BmNPV/SOD) were collected and dried with a vacuum dryer. To study the function of silkworm larvae powder containing superoxide dismutase and potential practical development, we investigated the safety assessment and effects on immune activity of mice such as the growth of immunity-related organs, delayed-type hypersensitivity (DTH) and charcoal particle clearance ability. The mean body weights in treated mice were significantly heavier than that of the control, meanwhile, the ratio of the thoracic gland/body weight in treated mice was significantly enhanced after 30 days treated with silkworm larvae powder containing manganese superoxide dismutase. The treated mice resulted in a profound activation of the DTH and charcoal particle clearance, and indicated the treated mice have stronger phagocytic activity to exogenous materials. Our data also indicated the feeding treatment was safe with 360 folds of recommended human dosage in acute toxic test. In long-term test, there were no effects of silkworm larvae powder containing SOD on treated mice's growth and inside organs as long as 90 days. Further the electronic microscope investigation showed the intestine, liver, splenocyte and stomach in mice were no obvious changes both in organs and sub-organs such as nucleus, endoplasmic reticulum, mitochondrion, Golgi and peroxisomes after treated for as long as 90 days. We investigated the effects of silkworm larvae powder containing SOD on the immune system of mouse and employed a proteomics approach to examine this phenomenon. Our data on the effects of continuous treatment with SOD-containing silkworm larvae powder showed that the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control. The results of PFC assay also revealed that antibody production was higher in all three treated groups than controlled mice. We investigated the phagocytosis of mouse macrophages. The SOD treatment led to a dose dependent increase of phagocytic activity. We identified six proteins that related to immunity of mice. The data showed all these six matched proteins related immunity presented the increase of expression level in plasma of mouse administrated with silkworm powder including SOD compared to that of control. These findings demonstrate that administration of silkworm larvae powder containing SOD results in enhancement of immunity activities in the mouse. The results also suggested that the SOD expressed in silkworm maybe have potential application in medicine.We used the bacmid DNA developed in our laboratory for direct injection in silkworm larvae, thereby avoiding the preparation of high-titer viral stocks for infecting larvae. Using silkworms or pupae are 10-100-fold higher than those using Bombyx mori cells or conventional insect cells, indicating that the silkworm or its pupa is one of the most suitable systems for large-scale production of eukaryotic proteins. In this study, we cloned the human insulin gene using human genomic DNA as template, and constructed the recombinant Bacmid DNA including human insulin and FLAG tag. The silkworm larvae were infected with above recombinant Bacmid DNA, and the insulin were purified and identified from the infected worm haemolymph. Several kinds of reBmMNPVbacmid, which are cysteine protease gene deletion (CPD-BmMNPV bacmid) and cysteine protease-and chitinase-deficient (CPPD-BmMNPV bacmid) including human insulin and FLAG tag were constructed. The silkworm larvae were infected with above recombinant Bacmid DNA, and the expressed insulin were purified and identified from the infected worm haemolymph. The highest expression was shown in CPPD-BmMNPV bacmid that is about two times of the wild type of reBmMNPVbacmid, reaching 15.827ng/ml haemolymph. The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 (E) and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. Two different DNA vaccine constructs, expressing GP5+γ, GP5 and M proteins+γsimultaneously (ORF5/ORF6/y) were constructed. Co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.