| Goose astrovirus(GoAstV)is a novel pathogen that mainly attacks 4-21-day-old goslings and is characterized by visceral and joint gout.The viral capsid protein ORF2 is the main protective antigen protein of GoAstV.At present,there are no effective vaccines and drugs for the prevention or treatment of the disease.Therefore,early detection and accurate diagnosis are important means for preventing the epidemic and spread of the disease.Traditional electron microscope inspection,enzyme-linked immunosorbent assay,and RT-PCR-related molecular biology methods have certain advantages and disadvantages.Therefore,it is necessary to establish a rapid,simple,specific and efficient diagnostic method to detect the virus.POCT(point-of-care testing)refers to a testing method that is carried out at the sampling site,through the test paper,combined with portable analytical instruments to quickly obtain the test results.The principle of this method is to use fluorescent microspheres to label antibodies.Through immunochromatography and capillary action of antigens,a fluorescence reader is used to read the fluorescence intensity of the T and C lines set on the test board and calculate the T/C value.The standard curve built into the portable analytical instrument calculates the content of the antigen to be tested in the sample.1.In order to obtain high-purity soluble GoAstV antigen,the recombinant plasmid pGEX-4T-1-ORF2 expressing GoAstV partial capsid protein ORF2 nucleic acid fragment was transformed into Escherichia coli BL21 competent cells;after the optimization of the conditions,the maximum expression level of GoAstV-ORF2 fusion protein was obtained by inducing at 30 ? under IPTG concentration of 1.0mmol/L for 5h.After induction,pGEX-4T-1-ORF2 fusion protein was found to be expressed in large amounts in the supernatant.After purification,SDS-PAGE showed a single band with a size of 49.1 KD;the concentration of GoAstV-ORF2 fusion protein was 4.015 mg / ml as determined by the BAC protein concentration detection kit.2.In order to prepare a polyclonal antibody that binds well to GoAstV antigen,two New Zealandwhite rabbits were immunized with the purified GoAstV-ORF2 fusion protein by conventional immunization procedures to prepare rabbit anti-GoAstV-ORF2 polyclonal antibodies.The titer of the polyclonal antibody is 1:25600;The anti-GoAstV-ORF2 polyclonal antibody reacts well with the p ET-32a(+)-ORF2 fusion protein by Western Blot and shows specific fluorescent signals with fixed GoAstV-positive paraffin specimens byindirect immunofluorescence histochemical tests.These results indicate that the rabbit anti-GoAstV-ORF2 polyclonal antibody prepared has good reactivity with GoAstV antigen.3.In order to establish a POCT method for detecting GoAstV,the rabbit anti-GoAstV-ORF2 polyclonal antibody was purified using Protein A/G-Sephrose FF affinity ch-romatography.The effective Ig G concentration was 2mg/m L after purification;fluorescent microspheres were used to label rabbit anti-GoAstV-ORF2 polyclonal antibody and use it as a capture pad and quality control line capture antibody;the mouse anti-GoAstV-ORF2 polyclonal antibody was used as a capture antibody and as a detection line for assembly POCT fluorescent microsphere immunochromatographic detection.A standard curve was drawn after diluting the test paper with the purified GoAstV-ORF2 fusion protein.The positive and negative GoAstV-infected sera were dropped into the POCT test paper,and the portable analytical instrument was used to test standard curve.The results showed that the standard curve meets the requirements,but there are certain differences in specificity and stability.In this study,a high-purity soluble GoAstV-ORF2 fusion protein and its rabbit anti-GoAstV polyclonal antibody were successfully prepared.Based on this,a method for quantitative detection of GoAstV by POCT fluorescent microsphere immunochromatography was established. |
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