Construction Of Expression Vector And Heterologous Expression Of Exogenous Gene In Lactobacillus Casei

Lactic acid bacteria(LAB) is one of the traditional industrial strains and receives much concern on its food grade security features.At present, some recombinant lactobacillus producing proteins, enzymes or metabolite have been reported. Compared with Escherichia coli and Pasteur pichia expression system, LAB especially the Lactobacillus expression system have not been well developed.This study took Lactobacillus casei MCJΔ1 as research object. On the basis of Intracellular expression vector p ELX1,12 proteins were tested the expression of generality of p ELX1,such as Epidermal Growth Factor(EGF), soybean peptides lunasin, Chitosan excision enzyme(Csx), Chitosan incision enzyme(Csn), Chitosan internal and external enzyme(Csx-n)from aspergillus niger, nattokinase(NK) from Bacillus subtilis, Mannanase(Man) from B. pumilus, β-D-glucuronidase(Gus A) from E.coli K12, β-galactosidase(β-gal)from Lb. casei ATCC 334, 6-phosphoric acid-β- galactosidase(Lac G) from Lb. casei ATCC 393, S-Adenosyl-L-Methionine Synthetase(Smat) from Sulfolobus islandicus and hand-foot-mouth disease antigen protein(VP1). Four recombinant proteins, namely Man, Gus A, β-gal and Lac G were successfully detected by SDS-PAGE and/or Western blot. The determination of enzyme activity, the maximum mannanase activity was about 23 U/mg total protein and the Gus A was 75 U/mg total protein. After analysing the Man property in different growth period ofp ELX1-Man-MCJΔ1,Results indicate that Man is more suitable for quantitative report gene than Gus A.Two expression vectors, secretory expression vector p ELSH and cell surface-anchor expression vector p ELWH were constructed based on the use of secreted usp45 signal peptide(SPusp45) from Lactococcus lactis and Slp A protein of S layer protein from Lactobacillus acidophilus NCFM respectively. When using Man as the report gene, a large amount of recombinant Man protein was detected in the supernatant of p ELSH-Man varient,but not the same as p ELWH-Man variant which hold the protein only and in the surface of cell. The determination of enzyme activity, the maximum secreted mannanase activity was about 8800U/L for p ELSH-Man varient and 28 m U/5×109 cfu in the cellsurface for p ELWH-Man varient.In conclusion,heterologous protein was successfully Secretion and anchor expressed in L.casei by p ELSH and p ELWH. Construction of this L.casei expression system makes it possible for more foreign proteins expressed in L.casei and provides theoretical supports for L.casei as the Living bacterium vaccine antigen delivery carrier.