以枯草芽孢杆菌递呈VP28对南美白对虾免疫相关基因表达和细胞特异性吞噬的影响

IMMUNE-RELATED GENES EXPRESSION AND PHAGOCYTOSIS AGAINST WHITE SPOT SYNDROME VIRUS AFTER ORAL DELIVERY OF VP28 USING BACILLUS SUBTILIS AS VEHICLES IN LITOPENAEUS VANNAMEI

以枯草芽孢杆菌(Bacillus subtilis)为活载体口服递呈对虾白斑综合征病毒(WSSV)囊膜蛋白VP28,评价其抗病毒感染能力、对南美白对虾免疫相关基因表达以及血淋巴细胞对病毒特异性吞噬的影响。经口服免疫枯草重组菌株B.subtilis-VP28攻毒后,对虾的相对存活率达83.3%。为探讨重组菌株的抗病机理,比较研究了免疫相关基因—proPO(酚氧化酶原)、Peroxinectin(PE)和脂多糖-β-1,3-葡聚糖结合蛋白(LGBP)基因的表达差异,并进一步分析了血淋巴细胞吞噬活性和特异性。结果表明,B.subtilis-VP28菌液能显著提高(P<0.05)对虾proPO、PE和LGBP mRNA的表达水平和血细胞对WSSV的吞噬活性,B.subtilis组对免疫相关基因也有一定的激活作用,而B.subtilis-VP28发酵上清液则能增加血细胞吞噬活性;此外,B.subtilis-VP28菌液组血细胞对WSSV具有特异性吞噬作用。研究为枯草重组菌株B.subtilis-VP28抗WSSV感染作用及其作为特殊功能水产微生态制剂的应用提供了一定的科学依据。

The regulation of immune-related genes expression and phagocytosis of White Spot Syndrome Virus（WSSV） were evaluated by oral delivery of VP28 using Bacillus subtilis as vehicles in Litopenaeus vannamei.In our initial experiment,by oral delivery of B.subtilis spores harboring VP28（B.subtilis-VP28） to L.vannamei,the extremely high survival（Relative Percent Survival： 83.3%） upon challenge with WSSV can be observed.The differences of genes expression levels of proPO,Peroxinectin（PE） and lipopolysaccharide-and beta-1,3-glucan-binding protein（LGBP） were demonstrated among experimental groups of B.subtilis-VP28 bacterial spores,B.subtilis-VP28 supernatants,B.subtilis and control.The result showed that immune-related genes（proPO,PE and LGBP） were significantly（P〈0.05） upregulated in both B.subtilis-VP28 bacterial spores and B.subtilis feeding groups compared to B.subtilis-VP28 supernatants and the control groups.The phagocytosis in groups of B.subtilis-VP28 bacterial spores and B.subtilis-VP28 supernatants were enhanced significantly（P〈0.05）.Besides,the percentage of haemocytes phagocytosing WSSV（46.8±3.9） in shrimp previously fed with B.subtilis-VP28 was significantly higher（P〈0.001） than that of phagocytosing TSV（an unrelated virus of shrimp）（14.9±2.2）.This suggests that the heightened phagocytic activity,and thus the high-level survival of shrimp after B.subtilis-VP28 ‘vaccination’ are selective or specific towards WSSV.These results may raise the application prospective of this recombinant B.subtilis spores against WSSV infection in shrimp farms.