三疣梭子蟹LGBP基因的重组表达及微生物结合实验

摘要

将扩增得到的三疣梭子蟹LGBP基因开放阅读框与表达载体pET-22b(+)连接,转化E.coil BL21(DE3)plysE后IPTG诱导表达.经SDS-PAGE检测,发现诱导组比空载体和未诱导组多出一条分子量约为41 ku的表达产物,与预测的重组蛋白分子量大小基本一致.重组质粒在不同IPTG浓度和不同温度条件下诱导表达产物的SDS-PAGE分析结果显示,低浓度0.4 mmol/L IPTG和30℃诱导能有效减少菌体蛋白的本底表达.用纯化的重组蛋白连续免疫小鼠,4周后得到抗血清,经Western-blotting检测,其具有很好的特异性.微生物结合实验表明,重组表达的pET-LGBP具有较强的生物活性,其与丹麦啤酒酵母、巨大芽孢杆菌、副溶血弧菌、溶藻弧菌、大肠杆菌等均有结合能力.%The lipopolysaccharide-and beta-1, 3-glucan-binding protein (LGBP)is a pattern recognition receptor which is fundamental for the innate immune response of crustaceans.A pair of primers were designed according to LGBP cDNA and were used for ORF amplification.Then the fragment was subcloned into pET-22b( + )and expressed in Escherichia coli BL21 (DE3)plysE.We got a 41 ku clear visible band in the expected position by SDS-PAGE detection, which indicated that it was the same as the expected molecular weight, and the negative controls were blank.Expression of recombinant protein induced under different conditions indicated that lower concentration of IPTG (0.4 mmol/L) and lower temperature 30 ℃ could reduce the expression of background effectively by SDS-PAGE detection.The purified recombinant protein was used as antigen to prepare antiserum in mouse directly.Four weeks later, a polyclonal antiserum was obtained and it was specified by Western-blotting analysis.At last, three types of Gram-negative bacteria ( Vibrio parahaemolyticus, V.alginolyticus, E.coli), four types of Gram-positive bacteria ( Bacillus subtilis,B.megaterium,B.cereus, S.aureus )and one fungus (Sac-charomyces cerevisiae )were utilized to analyze pET-LGBP binding activity.Our data suggested that pET-LGBP was able to bind S.cerevisiae, B.megaterium, V.Parahaemolyticus, V.alginolyticus and E.coli.