Enzyme-linked immunosorbent assay (ELISA) antibody detection kit for novel porcine circovirus type 3 (PCV3), and application of ELISA antibody detection kit
A porcine circovirus and antibody detection technology, applied in the direction of biological testing, material inspection products, etc., to achieve the effects of long storage time, good stability, good specificity and sensitivity
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Embodiment 1
[0054] Embodiment 1 expresses recombinant Cap protein by escherichia coli
[0055] 1. Gene amplification
[0056] Using the PCV3-China / GD2016 virus strain (PCV3-China / GD2016 has been disclosed in Genomecharacterization of a porcine circovirus type 3 in South China, Changxu Song, Transbound Emerg Dis.2017Mar 13. in the paper) as a template to amplify the Cap gene, the reaction system As shown in Table 1, the reaction procedure is shown in Table 2.
[0057] Table 1 Reaction system for Cap gene amplification
[0058]
[0059] Among them, the upstream primer is: 5'-GGCggatccATGAGACACAGACCTATATTC-3', the downstream primer is: 5'-GGGctcgagGAGAACTGACTTGTAACGAAT-3', and the lowercase letters indicate the enzyme cutting sites BamHI and XhoI respectively.
[0060] Table 2 is used for Cap gene PCR reaction procedure
[0061]
[0062] (3) Gel electrophoresis of PCR amplification products
[0063] Refer to the method in the "Molecular Cloning Experiment Guide" to prepare 1.2% aga...
Embodiment 2
[0132] Example 2 Baculovirus expression of recombinant Cap protein
[0133] 1. Gene amplification
[0134] With embodiment 1.
[0135] 2. Cap target fragment gel recovery
[0136] With embodiment 1.
[0137] 3. Cap fragment and pFastBac-HTB double digestion
[0138] The obtained Cap gene fragment and the pFastBac-HTB plasmid were respectively digested with BamHI and XhoI, and the digestion system is shown in Table 6 and Table 7.
[0139] Table 6 Double digestion reaction system of pFastBac-HTB plasmid
[0140]
[0141]
[0142] Table 7 Double digestion reaction system of Cap gene fragment
[0143]
[0144] Incubate in a metal bath at 37°C for 20 min. After the reaction is complete, the digested product is analyzed by agarose gel electrophoresis according to the molecular cloning manual, and the target fragment is recovered. For the recovery method, refer to the manual of the gel recovery kit (Omega Company).
[0145] 4. Cap fragment and pFastBac-HTB connection ...
Embodiment 3
[0202] The formation of embodiment 3 detection kits
[0203] 1. Preparation of an ELISA plate with purified PCV3Cap protein as the coated antigen
[0204] (1) Solution preparation:
[0205] Coating solution (sodium carbonate buffer solution with a pH value of 9.6): 1.59g Na 2 CO 3 , 2.93g NaHCO 3 Dissolve in 900mL double-distilled water until completely dissolved, dilute to 1L, sterilize by filtration, and store at 2-8°C for later use.
[0206] Washing solution (20 times concentrated PBST solution): weigh 8.71g NaCl, 0.26g KH 2 PO 4 , 2.89g Na 2 HPO 4 12H 2 Dissolve O in 900mL double-distilled water until it is completely dissolved, add 0.5mL Tween-20, dilute to 1L, filter and sterilize, store at room temperature (15-25°C) for later use; dilute to 1 times when used.
[0207] Blocking solution: Take 3% gelatin, dissolve in 800mL of washing solution diluted to 1 times, after completely dissolved, dilute to 1L, store at 2-8°C for later use.
[0208] (2) Dilute the recombi...
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