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Background Tuberculosis is a chronic infectious disease spread through the respiratory tract, occurs mainly in the lungs, is a single pathogen infection important infectious cause the highest mortality worldwide [1]. Retrospective survey results show that our TB misdiagnosis rate of up to 24%, and urgently need to improve the laboratory diagnosis of tuberculosis rate. Therefore, the use of simple, rapid, accurate laboratory tests as soon as possible to make a diagnosis of tuberculosis is essential to control the TB epidemic. The gold standard for the diagnosis of tuberculosis bacteriology low, but positive rate of smear and culture, and bacterial culture is also more time-consuming, and can not make a timely diagnosis of tuberculosis [2]. Body by TB infection, the body first appeared tuberculosis antigens detect tuberculosis antigen important early diagnostic value, and the TB antigen can be detected as Mycobacterium tuberculosis direct evidence, and avoids TB patients because of low immune response cause detection of humoral immunity or cellular immune detection of \Earliest of Mycobacterium tuberculosis Mycobacterium tuberculosis antigen is cultured in a liquid medium containing glycerol, sterile filtered, and concentrated prepared from known as the old tuberculin. Since 1932, the application of protein purification means further purification old tuberculin, the resulting product is the tuberculin protein derivative (purified protein 衍生, of tuberculin, PPD). PPD is mainly used for the tuberculin skin test (tuberculin skin test), due to its complex antigen components, most of the ingredients shared by non-tuberculous mycobacteria and BCG (BCG), and therefore low specificity, and can not distinguish between infection and BCG vaccination open tuberculosis and systemic tuberculosis infection late insensitive. Culture filtrate protein 10 (culture filtrate protein 10, CFP10) is found in the culture filtrate of a molecular weight of about 10KDa highly specific antigens from Mycobacterium tuberculosis in recent years, specific to the human type of Mycobacterium tuberculosis and Mycobacterium bovis BCG (BCG) and non-pathogenic mycobacteria lack of good immunogenicity, can be used as a specific candidate antigens for diagnosis of tuberculosis, but usually TB patients serum or body fluids (sputum, pleural effusion, cerebrospinal fluid, joint effusion The) in CFP10 content is very low, detected by ELISA, colloidal gold immunochromatographic method, it is difficult to achieve the desired results. Time-resolved fluorescence immunoassay technology (time-resolve fluoroimmunoassay, TRFIA) new development since the 1980s a new type of marker analysis technology, has its unique advantages compared with other immune markers analysis techniques. It overcomes the radioimmunoassays (Radioimmunoassay, RIA) pollution problems caused radioisotope; overcome the disadvantage of enzyme instability of the enzyme immunoassay (Enzyme immunoassay, EIA); Moreover, since the unique fluorescence characteristics of lanthanide technology and the wavelength resolution TRFIA detection and time delay can be good to eliminate the interference of background fluorescence and dissociation enhancement technology, the fluorescent signal amplification million times, making it more sensitive than ordinary fluorescent (Fluoroimmunoassay, the FIA ) several orders of magnitude higher, with high sensitivity (10-18 mol / holes) is simple, easy to automate a wide range of standard curve, from samples of natural fluorescence interference markers preparation simple and stable, non-radioactive contamination, multi-label characteristics, has a great potential for clinical application minim analytical techniques. Through genetic engineering techniques, we prepared a CFP10 three recombinant proteins the CFP10 and streptavidin labeled CFP10/SA, the purified recombinant antigen renaturation identified, the application of time-resolved fluorescence immunoassay technology to double antibody sandwich create a reaction mode and optimize the conditions. CFP10, CFP10/SA analysis to compare sensitivity, stability, and filter out the optimal target protein as a detect the a CFP10 reference standard, and the initial establishment of the method of time-resolved fluorescence immunoassay detection CFP10. The purpose prepared a CFP10 three recombinant proteins the CFP10 and streptavidin labeled CFP10/SA, selected from a suitable target protein as detection a CFP10 reference standard, and the initial establishment of the time-resolved fluorescence immunoassay detection CFP10 method. Method 1. Recombinant plasmid multicloning site primers designed to tuberculous mycobacterial standard strain H37RV whole genome as the template according to a CFP10 the gene sequence and the selected carrier, adding PCR reagents, CFP10 fragment was amplified, according to the design of the restriction sites were digested pET21a-SA vector pET24b, pET24b-SA carrier connections, transformed into DH5α competent cells, several monoclonal colonies were picked from the transformed resistant plate colony PCR and double digestion selection and identification of successful delivery company bidirectional sequencing. 2 recombinant protein expression and purification of the correct sequencing of plasmid transformed into E. coli the Rosetta three monoclonal colonies were picked inoculated in 3mlLB standard medium, grown to OD600 = 0.4-0.6, and added to a final concentration of 1 mmol / L of IPTG, were selected at different temperatures and different induction time for culture, different experimental conditions on the amount of recombinant protein expression, and to optimize the expression conditions. Under optimal culture inducing conditions, a large number of expansion bacterial expression, cells were collected and added to lysis buffer, and the ice bath was sonicated bacteria, by SDS-PAGE electrophoresis analysis of the form of expression of the protein, and inclusion body expressed protein to five chamber invention step washing method be washed. Will sufficiently dissolve the inclusion bodies were washed 8M urea nickel metal chelate chromatography column (Ni-NTA), after separation and purification, for soluble recombinant protein, is collected sonicated supernatant directly after filtration by affinity and layers analytical column purification. Refolding of the recombinant protein by Western blotting The purified denatured protein concentration adjusted to 0.2 mg / mL, greater than 50 times the volume of dialysis fluid (50 mmol / L Tris-HCl and 5M urea, 0.5M L-Arg , 0.1% PEG8000, 0.5M EDTA), 4 ° C urea gradient (5M-3M-2M-1M-0M Urea) dialysis, and the medium was changed once every 4h to 0M urea, and finally with 50 mmol / L Tris-HCl ( PH8.0) slow dialysis for 12-24h, the supernatant was collected by centrifugation. And dialysis bag and protein solubility was observed in the dialysis process, the turbidity or precipitate forms, refolded protein by SDS-PAGE electrophoresis renaturation effect. The recombinant protein was separated by SDS-PAGE electrophoresis, proteins were transferred to PVDF membranes using wet electroporation method. 0.2% Ponceau staining observed transfer effect, satisfied with blocking solution (5% nonfat dry milk, PBST preparation) 37 ° C incubated for 2h, wash the film, adding blocking solution diluted mouse-CFP10 monoclonal antibody, 37 ℃ incubated 2h, the membrane was washed by adding PBST diluted secondary antibody HRP-labeled goat anti-mouse IgG, 37 ° C incubated for 1h, after the film is fully washed by adding DAB reagent color, color when appropriate, and deionized water to terminate. Time-resolved fluorescent immunoassay detection the CFP10 reference product screening identification application time-resolved fluorescence immunoassay technology, double antibody sandwich method to establish the response pattern and coated antibody and labeled antibody concentration optimization. Then of CFP10 SA/CFP10 in analytical sensitivity, stability compared to select the right target protein as detection a CFP10 reference standard, and online reference standard, precision and specificity analysis evaluation. Results 1. Recombinant plasmid constructed three recombinant plasmid DNA sequencing, sequencing results included in the GeneBank SA and CFP10 sequence alignment, are fully consistent and non-reading error. Recombinant protein expression, purification, sequencing the plasmid was transformed into E. coli the Rosetta induced expression, cells were collected by ultrasonic sonicated, SDS-PAGE electrophoresis results show SA-CFP10 and CFP10-SA, the molecular weight of about 30KDa with theoretical values, the expression levels were about 25% and 30% of the total bacterial protein. CFP10 basically consistent with molecular weight in 10KDa around with the theoretical value, the expression level of total bacterial protein of about 35%. The fusion protein SA/CFP10 mainly exist in the form of inclusion bodies broken bacteria after precipitation, and CFP10 recombinant protein in a soluble form of sonicated supernatant. After the fusion protein SA/CFP10 washed inclusion bodies After filtration of the soluble CFP10 recombinant protein are purified by Ni-NTA, His-tag, the SDS-PAGE electrophoresis results show that the purification effect of the three recombinant proteins were better, purity can reach above 90%. Refolding of recombinant proteins by Western blotting the fusion protein CFP10/SA purified placed in a dialysis bag, dialysate urea gradient the 4 ℃ dialysis refolding, SDS-PAGE electrophoresis results to see non-reducing strip on trimer (streptavidin SA itself may be formed tetramer). Soluble expression of CFP10 slight degeneration in 2M, equivalent to the refolding conditions renaturation CFP10/SA. Three recombinant proteins can be identified by Western blotting, and imported CFP10 monoclonal antibodies that specifically react. Time-resolved fluorescence immunoassay detection the CFP10 reference product screening identification we have established a time-resolved fluorescence double antibody sandwich method, and coated antibody and the labeled antibody concentration optimization, orthogonal experiment to determine the optimal conditions: coated The antibody concentration 3μg/mL labeled antibody concentration of 0.8μg/mL. Homemade of CFP10-SA SA-of CFP10 and CFP10 in analytical sensitivity, stability compared to that CFP10-SA is the best its linear range 0-80ng/ml, and minimum detectable concentration of up to 0.02ng / ml, were placed in the 37 ℃ 7 days, 14 days, 21 days, 28 days, the maximum degradation rate of less than 8%, is optimum. Analysis through its precision, intra CV lt; 6%, inter-assay CV lt; 5%, good precision. Specific experimental results show that the, CFP10-SA and SA-IL15, SA-GM-CSF, the ESAT6 SA-ESAT6 ESAT6-SA had no cross-reactivity and specificity good. Draw the the dual logarithmic function TRFIA standard curve, R2 = 0.9997, well, and has good repeatability. The successful screening of the reference standard, and double-antibody sandwich time-resolved fluorescence immunoassay detection the CFP10 method of initial establishment, culture filtrate protein 10 time-resolved fluorescence analysis test kit and its clinical application to lay a foundation for further study of Mycobacterium tuberculosis . Conclusion 1. Successfully prepared the CFP10-SA, SA-CFP10 and CFP10 recombinant protein. Initial establishment of the time-resolved fluorescence immunoassay detection a CFP10 method and filter out the optimal reference product CFP10-SA
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