Hybridoma cell strain capable of secreting anti-CFP-10 antibody, antibody thereof and application thereof
A technology of CFP-10 and hybridoma cell lines, which is applied in the field of hybridoma cell lines, can solve the problems of not being able to meet the actual needs of specific detection of mycobacterial infection, high false positive test results, and low accuracy, and achieve The effect of improving animal welfare, shortening the detection cycle, and being simple and easy to operate
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0067] The aforementioned method for preparing the anti-CFP-10 antibody may include the following steps: cultivating the aforementioned antibody expression system under conditions suitable for expressing the aforementioned antibody, thereby expressing the aforementioned antibody, and purifying and isolating the aforementioned antibody.
[0068] Appropriate conditions for expressing the antibody should be known to those skilled in the art. Those skilled in the art can select a suitable medium based on experience and culture under conditions suitable for the growth of host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isol...
Embodiment 1
[0115] Example 1 The acquisition of hybridoma cell lines and the preparation of monoclonal antibodies
[0116] Obtaining the hybridoma cell line with the preservation number CCTCC NO: C2019191.
[0117] 1. Expression of recombinant BL21(DE3)-pET-30a(+)-cfp10 and purification of fusion protein
[0118] Synthesize a pair of primers:
[0119] Primer 1: 5'-AATGGATCCATGGCAGAGATGAAGACC-3' (BamH I) (SEQ ID NO: 17); Primer 2: 5'-ATTAAGCTTTCAGAAGCCCATTTGCGA-3' (Hind III) (SEQ ID NO: 18). The cycle parameters were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 50 s, annealing at 59°C for 50 s, extension at 72°C for 1 min, 25 cycles; 10 min at 72°C. The CFP-10 gene was amplified using Mycobacterium tuberculosis H37Rv as a template.
[0120] Ligation, transformation, plasmid extraction, electrophoresis identification, and the correctly identified recombinant plasmid was named pET-30a(+)-cfp10; the recombinant bacteria was named BL21(DE3)-pET-30a(+)-cfp10. Afterwards, th...
Embodiment 2
[0181] The establishment of embodiment 2 competition ELISA method
[0182] 1 Determination of optimal antigen coating concentration and enzyme-labeled antibody concentration
[0183] The titration test was carried out according to the orthogonal matrix method, the coating antigen concentration was 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 μg / ml, and the dilution factor of the enzyme-labeled antibody was 1:5000, 1:6000, 1 :7000, 1:8000, 1:9000, 1:10000. Overnight coating at 4°C, 100 μL / well. Wash 3 times with 1× washing solution, add PBST containing 2% BSA, 300 μL / well, block at 37°C for 2 hours, add 1:10 diluted positive serum and negative serum, 50 μL / well, and add enzyme at working concentration at the same time The dilution times of the labeled antibody were 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 respectively. The reaction was performed at 37°C for 0.5h, and after washing, TMB chromogenic solution was added, 100 μL / well. After 7 minutes at room temperature, add ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More