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Potential Mucosal Delivery System: Construction of Recombinant Lactic Acid Bacteria

Author: CaoXiaoMei
Tutor: ChenZuo
School: PLA Military Academy of Medical Sciences
Course: Biology
Keywords: Lactic acid bacteria Lactobacillus plantarum Lactococcus lactis Interferon omega The plague antigen- LcrV Delivery system Mucosa
CLC: Q78
Type: PhD thesis
Year: 2009
Downloads: 710
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The majority of pathogenic microorganisms enter the body through mucosal surfaces, the development of mucosal vaccines against the class of pathogens or therapeutic drug for the prevention or treatment of diseases ideal. Lactic acid bacteria (lactic acid bacteria, LAB) is a kind of food-grade security GRAS (Generally Recognized As Safe) microorganisms, and also the human intestinal probiotics on human health and nutrition benefits. Lactobacillus is the normal flora in the vagina of healthy women, from the Jieyin Road micro-ecological environment, which restricts other pathogenic bacteria colonization growth, but also vaginal probiotic. Lactic acid bacteria as mucosal immunity or treatment of drug delivery vehicle, the antigen or the treatment of drug directly to the mucosal surface, the vaccine or the development trend of protein drugs. Constructed in this study Lactobacillus and Lactococcus expression system intended to be used in the vaginal mucosa recombinant probiotics or gastrointestinal mucosal vaccine delivery vehicle. 1 Construction of expression the interferon omega restructuring Lactobacillus. Build Nisin Nisin as inducer NICE Lactobacillus plantarum inducible expression system (Nisin-controlled expression, NICE). Nisin is an antimicrobial peptide of 34 amino acids, and its synthesis is a two-component nisK (protein kinase) and nisR (regulatory proteins) adjustment control. This study from Lactococcus lactis the Lactococcus lactis 1.2030 genomic DNA was cloned into nisA promoter, nisR, nisK, the gene construct with the E. coli replicon-Ori and Lactobacillus replicon P256, erythromycin resistance marker for shuttle plasmid pNIS300. The the interferon omega the gene in PnisA promoter downstream, to build pNIS300-omega expression plasmid. After induction of expression, found that the amount of protein expression in Lactobacillus plantarum weak. Forthwith by the inducible expression system of another antimicrobial peptide similar to the NICE system, i.e. to bacteriocin SapIP is induced by substance the SAPK and sapR regulate gene expression plasmid pSIP300. Interferon-omega gene in downstream of a PsapA-promoter, composed of interferon expression plasmid pSIP300-Omega, were amplified in E. coli DH5, plasmid was extracted, respectively, was transformed into Lactobacillus plantarum: Lacbacillus plantarum 1.3 Lacbacillus plantarum 1.11 Lacbacillus plantarum 14917. With the Bender MedsystemsTM human IFN-ωELISA BMS233TEN kit detection, recombinant bacteria Lacbacillus plantarum 1.11/pSIP300-ω able to express IFN-omega. 2 to construct the proposed restructuring for oral or nasal mucosa immune Lactococcus lactis and Lactococcus lactis. The process of acid-induced P170 promoter by H and Growth of its own metabolism, in Lactococcus growth process, the medium pH values ??falling, when the pH is between 5.0-6.5, having an active promoter, is the bacterial cells from the exponential growth phase when the transition to the platform, and neither need the addition of an inducer, the cell growth phase and the production of exogenous protein can be separated from. Using SP310mmut2 signal peptide, the signal peptide is derived from Lactococcus lactis, and the enzyme cleavage site plus four amino acids AERS, these four amino acids are conducive to the secretion of heterologous proteins. The placed the AERS 4 amino acids after removal of the signal peptide of Y. pestis LcrV antigen structural gene, form recombinant plasmid pAMJ397-V power to transform competent state Lactococcus lactis and Lactococcus lactis PSM565. The recombinants were identified by PCR, cell culture, detection of SDS-PAGE, Western-blot identification, in Lactococcus lactis PSM565/pAMJ397-V medium supernatant 38kD plague antigen LcrV protein. With DEAE anion column, the proteins were preliminary purification.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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