| Hog Cholera (HC) is an acute and highly contagious disease that has been a great threat to the global swine industry. This disease is caused by classical swine fever virus (CSFV), which is also named as Hog cholera virus (HCV) infection. Large-scale epidemic spread of HC has been controlled due to various comprehensive measures taken in recent years. However, occasional sporadic HC prevalence occurs constantly in our country and results in significant economical lost. Statistical data indicates that annually 3% pigs, 90% of them are young pigs, are dead of HC in China. To make it even worse, extensive HC spreading tendency has been detected in some America, Asia, Europe countries or regions. HC cases were reported in even some countries such as France, Netherland, Germany and Belgium that had declared extinction of HC. Glycoprotein E2 of CSFV is the primary antigenic protein with high variability, which can induce virus-neutralizing antibodies against CSFV and resisted lethal CSFV challenge. This protein has been studied widely all the word as an important protective antigenic protein.The DNA sequence encoding swine fever disease virus (CSFV) glycoprotein E2 antigenic epitope and Escherichia coli Heat-labile Enterotoxin B subuint (LTB) gene with codons preferred by the methylotropic yeast pichia pastoris were synthesized and inserted into pPICZaA expression vector. After being linearized by digestion, the vector with AOX1 promoter and a -factor secretion signal sequence was transformated into Pichia pastoris (GS115) by electroporation to integrate with the genome. The transformants with high copies were screened by Zeocinn? and were induced with methonl to express the recombinant protein. SDS-PAGE and Western blot analysis showed that the fusion protein successfully secreted into the culture medium and retained the antigenicity associated with LTB and CSFV antibodies.The optimized expression conditions of the acquired positive recombinant yeast strains P. pastoris GS115- pPICαA-E2-LTB was established by studying the relations between expression yield and growth conditions with different induction time, strain density, pH value and dose of methanol, respectively. And the conditions that the strain zymolysised in fermenter (NBS BIOFLO 4500) with higher density was optimized, and then the recombinant protein was purified by stepwise precipitation with ammonium sulfate and HPLC. The optimal conditions of the recombinant protein expression were planted in medium with 30℃, pH 6.0 and incubated 96h with 1.0% methanol. After induction under optimal conditions with high density,the maximum yield of recombiant protein could reach to 1g/L theoretically. In this study, yield of E2-LTB protein was 400mg/L, and the concentration of E2-LTB was 95%. It indicated that the strain was character of good stability, higher output and adapt to product cosmically.A positive recombinant strain Pichia pastoris, expressing swine fever virus (CSFV) glycoprotein protein E2 epitopes and E. coil heat-labile enterotoxin B Subunit (LTB), was used to produces mucosal vaccine E2-LTB. The antibody and cytokines response to E2-LTB vaccine were evaluated by ELISA. Remarkably higher levels of antiviral IgA and IgG antibodies were induced in serum, nasal wash and lung wash by routes of intranasal and perorall vaccination, respectively. All antibodies induced by intranasal rout were higher than that induced by perorall rout. E2-LTB could directly enhance the expression of T-cell-mediated interleukin-2 (IL-2), IL-5 and IFN-γin both the serum of mouse and the cultured mouse spleen cells. In virus challenge experiments, all the rabbits were challenged with 100MID50 HCLV. E2 Eimmunized Rabbits showed a mild increase of body temperature, while no E2-LTB Eimmunized rabbit increased its body temperature. On the other hand, the body temperature of control rabbits increased, but then recovered normally. These results demonstrated that the E2-LTB could induce an efficient immune protection against HCLV infection, and might provide a new kind of vaccine for CSFV.An indirect ELISA Kit was constructed to detect antibody against CSFV by coating the wells of 96-well plate with purifted E2 protein.Various factors and conditions of ELISA were explored,and the optimal reaction conditions of ELISA were determined.The optimal concentration of recombinant E2 protein for plate coating was 2.5μg/mL,and the optimal coating condition of recombinant E2 protein for ELISA was at 4℃for 24h. The blocking agents were l% BSA, 0.02% thimerosal and 2mMEDTA, and the blocking time was 1 hours at 37℃. The dilution of serum sample was l:100,and the dilution of HRP-labeled rabbit anti-porcine lgG was l:1200. The samples for ELISA were incubated at 37℃for 20min before terminated with the stopping solution.The indirect ELISA Kit by the purified recombinant nucleoprotein had good specificity for the detection of CSFV antibody in serum.The difference value among wells in a plate and among plates for ELISA was both less than 6%,which showed the assay had a good retrievality.60 serum samples from swines were detected for the antibody to CSFV by using our developed ELISA and the IDEXX CSFV Antibody Test Kits simultaneously. It was found that our developed ELISA had the highly sensitivity and correspondence, and the Cut off value of the kit is 0.15 between positive serum and negative serum. The correspondence between the ELISA kit and the IDEXX kit was 83.3%...
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