| White spot syndrome virus (WSSV), which was first discovered in northern Taiwan around 1992, is currently the most serious viral pathogen of shrimp worldwide. It causes up to 100% mortality within 7 to 10 days in commercial shrimp farms, resulting in large economic losses to the shrimp farming industry. Due to the current intensity of aquaculture practices and the broad host range of WSSV, novel control strategies including vaccination against this virus would be highly desirable. In this study, B. subtilis-VP28 engineering strain and VP28-IgY antibody based on WSSV VP28 protein were constructed and delivered orally to crayfish, Procambarus clarkii. The protective effects of them were demonstrated after challenge. A pilot study on potential mechanism of B. subtilis-VP28 and VP28-IgY with resistance to WSSV infection was also discussed. The main research contents and results were as follows:1 Construction of novel shuttle secreted-expression vector pBESThe novel shuttle secreted-expression vector pBES was constructed by introducing of the strong promoter PEσ43 and a novel natural signal peptide into the shuttle clone vector pBEC which stems from vectors B. subtilis pUB110 and E. coli pGEM3. In order to evaluate the secreted efficiency of pBES, the reporter gene ofβ-glucanase was inserted. Time-course analysis of extracellular enzyme quantities and activities were demonstrated by SDS-PAGE andβ-glucanase detection assay. The total enzymatic activity in the culture supernatant was 283.45 U/ml, while the extracellular part was 116.95 U/ml. Thus, the percentage of secreted vs totalβ-glucanase amount was 41.26%.2 The relationship of amino acid characters on signal peptide N-& H-regions and secreted efficiencyFor alteration of charged residues at N domain as well as hydrophobic residues on H domain of the signal peptide, fifteen mutants were designed by primer-directed site mutagenesis. It was revealed that more positive charged residues at the N-region improved the processing efficiency of signal peptide slightly, whereas signal peptide hydrophobicity could be an important factor for secretion ofβ-glucanase precursor. The H region of high hydrophobicity in combination with the four positively charged residues present in N region might result in a tighter binding of the signal peptide to the existing signal recognition factors, leading to difficult release of the enzyme precursor. By screening of the mutants, two novel signal peptides were confirmed with high secreted efficiency, which were H3N2 (MRARKIAGMATSGGVAQPSSAV A) and H3N23 (MRRRKIAGMATSGGVAQPSSAVA). The percentages of secreted vs totalβ-glucanase amount were 80.28% and 80.66% conducted by H3N2 and H3N23, respectively. Therefore, the highly secreted expression vectors pBES-H3N2 and pBES-H3N23 of B. subtilis were constructed based on the promoter PEa43 and H3N2/H3N23 signal peptides.3 Highly secreted expression of vp28 gene and time-course analysisThe vp28 gene was inserted into the vectors pBES-H3N2 and pBES-H3N23, and introduced into the host B. subtilis WB600, respectively. The antigen protein VP28 was successfully secretion, and its immunogenicity was confirmed by Western blot using rabbit anti-VP28 IgG. Time courses for secreted recombinant VP28 in B. subtilis WB600 were analyzed by SDS-PAGE. The vp28 gene was continuously expressed during the exponential and stationary growth phases. On extrapolation by BandScan, the amount of VP28 in the growth medium accumulated to a maximum level (45.6 mg/L and 47.4 mg/L in B. subtilis (pBES(H3N2)-VP28) and B. subtilis (pBES(H3N23)-VP28), respectively) after 22 h of cultivation, declined slightly at 23 h, and then remained at a relatively stable level for further incubation.4 Preparing of specific VP28-IgY egg yolk antibody and its stability analysisSix layer hens (HY-LINE Variety White) were immunized with the purified recombinant VP28 protein expressed from E. coli. The titer and time-course study of specific VP28-IgY were determined by ELISA. The results showed that the special antibodies titer firstly presented in the egg yolk at the 14th day (P/N=2.555,1:400), reached the high point at the 56th day (P/N=14.920,1:3200), and kept stable level at the 120th day (P/N=9.265,1:1600). The specific VP28-IgY could be purified by one-step of distilled water dilution combined with two-step of saturation sulfate ammonium precipitation (50%& 33%). The average molecular weights of heavy chain and light chain were determined by SDS-PAGE as 60 kDa and 25 kDa, respectively. In the study of stability analysis, it was showed that VP28-IgY had the relatively high resistance to heat (<70℃), acid (pH 3.0-10.0), pepsin and osmotic pressure.5 Protective effects of B. subtilis-VP28 and VP28-IgY in crayfish by oral deliveryThe B. subtilis-VP28 performed as an active immune manner of protection. The relative percentage survival (RPS) values of the groups rVP28-bs (form of spores) were remarkably higher than those of the groups rVP28-bv (form of vegetative cells). Challenged at 3,14,28 days post-vaccination, the RPS values of the former were 65.4%,78.3% and 59.7%, respectively, while those of the later were 38.2%,51.7% and 36.8%, respectively. The VP28-IgY performed as a passive immune manner of protection. The RPS values of 0.01%,0.05% and 0.1% supplementation groups were 48.3%,86.0% and 88.3%, respectively. In conclusion, the highest RPS value of B. subtilis-VP28 immunized crayfish could reach 78.3% in the form of spores delivery challenged at the 14th day post-vaccination. The minimum additive amount of VP28-IgY in relatively high RPS (86.0%) was 0.05%.6 A pilot study on mechanism of protective effects conducted by B. subtilis-VP28 and VP28-IgYThe B. subtilis-VP28 significantly increased the activities of AKP, ACP, SOD, PO, T-AOC, CAT and GSH-Px (P< 0.05), while decreased the levels of iNOS, O2-and MDA(P< 0.05). It was indicated that B. subtilis-VP28 had the role of improving the immune function and antioxidation system of crayfish. Observed by Optical and transmission electron microscope, the VP28-IgY neutralized majority of WSSV virions in the early stage of infection, and eliminated the remained virions diffused into the tissues in the late stage. Both B. subtilis-VP28 and VP28-IgY could decrease the activity of Caspase-3 (P< 0.05) compared to the group of WSSV infection. Thus, B. subtilis-VP28 and VP28-IgY could neutralize and eliminate the WSSV by the manners of active and passive immune, respectively, as well as block apoptosis induced by WSSV.
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