Fusion protein for detecting pcv2, preparation method and application
A technology of fusion protein and coding gene, which is applied in the field of PCV2 virus prevention and diagnosis, can solve the problems of time-consuming, limited application, high detection cost, etc., and achieve the effect of high yield
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Embodiment 1
[0031] Example 1 is used to detect the preparation of the fusion protein of PCV2
[0032] 1. Design and synthesis of fusion protein gene
[0033] Fusion protein A with small molecular weight was artificially designed for detection of PCV2. The fusion protein is formed by linking the anti-human erythrocyte H antigen nanobody and the anti-PCV2 nanobody through a Linker sequence. The amino acid sequence of fusion protein A is shown in SEQ ID No:2. The coding gene of fusion protein A is designed, and the nucleotide sequence is shown in SEQ ID No:1. The gene encoding fusion protein A was synthesized by Nanjing GenScript Biotechnology Co., Ltd.
[0034] Alternatively, the anti-human erythrocyte H antigen antibody and the anti-PCV2 antibody are connected through a Linker sequence to form a fusion protein B with a larger molecular weight. The amino acid sequence of fusion protein B is shown in SEQ ID No: 4, and the coding gene is shown in SEQ ID No: 3. The gene encoding fusion pr...
Embodiment 2
[0046] Example 2 Verification of the bifunctional properties of fusion protein A
[0047] The red blood cell agglutination test verified the bifunctional properties of the fusion protein A after renaturation. Specific steps are as follows:
[0048] (1) Wash fresh type O human erythrocytes twice with 0.1M, pH7.4 PBS buffer and resuspend to make a 10% (volume percent concentration) suspension. Take this suspension and slowly add it to an equal volume of 1 % (volume percentage concentration) glutaraldehyde (25% glutaraldehyde diluted with 0.1M, pH7.4 PBS buffer) solution, 50rpm, 37 ℃ for 1h, and then with 0.1M, pH7 .4 washed with PBS buffer for 3 times, and finally resuspended with 0.1M, pH7.4 PBS buffer to form a 20% suspension, adding NaN with a final concentration of 0.1% (mass percentage concentration) 3 Anti-corrosion to obtain the formaldehyde-type O human erythrocyte mother solution, and store it in a refrigerator at 4°C for later use. Centrifuge the mother liquor of fo...
Embodiment 3
[0051] Example 3 Specific verification of fusion protein A
[0052] Porcine epidemic diarrhea virus (PEDV CV777, Genbank NO.: AF353511), porcine Japanese encephalitis virus (JEV RP9, Genbank NO.: DQ648597.1), porcine reproductive and respiratory syndrome virus (PRRSV JX-1, Genbank NO.: AF331831), PCV2 virus (PCV2-DBN-SX07, Genbank NO.: FJ660968) each 50 μ l was added dropwise in the hemagglutination plate, and then 50 μ l sensitized human type O red blood cells were added dropwise to carry out the hemagglutination test (same as in Example 2), adding PBS buffer to replace the virus in the control. The result is as image 3 As shown, in the erythrocyte agglutination test, only the wells where PCV2 virus was added showed strong agglutination, and the wells where other viruses were added did not observe agglutination. The results showed that: the fusion protein A had strong specificity, and only when it interacted with human O-type red blood cells and PCV2 antigen at the same ti...
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