A kind of anti-porcine epidemic diarrhea virus nsp13 protein monoclonal antibody and its application
A porcine epidemic diarrhea and monoclonal antibody technology, applied in the field of bioengineering, to achieve the effect of mild conditions and simple process
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0036] Embodiment 1: Recombinant expression of porcine epidemic diarrhea virus nsp13 protein:
[0037] According to the nucleotide sequence information of porcine epidemic diarrhea virus CH / JX / 01 / P30 strain, a pair of primers for amplifying the nsp13 gene were designed. The primers were synthesized by Qingke Bioengineering Co., Ltd. The upstream primer sequence is: nsp13-F: 5'-TTTCTCGAGtctgcagggctttgtgttgt-3', the restriction site is XhoI; the downstream primer sequence is nsp13-R: 5'-CCGGAATTCttatgcaaatcagac-3', the restriction site is EcoRI. The amplified nsp13 target gene has a full length of 1791bp, and the nucleotide sequence of the nsp13 gene is shown in SEQ ID NO.2. Extract viral genomic RNA, reverse transcribe and synthesize cDNA, amplify the full-length nsp13 gene with primers nsp13-F and nsp13-R, detect and recover the PCR product by 1% agarose gel electrophoresis, and amplify a protein with a size of about 1791bp by PCR nsp13 gene, consistent with the expected resu...
Embodiment 2
[0040] Embodiment 2: Preparation of anti-porcine epidemic diarrhea virus nsp13 protein monoclonal antibody:
[0041] The recombinant porcine epidemic diarrhea virus nsp13 protein purified and prepared as in Example 1 was used as an immunogen to immunize mice for the preparation of monoclonal antibodies. Select 6-week-old BABL / c female mice to immunize with recombinant nsp13 protein. For the first immunization, the recombinant nsp13 protein was used as the immunogen, mixed with complete Freund's adjuvant at a ratio of 1:1, and injected subcutaneously with an antigenic amount of 200 μg / rat (200 μl / rat). Two weeks later, the same antigen was treated with incomplete Freund's adjuvant by subcutaneous injection at a dose of 100 μg per mouse, and the immunization was repeated every two weeks, for a total of three immunizations. Before each immunization, blood was collected from the orbit, and the antibody titer was identified by ELISA. 3-4 days before fusion, intraperitoneal inject...
Embodiment 3
[0055] Example 3: Anti-porcine epidemic diarrhea virus nsp13 protein monoclonal antibody ascites and BABL / c mouse fourth immune serum and BABL / c mouse negative serum ELISA titer identification:
[0056] The ascites prepared in Example 2 and the positive serum and negative serum collected before fusion were respectively used as the primary antibody, and the titer was detected by indirect ELISA method. The specific method is as follows:
[0057] (1) Coating: The recombinant nsp13 protein was coated on an ELISA microtiter plate with a coating amount of 7.5 μg / mL, and coated overnight at 4°C.
[0058] (2) Washing: wash 3 times with PBST containing 0.05% tween-20, 300 μL / well, 3 minutes each time.
[0059] (3) Blocking: incubate with 5% skimmed milk powder at 37° C. for 2 hours, 300 μL / well.
[0060] (4) Washing: wash 3 times with PBST containing 0.05% Tween-20, 300 μL / well. 3 minutes each time.
[0061] (5) Add primary antibody: ascitic fluid, positive serum and negative serum ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More