Prokaryotic Expression Of Listeria Monocytogenes In1A Gene Fragments And Identify The Major Functional Area

Listeria monocytogenes(LM) is a zoonotic facultative intracellular parasite bacteria. In the20th century, It was distributed one of the four most important foodborne pathogens by the World Health Organization WTO widely.It has some characteristics,such as widely distributed,more susceptible populations, cause sepsis, abortion, meningitis and gastroenteritis symptoms in humans and animals, also cause miscarriages and causing neonatal sepsis through the placenta. InlA gene is important virulence genes of LM, InlA (internalized hormone A) was consisted of800amino acid residues, and it is the necessary when LM invasion of human epithelial cells. Its receptor was E-cadherin, which was the first identified virulence factors of bacterial invasion, was essential of the non-phagocytic cells, the unique of LM,is not stored in the surface of non-pathogenic Listeria.Preparation of InlA high affinity and highly specific antibodies are the base of study which segment of InlA protein is more important when LM invading cells. In this study, to clone LM specific membrane surface proteins InternalinA (InlA)gene, and obtain LM specific membrane surface proteins InlA antibody, then analysis composition of amino acid and protein structure. To obtain polyclonal antibodies by immunizing rabbits. This study laid the material foundation for the further research and analysis the role of InlA paragraphs proteins when LM invading in the cells.Use of biological software design LM InlA gene primers, amplified LM08-5923strain InlA gene by PCR. To break the InlA gene by Genebank description InlA amino acid sequences,which are segmented into two fragments gene InlA-1(1bp-1488bp) and InlA-2(1315bp-2403bp), were cloned into the prokaryotic expression vector pGEX-6P-1, co-transformed the recombinant plasmid and chaperone plasmid pG-KJE8into BL21. To induce the expression with final concentration of lmg/mL L-arabinose and lmmol/L of IPTG, to analyze the expression product by SDS-PAGE. To purify the products and then immunize rabbits, and prepare polyclonal antibodies. To detect the titer of polyclonal antibody and the reactivity of polyclonal antibodies with natural InlA by ELISA. To analysis reactivity that antibodies with the expression of products and natural InlA by Western blot. Select254-7weeks old BALB/C mices were randomly divided into five groups (PBS group, challenged group, immune InlAl group, immune InlA2group and immune InlAl+InlA2group), the immune team using purified proteins InlAl, InlA2and two together immunized mice, after the third immune14days given orally LM (inoculum reached about10CFU/mL), while giving the challenged group and PBS group fed the same conditions, the mice were sacrificed after gavage60h, To remove the large intestine of mice sterile, after washing with sterile PBS, homogenized, diluted the homogenized liquid based on concentration. Coated on the BHI (L+G)plate and count the colony, and compare LM colonization situation in colorectal epithelial cells after oral quantitative LM by this method, and to analyze the role of paragraphs InlA protein when LM invasion cells.Results:With positive plasmid and chaperone plasmid pG-KJE8expression system expressed InlA segment protein, molecular weight of the fusion protein approximately79ku and64ku. To purify the protein by GST-Sefinose TM Resin purification kit, then western blot analysis, the results showed that the two recombinant proteins were able to react with the two polyclonal antibodies, there were specific bands in the expected location, so the proteins with reactogenicity; The antiserum titers were obtained by immunizing rabbits, serum titer of InlA2(new flag) was1:4096000, The highest titer of InlA1(LRRs) was1:1024000; And polyclonal antibody can react with InlA natural proteins of Listeria monocytogenes. The results of ELISA show that the two kinds of recombinant proteins were able to induce an antibody response, which recombinant InlA-2induced higher antibody titer, has a strong binding capacity with InlA natural protein of Listeria monocytogenes. Animal experiments indicate that after challenge LM colonization amount in the large intestine epithelium cells, the immune team was significantly lower than challenged group, and LM colonization amount in the large intestine epithelium cells, InlA1immune team less than InlA2immune team, indicating InlA1(LRRs) segment plays a major role in the LM invade non-phagocytic cells.