Monoclonal antibody against porcine epidemic diarrhea virus N protein and application thereof
A porcine epidemic diarrhea and monoclonal antibody technology, which is applied in antiviral immunoglobulins, antiviral agents, antibodies, etc., can solve the problems of labor consumption, inability to be widely used, time-consuming diagnostic techniques, etc., and achieve good application prospects and good reactive effects
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[0035] Example 1 Recombinant expression of porcine epidemic diarrhea virus N protein
[0036] Based on the nucleotide sequence information of the porcine epidemic diarrhea virus JS-2013 strain, a pair of primers for amplifying the full length of the N gene (1326 bp, the full length of the N gene sequence is shown in SEQ ID NO. 2) was designed by Shanghai Shenggong Biological Engineering Co., Ltd. Co., Ltd. synthesis. The sequence of the upstream primer is: N-F: 5'-GCTCGGTACCCTCGAGATGGCTTCTGTCAGTTTTCAGGATC-3' (SEQ ID NO. 3); the sequence of the downstream primer is N-R: 5'-ATTCGGATCCCTCGAGTTAATTTCCTGTGTCGAAGATCTCG-3' (SEQ ID NO. 4), and the restriction site is XhoI. Extract viral genomic RNA, reverse transcription to synthesize cDNA, amplify the full-length N gene with primers NF and NR, and use 1% agarose gel electrophoresis to detect and recover the PCR product. Amplify the N gene with a size of about 1300bp by PCR. Expected results match ( figure 1 A). The vector pColdIDNA wa...
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[0039] Example 2 Preparation of mouse monoclonal antibody against N protein of porcine epidemic diarrhea virus
[0040] The recombinant porcine epidemic diarrhea virus N protein purified and prepared as in Example 1 was used as an immunogen to immunize mice to prepare monoclonal antibodies. 6-week-old BABL / c female mice were selected to immunize and express purified N protein. For the first immunization, purified N protein was used as the immunogen, mixed with Freund's complete adjuvant at a ratio of 1:1, and then injected into the abdominal cavity at an amount of 50ug / mouse (200ul / mouse). Two weeks later, they were injected into the abdominal cavity again, treated with Freund’s incomplete adjuvant to treat the same dose of antigen, and repeated immunization every two weeks. Before each immunization, blood was collected from the orbit, and the antibody titer was identified by ELISA. The third immunization started without adjuvant. 3 to 4 days before fusion, pure antigen was in...
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[0054] Example 3 ELISA titer identification of monoclonal antibody against N protein of porcine epidemic diarrhea virus
[0055] As the ascites prepared in Example 2, as the primary antibody, the indirect ELISA method is used to detect the ELISA titer of ascites. The specific method is as follows:
[0056] 1. Coating: Purified recombinant porcine epidemic diarrhea virus N protein and pColdIDNA expression vector His tag protein were respectively coated on ELISA plate with 200ng / 100μL per well, and coated overnight at 4℃.
[0057] 2. Washing: Wash 3 times with PBST containing 5‰tween-20, 200μL / well. 5 minutes each time.
[0058] 3. Blocking: Incubate with 5% skim milk at 37°C for 2 hours, 200μL / well.
[0059] 4. Washing: Wash 3 times with PBST containing 5‰Tween-20, 200μL / well. 5 minutes each time.
[0060] 5. Add the primary antibody: Dilute the collected ascites with 5% skim milk according to 1:10000, and add the enzyme-labeled plate coated with recombinant porcine epidemic diarrhea vi...
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