A kind of recombinant Lawsonia intracellularis porcine hsp60 protein monoclonal antibody and its application
A monoclonal antibody, Lawsonia technology, applied in anti-bacterial immunoglobulin, application, immunoglobulin and other directions, can solve the problem of less research, achieve small molecular weight, high specificity, suitable for commercial large-scale production. Effect
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Embodiment 1
[0063] Example 1: Expression and purification of Lawsonia intracellulare Hsp60 protein
[0064] 1) PCR amplification of Hsp60 gene
[0065] According to the Hsp60 gene sequence of Lawsonia intracellulare in GenBank, DNAStar biological software was used to analyze, select the region with good antigenicity, and entrust Shanghai Sangon Bioengineering Co., Ltd. to synthesize a Hsp60 gene with EcoR I and Sal I restriction sites Sequence, the expected target fragment length is 1647bp, the Hsp60 gene-specific primer sequence is F1 5'CG-GAATTC-ATGGCTTCTAAAGAAATC3'; R15'GC-GTCGAC-CTAGTACATACC GTCCAT 3' (the underlines indicate EcoR I and Sal I restriction sites respectively) . The 25 μL PCR reaction system is: 2 μL of ileitis-positive pig fecal DNA, 12.5 μL of Mix, 1 μL of 10 μmol / L upstream and downstream primers, ddH 2 O 8.5 μL. PCR amplification conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C fo...
Embodiment 2
[0075] Example 2: Preparation of monoclonal antibody against Lawsonia intracellulare Hsp60 protein
[0076] 1) Immunogen preparation and animal immunization procedures
[0077] The purified Hsp60 protein was mixed with complete Freund's adjuvant at a ratio of 1:1, and emulsified into an immunogen. The immunogen was immunized with 7-week-old Bab / C mice by multi-point subcutaneous injection on the back. waived. Ten days after the third immunization, blood was collected from the orbital venous plexus of the mice, the serum was separated, and the antibody level in the serum was determined by indirect ELISA. The mouse with the highest antibody level was selected for shock immunization 3 days before fusion, that is, the purified Hsp60 protein was directly injected intraperitoneally without adding any adjuvant to the immunogen, 50 μg / mouse.
[0078] 2) cell fusion
[0079] Preparation of feeder cells
[0080] (1) 8-week-old Bab / C mice were sacrificed by cervical dislocation and ...
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