Expression Of Vibrio Cholerae Outer Membrane Protein W And U And Analysis Of Their Antigenicity

Vibrio cholerae belongs to the gram-negative bacteria, has been identified more than 200 different serogroups based on bacterial antigens. Vibrio cholerae distributes in rivers, lakes and coastal waters, and is a member of natural water environment, can cause water-or food-borne illness outbreaks. It has reported that there are 200 million cases of cholera caused by O1 and O139, non-O1, non-O139 is the main regional outbreak of diarrhea, so detection of vibrio cholerae is the key to control disease.Currently, several methods are used to detect V. cholerae, such as PCR, serological method, isolation and culture method, etc. However, each of them has limitations for day to day usage. Immunogold labeling technique is a new solid-phase immunoassay technology, which includes labeling the antibody with colloidal gold and subsequent localization and even quantification of the antibody-specific antigen or antigenic substance under the electron microscope or light microscope. It has the advantage of simplicity, high sensitivity and repeatability. It does not require special equipment and reagents and is popular in the clinical tests, animal and plant quarantine and food safety.OmpW and OmpU are important outer membrane proteins of V. cholerae. Both of them have good antigenicity. When vibrio cholerae infected the body, they can irritate the production of the antibodies of OmpW and OmpU, which can be used for cholera vaccine. OmpW and OmpU of vibrio cholerae are the potential adhesion factors, may play a role in the process of bacterial invasion and virulence, but their precise functions need further researching. OmpW is the species-specific antigen of vibrio cholerae. The expression of OmpW depends on the environmental and pressure conditions. OmpU is the porin of vibrio cholerae as a hydrophilic and low molecular weight material input and output channels. When V. cholerae grows in the normal condition, OmpU can compose 30 % of outer membrane proteins, but it also can achieve 60 % or more in the salt environment. The function of OmpU is similar to OmpF of E. coli. The homologous nucleotide sequences do not exist between OmpU and OmpF, and the antiserum of OmpU does not have cross-reaction with OmpF.In this study, based on the morphological identification, we used the API 20NE and PCR to evaluate the colloidal gold test strip, while the purified recombinant V. cholerae outer membrane proteins OmpW and OmpU were used to immunize New Zealand white rabbits and polyclonal antibody were isolated to produce colloidal gold immunochromatographic test strips for rapid detection of V. cholerae based on the double antibody sandwich method. We also optimized the key steps for using this test strip, which built the foundation for the commercialization of colloidal gold test strips for V. cholerae detection. The results of this study are as follows:1. Identification of the stocked vibrio choleraeV. cholerae in the samples was enriched first in the alkaline peptone solution and then cultured in trypsin soybean agar plates for 12 hour. When V. cholerae grew well, colonies were round, convex and had smooth surface. They were stained red by Gram's staining. Bacteria bodies were short, slightly curved or comma-rounded in shape. On the top of the morphological study, the the standard identification system for non-enterobacteriaceae and gram-negative non-fastidious bacteria, API 20NE identification system, was used to verify the identity of V. cholerae and the results were 27stains of V. cholerae. The results were verified by PCR method using primers to recognize the species-specific antigen ompW gene of V. cholerae, indicating that API 20NE can be used for initial detection of V. cholerae. At last, this 27 stains of V. cholerae strains were typed by the diagnostic antisera of O1 (Ogawa and Inaba) and O139 group.2. Cholerae antigen genetic engineering and identification of polyclonal antibodiesThe ompW gene without the signal sequence and the ompU gene were amplified by PCR from the genomic DNA of V. cholerae 860069 strain (should give primer sequences). The amplified products were 588 bp and 1026 bp on agarose gel eletrophoresis. The amplified products were inserted into the pMD18T vector to construct the cloning plasmids pMD18T-ompW and pMD18T- ompU. E. coli stain JM109 was transformed with the cloning plasmids and the plasmids from the transformants were confirmed by PCR. The plasmids were digested with BamH I and EcoR I. The double-digestion products were recovered and ligated with the expression vector pET32a digested with BamH I and EcoR I. E. coli BL 21(DE3) strain was transformed with resulting expression plasmids pET32a-ompW and pET32a-ompU after their sequences were verified by DNA sequencing. Protein expression in the engineered E. coli strains was induced with 1mM IPTG for 5h in 37℃. Due to the incorporation of thioredoxins, recombinant OmpW and OmpU (r-OmpW and r-OmpU) were about 41 kDa and 57 kDa in size. The expression levels of r-OmpW and r-OmpU reached as high as 53% and 57% of total protein. After purification, the purity of r-OmpW and r-OmpU were over 90%. Western Blot analysis showed that r-OmpW and r-OmpU reacted with the serum from rabbits immunized by V. cholerae, suggesting the good antigenicity of r-OmpW and r-OmpU.The purified r-OmpW and r-OmpU protein were used to immunize New Zealand white rabbits to raise antibody. The titers of the resulting antiserum were 1:32 determined by agarose double diffusion method. The antiserum against OmpW or OmpU recognized V. cholerae O1 classical or EL Tor groups and O139 serogroup in Western Blot analysis, indicating these antibodies have the potential to be used as the general detection antibody for V. cholerae. The antibodies against OmpW and OmpU were further purified by saturated ammonium sulfate precipitation and protein G affinity chromatography.3. Development of gold immunochromatography assay to detect vibrio cholerae.The colloidal gold test strips were made of the filter paper with proper pore size as carrier and included a detection region containing colloidal gold labeled rabbit anti-OmpW antibody or OmpU antibody and a quality control region coated with goat anti rabbit IgG. If a bacteria sample contained OmpW or OmpU antigen, a red line, the color from colloidal gold, was visible on the strip due to the formation of antigen-antibody complex. Both of the colloidal gold strips were positive results when detected 27 stains of V. cholerae. The detection rate is 100%. In the detection of V. alginolyticus, V. vulnificus, V. parahaemolyticus, the strip of OmpW antibody can produce positive results when detected V. parahaemolyticus (860160) or V. vulnificus (870101) at the concentration of 1×108cfu / ml.