| 1. Background and purposeEnterohemorrhagic Escherichia coli (EHEC)0157:H7is a new type of food-borne pathogenic bacteria, spread by the food mainly, and the cause of food poisoning. The main clinical manifestation are hemorrhagic colitis (HC), thrombotic thromobocytopenie porpura (TTP) and hemolytic uremic syndrome (HUS), and so on.Since1982, the United States first outbreak EHEC0157:H7after infection, then all over the word appeared to sends out and explosive epidemic cases successively. Both the pathogenic rate and mortality are high. Our country first separated the EHEC0157:H7from the patient with hemorrhagic colitis in1986. And now, more than10provinces have separated the EHEC0157:H7from food, poultry, livestock and stool which came from the diarrhea patients. EHEC O157: H7have become a serious threat to public health and human health. How to detect it quickly and to prvent and control the infection effectively are always a difficult problem that the scholars pay close attention to.At present, there are many methods to detect the EHEC0157:H7. The traditional separation culture method has the advantage of low cost and is widely accepted. But it needs to separate training, many steps for biochemical and serum indentification, so the operation is complex and the cycle is long. PCR is one of the most commonly method to detect EHEC0157:H7in recent years. This method could amplification trace specimens, but it needs special equipment and the testing cost is high, also the specific and simplific have deficents. Therefore, establishing a rapid, simple and accurate testing method is very important to the epidemiological survey and prevention and control work for the EHEC0157:H7infection.LAMP is a new nucleic acid isothermal amplification technology, is established by Notomi et al Japanese scholars. It designes4or6pairs of special primer according to the target genes6or8region, then usees a chain replacement DNA polymerases (BstDNA polymerase) in isothermal conditions (about65℃), and keeps the temperature for dozens of minutes, then the nucleic acid amplification reaction can complete. It can amplification the target DNA highly efficient, fastly, sensitively and specificly. The method is simple, low cost, suitable for large-scale sample testing, and now is widely used in disease diagnosis, pathogenic microorganism inspection and the animal embryos in the gender identification, etc.Our study choose the rfbE gene (GenBank:S83460.1) of EHEC0157:H7, and used online primer design software Primer Explorer4.0to design LAMP primer. We optimized the amplification condition and established the LAMP reaction system. We also used PCR detection method simultaneously, and then compared these two kinds of testing methods among specificity, sensitivity and the results of the actual sample in order to prove the reality of the LAMP is feasible.On the other hand, there is lack of effective prevention and control method for the EHEC0157:H7infection. Due to the antibiotic therapy can lead to EHEC0157:H7cell walls to dissolve and promote shiga toxin (Stx) release, then can increase the risk of patients developed HUS, so the clinical therapy is given priority to the prevent and conservative therapy. There is no effects of drug treatment. So developing a safe and effective vaccine to prevent EHEC0157:H7infection has the important meaning.At present, the domestic and foreign researchers have done a lot of work for developing EHEC0157:H7vaccine, which mainly in the following aspects:the polysaccharide combined vaccine, bacteria ghost vaccine, live vaccine carrier, genetic engineering unit vaccine, transgenic plants vaccine and Stx endotoxins vaccine, etc. Although the above vaccine researches have get some progress, even some have already entered clinical trials, but there are also some flaws. So far, there is no ideal vaccine for clinical application.In recent years, the research of Lactic acid bacteria as a carrier to express and transfer purpose antigens cause for concern. The bacteria is safety, non-toxic, and is generally recognized as safe (GRAS). With the separation of all kinds of expression regulation components of the lactic acid bacteria, researchers have developed a series lactic acid bacteria carriers, such as the cloning vector, expression carrier and integrating carrier, and the field of lactic acid bacteria efficiently express system construction of food grade and application is the frontier and hot spots. Our research focuses on the establishing the food grade lactobacillus acidophilus expression system, and used as the EHEC O157:H7vaccine antigens delivery system.The food grade lactobacillus acidophilus expression system must have the following reguirements:1. The carrier must be food grade, shall not contain the non-food grade functional DNA fragments;2. The express host must be safe food grade microbial;3. The induction things must be food grade, like milk chain peptide, lactose, purine bacteria, pyrimidine, sucrose, etc, that can be people to eat.This study choose lactobacillus acidophilus as host bacterium expression carrier. The bacteria is non-toxic, and can adhere to mucosal epithelium closely, can synthesis and secrete exogenous protein, can directly contact with the immune system and cause the mucous membrane immune response. The bacteria don’t need to cultivate nor purify the purpose antigen. The oral way can used as immune inoculation way. So this bacteria is the ideal genetic engineering vaccine carrier.Using the bacteria nutrition defect type complementary principle, we selected LacF genes as markers, and built half nucleoside lactose enzyme defect type lactobacillus acidophilus (LacF-mutations) and half lactose glucoside enzyme complementary carrier (LacF+plasmid), and constituted the lactose inducing food grade lactobacillus acidophilus expression system. And this would be a foundation of developing food grade live vaccine carrier for preparing and preventing EHEC0157:H7infection. 2. Methods2.1According to the conservative district of rfbE gene sequences (GenBank: S83460.1) of EHEC0157:H7, we designed the specific LAMP primers, and optimized the amplification conditions, established25ul of LAMP reaction system.2.2We used39experimental strains as templates, and used LAMP and PCR amplification method respectively, then compare the specificity of this two methods. Diluted the overnight cultured EHEC0157:H7standard strain, LAMP and PCR amplification respectively, and compare the sensitivity of the two methods.2.3We took3samples from the32pork samples (EHEC0157:H7negative), and vaccinated with EHEC0157:H7, followed by LAMP and PCR detection, then compared the results with national standard method.2.4We amplification out LacF gene from the L. acidophilus ATCC4356genome, and connected to the PMD-19T, amplification LacF that both ends contain restructuring of the homologous recombination seguqence and connected to the son PUC-19. We amplication the mutants LacF, then connected to the above carrier, and substituted for the no righteous sequence (PUC-19:ΔLacF). We cloned the ΔLacF which gone through T-A cloning to knock down carrier PBR322, then transferred to lactobacillus acidophilus through electricity transformation. We choose seal method to select the mutations and Southern blot to identify.2.5We used pNZ9530plasmid as templates, and amplification RepA-RepC replicators and nisin promoter Pnis, then connected it to a skeleton plasmid pllac, synthetic MCS polyclonal site and cloned to the above plasmid. Then, We cloned LacF to this carrier, and contributed half lactose glucoside enzyme complementary plasmid which using LacF as selection marker. Finally, We transferred the plasmid to the mutations which constructed in last step and observed the growth in the lactose culture medium.3. Result3.1We successfully constructed the optimization of LAMP raction system.3.2The results showed that the specificity of LAMP method was higher than PCR when compared the specificity and sensitivity between LAMP and PCR method. The detection limit of LAMP method was101cfu/ml, an order of magnitude higher than PCR. 3.3For real samples, the results from LAMP method and PCR method are consistent with the conventional detection results. All of them tested only3positive samples.3.4This experiment got LacF defects homology fragments ΔLacF, and successfully constructed restructuring knock down carrier PBR322-ΔLacF, and screed and identified the half lactose enzyme defect type lactobacillus acidophilus.3.5Our study successfully constructed half lactose glucoside enzyme complementary plasmid which using LacF as selection marker. We transferred the plasmid to the mutations which constructed in last step and cultured in the lactose culture medium, mutations recovery of growth. This explained we successfully constructed the half lactose glucoside enzyme complementary defect type lactobacillus acidophilus expression system.4. ConclusionWe successfully established the LAMP detection technology which can detect EHEC O157:H7. Through the exploration of the reaction conditions, we optimized a complete reaction system and amplification procedures. The amplified product could be observed as white precipitation by the naked eyes, and could present strong fluorescence in the ultraviolet irradiation after join SYBR Green I, and could be observed the specific bands after gel electrophoresis.The specificity and sensitivity of LAMP method are better than PCR method through comparison. In the real sample test, both the two methods tested out the positive samples, and was consistent with notional standard method. This suggested LAMP method is realistic. So, LAMP technology is a simple, quick and high specific gene amplification technology. This will provide a new direction for rapid detect EHEC0157:H7infection.Our study successfully constructed lactose induction food grade lactobacillus acidophilus expression system by using bacteria nutrition defect type complementary principle. This laid the foundation of developing food grade live vaccine carrier for preparing and preventing EHEC0157:H7infection. |
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