[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.
Xu
Bo
B
The Key Laboratory of Food Science of Ministry of Education, Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China. xubo583@sohu.com
Cao
Yu-sheng
YS
Chen
Yan
Y
Guo
Xing-hua
XH
D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D005516:Food Microbiology; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D007114:Immunization; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011817:Rabbits; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins