表达堆型艾美耳球虫3-1E蛋白的三种重组乳酸乳球菌构建与鉴定  

Construction and Characterization of Three Recombinant Lactococcus lactis Expressing E. acervulina 3-1E Protein

作　　者：张丽丽[1] 马德星[1] 高铭扬[1] 王芬[1] 张跃[1] 王典[1] 黄宇晨[1] 李健[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《中国家禽》2016年第22期14-18,共5页China Poultry

基　　金：国家自然科学基金项目(30901061);黑龙江省自然科学基金资助项目(C201422);黑龙江省博士后启动金项目(LBH-Q14019)

摘　　要：为探索堆型艾美耳球虫(Eimeria acervulina)表面抗原3-1E基因在乳酸乳球菌L.lactis NZ9000中的不同表达形式,首先通过PCR扩增3-1E目的基因片段,克隆入pTX8048载体中,构建胞内表达质粒p TX8048-3-1E;将乳球菌未知分泌蛋白(Usp45)信号肽(SP)与3-1E基因片段融合(SP-Δ3-1E),并克隆入pTX8048中,构建分泌表达质粒pTX8048-SP-Δ3-1E;将化脓性链球菌M6蛋白细胞壁锚定序列(Cell wall anchor,CWA)克隆入pTX8048-SP-Δ3-1E中的Δ3-1E下游(去除Δ3-1E终止密码子,即NAΔ3-1E),构建表面表达质粒pTX8048-SP-NAΔ3-1E-CWA;将三种表达载体电转化到乳酸乳球菌L.lactis NZ9000中,构建三种乳酸乳球菌L.lactis NZ9000/pTX8048-3-1E(胞内表达菌)、L.lactis NZ9000/pTX8048-SP-Δ3-1E(分泌表达菌)及L.lactis NZ9000/pTX8048-SP-NAΔ3-1E-CWA(表面表达),通过Western blot来检测3-1E目的蛋白在三种阳性乳酸乳球菌中的表达。结果表明,构建的三种重组乳酸乳球菌均可表达目的蛋白。研究结果为进一步评估重组乳酸乳球菌的抗球虫免疫保护作用及后续非抗性标记乳酸菌体系建立奠定基础。In order to explore the expression of Eimeria acervulina 3-1E protein gene in L. lactis NZ9OOO,3-1E gene fragment was amplified by PCR, and then cloned into pTX8048 vector to construct the intracellular expression plasmid pTX8048-3-1E. The signal peptide （SP）of the unknown secretory protein Usp45 in Lactococcus was fused to 3-1E gene （SP-△3-1E）,and inserted into plasmid pTX8048 to construct the secretion expression plasmid pTX8048- SP-213-1E. The cell wall anchoring （CWA） sequence of M6 protein in Streptococcus pyogenes was inserted into the downstream of A3-1E in pTX8048-SP-△3-1E （terminator codon in A3-1E was deleted,and then named NA△3-1E）to construct the surface expression plasmid pTX8048-SP-NA△3-1E-CWA. The above three kinds of positive plasmids was electroporated into L. lactis NZ9000 to construct the three recombinant strains L. lactis NZ9000/pTXS048-3-1E （expressing intracellular target protein）, L. lactis NZ9000/pTX8048-SP-△3-1E （expressing target protein in secretion）and L. lactis NZ9000/ pTX8048-SP-NA△3-1E-CWA （expressing target protein on the surface of positive strain）. The results showed that the 3-1E protein expressing in different manner was all detected in the three recombinant positive strains using Western blot method. The results laid the foundation for evaluation of immune protection against Eimeria infection by immunized with the three recombinant Lactococcus strains,and for the establishment of non-resistance marker system of lactic acid bacteria.

关 键 词：乳酸乳球菌 胞内表达 分泌表达 表面表达 

分 类 号：S852[农业科学—基础兽医学]