Research On The Expression, Immunogenicity Of ActA And Colloidal Gold Strip Of Listeria Monocytogenes

Listeria monocytogenes (LM) is gram-positive, intracellular bacteria and also a food-borne pathogen for animal and human's listeriosis. LM infected human and more than 20 species of animals and was ranked as one of the four main food-borne pathogens in 1990's by World Health Organization (WHO). Recent years the incidence of listeriosis is rising all over the world. Although there is no report about listeriosis outbreak in China up to now, but LM could be detected from many foods. Therefore the main measures to prevent listeriosis are developing quick and specific method to detect LM and preparing effective vaccine.Colloidal gold detect technique give a new live to detect LM quickly. Colloidal gold detect technique is a new method to detect antigens or antibodies takes colloidal gold as the tracing marker. Colloidal gold strip is one of the main models and has many benefits, such as quick, sensitive, specific, stable, easy to perform with no requirement of special skill, reagents or equipment. At present it has been applied broadly in veterinary field to detect pathogens, toxin, medicine residue, and so on.Actin polymerizing protein (ActA) is surface protein, which mediates the polymerization of actin fibril so that propelling the bacteria to move intracellular and spread to adjacent cells. According to the genome sequence of actA gene reported in Gene Bank, one pair of specific primers were designed and synthesized. LM actA gene without signal peptide sequence was amplified by PCR and inserted in pMD-18T Simple Vector. After sequencing and enzyme digestion confirmation, the gene was then inserted in prokaryotic expression vector pGEX-3X to obtain the prokaryotic expressed plasmid pGEX-3X/ActA. The target gene was then expressed in the E.coli BL21(DE3) cells by inducted with IPTG. The size of fusion protein was consistent with the prediction by SDS-PAGE analysis. The fusion protein was purified with GSTrap FF column and the GST-tag was digested with Factor Xa. The purity of the purified ActA was over 90% by Image Master VDS software analysis. The content of the purified ActA was 0.86mg/mL. The purified ActA could react with polyclonal antibodies anti-LM by Western blot.Balb/c mice were immunized with the purified ActA emulsionized with FCA or FIA. After two weeks of immunization, the specific antibody level of mice serum was tested by indirect ELISA every week. The results indicated that ActA could effectively induce immunized mice to produce the specific antibody and the antibody level was highest at seventh week after immunization. The lymphocyte proliferative response and the subsets of the T lymphocyte of immunized mice were detected. The stimulation index (SI) of the immunity group and control group were 1.618±0.096 and 1.193±0.054 respectively. The proliferation response of the immunity group was higher than that of the control group (t-test, P<0.05). The mean of CD3+, CD4+, CD8+ , CD4+/CD8+ of the immunity group and the control group were 36.9%, 20.3%, 10.2%, 1.99 and 31.5%, 17.2%, 11.5%, 1.5 respectively. The mean of CD3+, CD4+, CD4+/CD8+ of the immunity group was increased by 17.14%, 18.03%, 32.67% respectively contrasted with control group, but the mean of CD8+ was decreased by 12.38%. These results indicated that ActA induced specific humoral immunity and cellular immunity. This study laid foundation for the development of immunology detection method and preparation of subunit vaccine of LM.The detection approach established by monoclonal antibody was more specific. The combination of monoclonal antibody and Colloidal Gold strip enhanced the specificity and sensitivity greatly. In this study the female BALB/C mouse were immunized by LM. After 4 times immunization, the splenocytes from immunized were fused with SP2/0 myeloma cells. The purified protein ActA was used as detecting antigen and the supernatant hybridoma clones were screened by indirect ELISA. Four hybridoma cell lines stably secreting McAbs against ActA were obtained, named 3G6, 2B4, 4E10 and 2B9. The of chromosome modes of the hybridoma cells were 80～90 and had good genetic stability. The immunoglobulin subclass of 2B4 was IgM, the others were IgG1. The specificity analysis showed that 3G6, 2B4, 4E10 were specific McAbs against LM and 2B9 was specific McAbs against LM and L.ivanovii. The ascites of 3G6, 4E10, 2B9 were purified by Caprylic acid一Ammonium sulfate and affinity chromatography. The antibody titers of ascitic fluid were 1:64000, 1:64000, 1:32000 respectively and the purities were 88.9%, 86.3%, 90.1% respectively. The affinity qualities of 3G6, 4E10 were 6.62×10⁷ M^-1, 5.67×10⁷M^-1 respectively. So 3G6 and 4E10 can be used to develop colloidal gold strip.The colloidal gold strip for detecting LM was developed using McAbs 3G6 and 4E10 based on colloidal gold immunochromatographic assay. The colloidal gold particles prepared by sodium citrate reduction method were uniform and well-distributed under the electron microscope. The colloidal gold strip was developed by using 3G6 labeled with colloidal gold as the detector, 4E10 and the purified goat anti-mouse IgG were blotted on the nitrocellulose membrane as the test line and control line respectively. The evaluation of specificity, sensitivity, stability and simulated samples of the colloidal gold strip were performed. The results showed that the strip has a high specificity and has no cross reaction with L.ivanovii, L.innocua, Escherichia coli, Salmonella, Bacillus subtilis, Staphylococcus aureus and Streptococcus. The sensitivity of LM pure culture was 3.9×10⁵CFU/mL. The expiration date was more than 120 days stored at 4℃. The sensitivity of the simulated samples including water, milk, pork, vegetable, freeze shrimp and bread without enrichment culture were 3.9×10⁵CFU/mL.