乳酸乳球菌食品级分泌性表达载体pSQZ的构建及报告蛋白的表达

文章编号:1002-2694(2008)03-0224-05

乳酸乳球菌食品级分泌性表达载体pSQZ

的构建及报告蛋白的表达

孙强正,熊衍文,叶长芸,徐建国

摘　要:目的　构建乳酸乳球菌(L actococcus lactis ,L lactis )食品级分泌表达载体,表达报告蛋白核酸内切酶(NucA )。方法　PCR 扩增乳酸乳球菌未知分泌蛋白(Usp45)编码基因的启动子、核糖体结合位点、分泌信号和成熟肽前11个氨基酸编码序列,克隆到载体pSH91中,构建食品级分泌性表达载体pSQZ ;克隆报告蛋白金黄色葡萄球菌NucA 成熟肽的编码序列

nuc A 到pSQZ 中分泌信号后,转化乳酸乳球菌MBP71,构建乳酸乳球菌分泌性表达系统L lactis MBP71/p SQZ 2nuc A ;采用

TB 2D 法和酶谱法检测乳酸乳球菌分泌性表达的NucA 活性。结果　PCR 扩增得到us p 45基因片段,酶切、连接到载体pSH91中构建了食品级表达载体p SQZ;将扩增的nuc A 基因片段克隆入载体p SQZ 并转化乳酸乳球菌,得到乳酸乳球菌分泌

性表达系统L lactis MBP71/pSQZ 2nuc A 。在TB 2D 平板上,L lactis MBP71/p SQZ 2nuc A 克隆子周围出现橙色光晕;在酶谱检测中,L lactis MBP71/p SQZ 2nuc A 上清和菌体在18kDa 位置均有橙色条带,但上清条带亮度和宽度大于菌体。结论　成功构建了乳酸乳球菌食品级分泌表达系统,实现了报告蛋白NucA 在乳酸乳球菌中分泌性表达,为进一步保护性抗原的分泌性表达研究奠定了基础。

关键词:乳酸乳球菌;食品级载体;分泌性表达;报告蛋白 中图分类号:R155.5　文献标识码:A

Construction of a food 2grade secretion expression vector

and the expression of reporter protein in L actococcus l actis

SUN Qiang 2zheng ,XION G Yan 2wen ,YE Chang 2yun ,XU Jian 2guo

(S tate Key L aboratory f or inf ectious disease p revention and cont rol ,N ational I nstitute f or Comm unicable Disease Cont rol

and Prevention ,Chinese Center f or Disease Cont rol and Prevention ,B ei j ing 102206,China )

A B S T RA C T :To construct a food 2grade secretion expression vector and its use in the expression of reporter protein nucle 2

ase (NucA ),a f ragment ,Usp45,containing the coding sequence of promoter ,ribosomal binding site ,secretion signal and the cod 2ing sequence for 11pre 2amino acids was amplified by PCR using the genomic DNA of ctis M G1363,and then inserted into the food 2grade vector p SH91,thus to construct a secretion expression vector pSQZ.In addition ,the coding sequence of NucA (nucA )was also amplified f rom S tap hy lococcus aureus chromosome and inserted into vector p SQZ under the control with pro 2moter usp45to construct a recombinant plasmid p SQZ 2nucA.Nuclease plate activity and zymogram assays were to be used to detect the NucA activity in the secretion expression of ctis .It was demonstrated that the secretion expression vector sys 2tem ctis MBP71/pSQZ 2nucA was successively constructed through the processes mentioned above and the quantity of Nu 2cA secreted into cultural supernatants was greater that that in bacterial lysates.Because all the f ragments used to construct the vector pSQZ were obtained from food 2grade bacteria ,the vector pSQZ can be regarded as a food 2grade secretion expression vec 2tor.These data can be used as the foundation for the further study on the secretion expression of the protective antigen in the o 2ral vaccine.

KE Y WOR DS :L actococcus lactis ;Food 2grade vector ;Secretion expression ;Reporter protein

通讯作者:徐建国,Email :xujg @ 作者单位:中国疾病预防控制中心传染病预防控制所,传染病预防

控制国家重点实验室,北京　102206

乳酸菌(l actic aci d bacteri a ,L AB )是一类可发酵糖产生乳酸的细菌总称,包括乳酸球菌、乳酸杆菌、双歧杆菌、肠球菌等二十几个属,广泛用于食品工业,用来生产和保存乳制品及其他食品,被公认为

是安全的食品级微生物(generally regarded as safe ,GRAS )。以乳酸菌的模式菌乳酸乳球菌(L l actis )