A bovine coronavirus chimeric antigen and colloidal gold immunochromatographic test paper card for detecting bovine coronavirus antibody
A chimeric antigen, coronavirus technology, applied in the directions of viruses/phages, viruses, viral peptides, etc., can solve the problems of difficult on-site large-scale application, unsuitable for grass-roots applications, and high equipment requirements, and achieve rapid diagnosis of calf diarrhea. Problems, easy to popularize and use at the grassroots level, and the effect of high practical value
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Embodiment 1
[0029] Example 1: Prokaryotic expression of the chimeric antigen of the present invention
[0030] 1. Construction of bovine coronavirus chimeric antigen gene recombinant expression plasmid pET28a-NS
[0031]According to the conservation and antigenicity analysis of the bovine coronavirus N protein and S protein sequences on the NCBI website, select a specific region of the gene to design the bovine coronavirus chimeric antigen gene (shown in SEQ ID NO: 2), and simultaneously optimize the sequence and use chemical The synthetic method synthesizes bovine coronavirus chimeric antigen gene. Digest the pET28a(+) vector with BamHI and XhoI. The enzyme digestion system is 50uL: 1uL each of BamHI and XhoI, 20uL of the vector fragment, 5uL of 10X digestion buffer, and 3uL of ddH2O2; the digested product is subjected to 1% agarose electrophoresis Gel recovery kit recovery. Then the digested vector was ligated with the chimeric antigen gene to construct a 10 μL ligation system: 5.5 μL...
Embodiment 2
[0036] Embodiment 2: Colloidal gold immunochromatography detection test paper card for preparing bovine coronavirus antibody
[0037] 1. Preparation of gold-labeled conjugates of SPA labeled with gold particles
[0038] Take 2ml of 40nm colloidal gold particles and adjust the pH to 6.8 with 0.1mol / L potassium carbonate solution, add 20ug Staphylococcus aureus protein A (SPA), mix quickly and mix at room temperature on a 3D rotator for 30min, then add the final concentration 1% BSA and mix on a 3D rotary mixer for 30min, centrifuge the gold standard solution at 12000r / min at 4°C for 10min, carefully discard the supernatant, wash the precipitate twice with 0.01M PBS buffer, and then centrifuge the resulting precipitate It is the purified SPA gold-labeled complex. The prepared colloidal gold-labeled SPA is resuspended in 0.01M PBS and stored at 4°C for later use.
[0039] 2. Nitrocellulose membrane testing line and quality control line spraying and colloidal gold bonding pad pre...
Embodiment 3
[0045] Embodiment 3: colloidal gold immunochromatography test paper card specificity test
[0046] Carry out cross-tests on known bovine Brucella positive sera, bovine foot-and-mouth disease positive sera, bovine viral diarrhea virus positive sera, bovine rotavirus positive sera, bovine herpes virus positive sera and bovine coronavirus antibody positive sera, see results figure 2 .
[0047] figure 2 The results showed that only the test line and quality control line of the bovine coronavirus antibody-positive serum sample developed color, showing a positive reaction, while when reacting with other sera, the test line did not show red, showing a negative reaction, indicating that the colloidal gold immune layer The test strips are highly specific.
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