乳酸乳球菌食品级诱导表达系统的构建及异源蛋白的表达

以α-aga基因为食品级选择标记构建了乳酸乳球菌食品级高效诱导细胞内和细胞壁锚定表达系统,并用这一表达系统表达了铜绿假单胞菌融合外膜蛋白基因OprF/H。首先以pRAF800和pNZ8048构建了含有α-aga、PnisA-MCS-TpepN和θ复制子的乳酸乳球菌食品级细胞内诱导表达载体pRNA48,再以pRNA48和pVE5524为出发载体构建了含有α-aga、PnisA-SPUsp45-nucA-CWAM6-t1t2和θ复制子的乳酸乳球菌细胞壁锚定诱导表达载体pRNV48。然后以食品级载体pRNA48和pRNV48为基础,构建了不含抗生素抗性选择标记的铜绿假单胞菌融合外膜蛋白基因的表达质粒pRNA48-OprF/H和pRNV48-OprF/H。利用nisin进行重组乳酸乳球菌菌株的诱导表达,通过SDS-PAGE和Western blot分析,检测到表达蛋白分别占细胞内可溶蛋白的9.6%和细胞壁锚定蛋白的9.8%,表达产物具有免疫原性,可与含OprF/H的乳球菌以及铜绿假单胞菌发生特异性的凝集反应。

Construction of food-grade inducible expression system in Lactococcus lactis and expression of fusion OprF/H from Pseudomonas aeruginosa

The food-grade inducible gene expression system in L. lactis was constructed for expression of cytoplasmic and anchored heterologous proteins. Gene alpha-aga encoding alpha-galactosidase was used as food-grade selectable marker instead of antibiotic resistance gene. Firstly, a food-grade cytoplasmic inducible expression vector pRNA48 containing alpha-aga, theta replicon from pRAF800, and PnisA-MCS-TpepN from pNZ8048 was constructed. Then the cell wall anchored expression vector pRNV48 containing alpha-aga, theta replicon, and PnisA-SPUsp45-nucA-CWAM6-tlt2 was constructed based on the plasmids pRNA48 and pVE5524, which was suitable for the heterologous proteins anchored to the cell wall of L. lactis NZ9000. The fusion OprF/H derived from Pseudomonas aeruginosa was cloned into plasmids pRNA48 and pRNV48 to construct the pRNA48-OprF/H and 3RNV48-OprF/H for the expression of OprF/H. OprF/H was produced by the recombinant strains when induced with nisin. The highest yield of active OprF/H was 9.6% of intracellular soluble protein and 9.8% of cell wall anchored protein in L. lactis NZ9000, respectively. The immunogenicity and specificity of the expressed protein from recombinant were tested by animal immunization and Western blot.