| Most pathogens enter into the body by mucosal surface, so development of mucosal delivery system for vaccines or medicines should be a very effective way to therapy a wide range of infectious diseases. Lactic acid bacteria (LAB) are considered as GRAS (Generally Regarded As Safe) microbe, play probiotic roles at the intragastric for the benefit to human and animals health care. Lactobacilli is a group of normal bacteria colonized on the female vagina's mucosal surfaces, restrains pathogens growth and regulates the microecology at the female vagina. So, it's a good ideal to develop LAB as mucosal delivery system for vaccines or medicines acted on the mucosal surface against pathogens. The molecular genetics research of LAB expression system is not so mature as other heterologous protein expression system. The object of this paper is to construct LAB's expression system for the vector of mucosal delivery system.1. Construction of recombinant Lacbacillus for microecology at the vagina.NICE(nisin-controlled expression) system was constructed in Lacbacillus plantarum. Nisin was a 34-amino acid anti-microbial peptide (lantibiotic). NisR and NisK belong to the family of bacterial two-component signal transduction systems. NisK is a histidine-protein kinase that resides in the cytoplasmic membrane and is proposed to act as a receptor for the mature nisin molecule. Upon binding of nisin to NisK it autophosphorylates and transfers the phosphate group to NisR, which is a response regulator that becomes activated upon phosphorylation by NisK. In this research, nisA promoter, nisR and nisK was cloned from Lactococcus lactis 1.2030 genome DNA respectively. Besides above of all theses essential elements, Ori replicon from E.coli and p256 from Lacbacillus, the antibiotic resistance marker erythromycin were composited of a shuttle vector. The enhanced interferonωwas linked respectively behind the promoter PnisA. So recombinant pSIP300-ωvector was constructed. Though, heterologous protein expression was a little lower. Another induced expression system similar to the NICE was constructed. Biotin sakacin A was inducer. sapK and sapR was the cognate regulatory system. IFN-ωwas linked behind with the promoter PsapA. The recombinant vector pSIP300-ωwas electrotransferred to Lacbacillus plantarum 1.3, Lacbacillus plantarum 1.11, and Lacbacillus plantarum 14917 respectively. In the result the recombinant Lacbacillus plantarum 1.11/pSIP300-ωcan express the IFN-ωby Bender MedsystemsTM human IFN-ωELISA BMS233TEN kit identification, in levels to several milligram per litre.2. Construction of recombinant Lactococcus lactis for oral or intranasal immunization.The promoter P170 used in the Expression System is regulated by pH and growth phase i.e. the activity is strongly upregulated at pH below 6.5 in the transition to stationary phase, without the need for addition of an exogenous inducer. Consequently, the growth phase is separated from the protein production phase. The SP used in this expression system, SP310mut2, is an optimized version of an SP from a native lactococcal protein. It has been shown that the sequence around the signal peptidase cleavage site affects the efficiency of protein secretion. Therefore, some of the expression vectors contain a sequence encoding four amino acids (AERS) after the cleavage site. Yersinia pestis LcrV antigen gene was cloned into vector pAMJ397, electroporation into Lactococcus lactis PSM565. PSM565 was derived from strain MG1363 (MG1363 is a prophage-cured and plasmid-free derivative of L.lactis ssp. cremoris NCDO712. It is one of the most widely used L.lactis strains in biotechnological research and as such the strain is well characterized genetically and physiologically.) by chemical mutagenesis using methanesulfonic acid ethyl ester. PSM565 is able to produce and secrete increased amounts of heterologous proteins. By PCR identification, bacterial inocula, SDS-PAGE detecting, and Western blotting analysis, a 38kD Yersinia pestis LcrV antigen was found in the supernatant of the culture medium, and recombinant LcrV was purified by DEAE-Sepharose F F.
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