Monoclonal antibody of Chlamydophila abortus macrophage infectivity potentiator and hybridoma cell thereof
A monoclonal antibody and hybridoma cell technology, applied in the field of hybridoma cells, can solve the problems of low sensitivity, unfavorable clinical promotion, high production cost, etc.
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Embodiment 1
[0034] Example 1: Preparation of Chlamydia abortitropica MIP protein
[0035] (1) Amplification of the coding gene sequence of Chlamydia aborophila MIP protein
[0036] Pick the culture of C.abortus strain (for the convenience of tracking, it is numbered as SX5, and the strain can be purchased from a common strain company), and the genomic DNA of SX5 is extracted using the Gram-negative bacterial genome extraction kit, and the DNA of SX5 is extracted. Genomic DNA is used as a PCR template to amplify each target gene.
[0037] The amplification primer sequence of PCR process is as follows:
[0038] Forward primer (the underlined part is the Ndel restriction site): 5'-AAC CATATG AAAAAAACAATGGTATTTAA-3';
[0039] Reverse primer (the underlined part is the Xhol restriction site): 5'-GGA CTCGAG TGA AGCTGTGTTTTTGTC-3'.
[0040] The PCR reaction system includes: 25 μL of 2×Power Taq PCR MasterMix, 2 μL of the corresponding upstream and downstream primers, 2 μL of DNA template, ...
Embodiment 2
[0045] Example 2: Animal immunization
[0046] BALB / c mice aged 6 weeks were immunized with purified Chlamydia abortus MIP protein as an antigen, and immunized 6 times at intervals of 1-2 weeks.
[0047] For the first time, the antigen was emulsified with an equal amount of Freund's complete adjuvant into water-in-oil for immunization. The immunization dose was 200 μg / rat, a total of 2 rats, and the immunization route was multi-point subcutaneous injection in the abdomen.
[0048] The second immunization was carried out two weeks later, and the third immunization was carried out at intervals of one week. Both times were administered with the same amount of Freund's incomplete adjuvant as the antigen, and the immunization dose was 100 μg per mouse.
[0049] Then the fourth immunization and the fifth immunization were carried out at intervals of one week, and the antigen solution was used for immunization, the dose was 100 μg / rat, and the immunization route was intraperitoneal i...
Embodiment 3
[0052] Example 3: Cell fusion of immune antibody cells and myeloma cells
[0053] Three days before fusion, the preserved SP2 / 0 myeloma cells were revived. Take the spleen of the above-mentioned immunized mice under aseptic conditions to prepare splenocytes, and absorb 1×10 8 splenocytes and 2×10 7 A suspension of myeloma cells was fused under the action of fusion agent PEG4000.
[0054] The fused cells were selected and cultured with HAT to suspend evenly, and added to a 96-well cell plate lined with feeder cells, 100 μL per well, placed in 5% CO 2 Cultured in an incubator. Observe and record cell growth every day.
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