[Construction of a food-grade secretion expression vector and use it for reporter protein expression in Lactococcus lactis].
We constructed a food-grade secretion expression vector and used it for reporter protein expression in live delivery vehicle L lactis MBP71. The p32 fragment, which containing the stronger p32 promoter, was amplified by polymerase chain reaction (PCR) with the plasmid pMG36e as template. After being purified, the p32 fragment was ligated with SPusp45 fragment amplified from genomic DNA of L lactis MG1363. The fusion fragment p32-SPusp45 was inserted into the food-grade vector pSH91 to construct a secretion expression vector, pSQ. The coding sequence of NucA (nucA) was also amplified from Staphylococcus aureus chromosome and inserted into pSQ under the control of p32 promoter to construct a recombinant plasmid pSQ-nucA. Nuclease plate activity assay and zymograme assay demonstrate that NucA was secretion expressed from L lactis harboring the recombinant plasmid pSQ- nucA, and the quantity of NucA secreted into supernamant was about ten times more than which in cell lysate. Results also indicate that expression efficiency of L lactis/pSQ-nucA was higher than that of L lactis/pSQZ-nucA, constructed by us earlier.
Sun
Qiangzheng
Q
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. zhlbsun@yahoo.com.cn
Xiong
Yanwen
Y
Ye
Changyun
C
Xu
Jianguo
J
D004268:DNA-Binding Proteins / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005516:Food Microbiology; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000201:enzymology
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D003851:Deoxyribonucleases; C052989:nucA protein, Bacillus subtilis