Lactococcus lactis食品级诱导表达系统构建及铜绿假单胞菌融合外膜蛋白的表达
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摘要
乳酸菌是在食品、医药等领域广泛应用的微生物，用乳酸菌构建以非抗生素抗性为选择标记的食品级载体的研究越来越为人们所关注。
NICE(The Nisin-Controlled Expression)系统是有效的食品级诱导表达系统。乳酸菌NICE系统是在乳链菌肽诱导下由nisA启动子控制目的基因的表达，含nisR和nisK的两组分调节系统的高效诱导表达系统。由于NICE系统的诱导剂、宿主菌和载体都是食品级的，构建乳酸菌NICE系统食品级表达载体，将集益生菌生物功能和外源基因表达产物活性于一体，在食品和医药方面有着很好的应用前景。
在目的蛋白生产和发酵中，由于乳酸菌分泌表达可使乳酸菌生产的异源蛋白质前体避免水解，异源蛋白质表达量更高，以同源的乳酸球菌SP_(Usp45)指导目的蛋白的分泌表达并且目的基因与SP_(Usp45)，Nuc和LEISSTCDA的融合体可提高分泌表达效率，因而乳酸菌分泌表达更有意义。
本研究利用食品级a-aga选择标记代替抗生素抗性选择标记，并利用nisA启动子、nisR、nisK和SP_(Usp45)／nucA／cwa_(M6)／t1t2基因，以乳酸球菌作为宿主菌，以乳链菌肽作为诱导分子，构建更为安全稳定的乳酸菌食品级高效诱导细胞质和细胞分泌表达的θ型复制载体系统。OprF／H为本实验室构建，是铜绿假单胞菌外膜蛋白组分F和H的融合蛋白，具有良好的免疫原性，为检测该系统的可行性，将OprF／H基因克隆入构建的载体中，在乳酸球菌中进行高效诱导细胞内和细胞壁分泌表达，为利用此两种载体表达各种外源性和内源性的功能基因提供理论和技术依据，同时也为研制有效的防治铜绿假单胞菌的新型疫苗提供思路。
从L.lactis SMQ719／pRAF800中提取到食品级载体pRAF800，根据公布的pRAF800的复制子序列，设计引物扩增rep基因，经限制酶EcoRI和NheI酶切，利用T4连接酶与pMG36e的Em~r连接。连接产物用电转化法转入L.lactis NZ9000中，经Em筛选、克隆子鉴定，获得重组质粒pEm~r：rep。用限制酶Hind III酶切pEm~r：rep，pEm~r：rep经绿豆核酸酶削平，T4连接酶连接，获得含Em~r的θ复制子缺失载体pEm~r：Arep。
然后以pNZ8048 DNA为模板，根据已发表的P_(nisA)和T_(pepN)序列，设计引物扩增出大小为312bp的P_(nisA)-MCS-T_(pepN)基因片段，再用限制酶XbaI酶切pRAF800
Lactic acid bacteria (LAB) are GRAS bacteria that are widely used in food industry and medicine. It is important to establish food-grade expression vectors systems for production of functional proteins. Food-grade means the vector bacteria and the selectable marker should be safe enough to meet the needs of food production.
The nisin-controlled expression (NICE) system of L. lactis is one of the most widely used expression systems of Gram positive bacteria. The NICE system for controlled heterologous and homologous gene expression in L. lactis has been proven to be very valuable.
In the production of protein with cells, the secretion of heterologous proteins are an efficient way to escape intracellular proteolysis, and the replacement of SP_(Nuc) by the homologous SP_(Usp45) and a fusion with SP_(Usp45), Nuc, and LEISSTCDA can enhance the production yields and the secretion efficiency of heterologous proteins in LAB. The construction of food-grade inducible gene expression system and engineering strains will intergate the biological function of beneficial bacteria with the activity of expressed products of heterologous genes, and will have great potential in food and medicine.
In the present study, we constructed a series of food-grade vectors harboring food-grade selectable marker a-aga, theta replicon, P_(nisA) -MCS-T_(pepN) and P_(nisA)-SP_(Usp45)-nucA-CWA_(M6)-tlt2 that were suitable for the production of cytoplasmic and secreted heterologous proteins in L. lactis. The vectors are controlled by the nisA promoter.
The theta replicon of the plasmid pRAF800 from L. lactis SMQ719 was amplified by PCR with the primers designed based on the pRAF800 sequence. The PCR products were digested with EcoRI and NheI and linked with the Em~r of pMG36e, then transformed into L. lactis NZ9000 by electroporation. The plasmid pEm~r:rep harboring the erythromycin resistance gene and theta replicon were screened on erythromycin plates. The repB-deficient companion plasmid pEm~r:△rep was constructed by enzyme digestion, deficiency and ligation, and could coextist with the plasmid with the same theta replicon in the media containing erythromycin only.
NICE(The Nisin-Controlled Expression)系统是有效的食品级诱导表达系统。乳酸菌NICE系统是在乳链菌肽诱导下由nisA启动子控制目的基因的表达，含nisR和nisK的两组分调节系统的高效诱导表达系统。由于NICE系统的诱导剂、宿主菌和载体都是食品级的，构建乳酸菌NICE系统食品级表达载体，将集益生菌生物功能和外源基因表达产物活性于一体，在食品和医药方面有着很好的应用前景。
在目的蛋白生产和发酵中，由于乳酸菌分泌表达可使乳酸菌生产的异源蛋白质前体避免水解，异源蛋白质表达量更高，以同源的乳酸球菌SP_(Usp45)指导目的蛋白的分泌表达并且目的基因与SP_(Usp45)，Nuc和LEISSTCDA的融合体可提高分泌表达效率，因而乳酸菌分泌表达更有意义。
本研究利用食品级a-aga选择标记代替抗生素抗性选择标记，并利用nisA启动子、nisR、nisK和SP_(Usp45)／nucA／cwa_(M6)／t1t2基因，以乳酸球菌作为宿主菌，以乳链菌肽作为诱导分子，构建更为安全稳定的乳酸菌食品级高效诱导细胞质和细胞分泌表达的θ型复制载体系统。OprF／H为本实验室构建，是铜绿假单胞菌外膜蛋白组分F和H的融合蛋白，具有良好的免疫原性，为检测该系统的可行性，将OprF／H基因克隆入构建的载体中，在乳酸球菌中进行高效诱导细胞内和细胞壁分泌表达，为利用此两种载体表达各种外源性和内源性的功能基因提供理论和技术依据，同时也为研制有效的防治铜绿假单胞菌的新型疫苗提供思路。
从L.lactis SMQ719／pRAF800中提取到食品级载体pRAF800，根据公布的pRAF800的复制子序列，设计引物扩增rep基因，经限制酶EcoRI和NheI酶切，利用T4连接酶与pMG36e的Em~r连接。连接产物用电转化法转入L.lactis NZ9000中，经Em筛选、克隆子鉴定，获得重组质粒pEm~r：rep。用限制酶Hind III酶切pEm~r：rep，pEm~r：rep经绿豆核酸酶削平，T4连接酶连接，获得含Em~r的θ复制子缺失载体pEm~r：Arep。
然后以pNZ8048 DNA为模板，根据已发表的P_(nisA)和T_(pepN)序列，设计引物扩增出大小为312bp的P_(nisA)-MCS-T_(pepN)基因片段，再用限制酶XbaI酶切pRAF800
Lactic acid bacteria (LAB) are GRAS bacteria that are widely used in food industry and medicine. It is important to establish food-grade expression vectors systems for production of functional proteins. Food-grade means the vector bacteria and the selectable marker should be safe enough to meet the needs of food production.
The nisin-controlled expression (NICE) system of L. lactis is one of the most widely used expression systems of Gram positive bacteria. The NICE system for controlled heterologous and homologous gene expression in L. lactis has been proven to be very valuable.
In the production of protein with cells, the secretion of heterologous proteins are an efficient way to escape intracellular proteolysis, and the replacement of SP_(Nuc) by the homologous SP_(Usp45) and a fusion with SP_(Usp45), Nuc, and LEISSTCDA can enhance the production yields and the secretion efficiency of heterologous proteins in LAB. The construction of food-grade inducible gene expression system and engineering strains will intergate the biological function of beneficial bacteria with the activity of expressed products of heterologous genes, and will have great potential in food and medicine.
In the present study, we constructed a series of food-grade vectors harboring food-grade selectable marker a-aga, theta replicon, P_(nisA) -MCS-T_(pepN) and P_(nisA)-SP_(Usp45)-nucA-CWA_(M6)-tlt2 that were suitable for the production of cytoplasmic and secreted heterologous proteins in L. lactis. The vectors are controlled by the nisA promoter.
The theta replicon of the plasmid pRAF800 from L. lactis SMQ719 was amplified by PCR with the primers designed based on the pRAF800 sequence. The PCR products were digested with EcoRI and NheI and linked with the Em~r of pMG36e, then transformed into L. lactis NZ9000 by electroporation. The plasmid pEm~r:rep harboring the erythromycin resistance gene and theta replicon were screened on erythromycin plates. The repB-deficient companion plasmid pEm~r:△rep was constructed by enzyme digestion, deficiency and ligation, and could coextist with the plasmid with the same theta replicon in the media containing erythromycin only.
引文
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88 廖立新，曹郁生，李国辉．一种快速粗提铜绿假单胞菌外膜蛋白的方法．江西医学院学报．2003，43(1)：60-61．
89 张万山，陈燕，曹郁生．铜绿假单胞菌外膜蛋白OprF和OprH基因扩增、融合、克隆及原核表达．江西医学院学报．2004，44(2)，5-9．
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92 刘同军，徐文琳，张玉臻．变溶菌(Mutanolysin)研究历史和发展前景．微生物学报，2000(40)2：224-227．