chi3l1 antibody and its application, nucleic acid, recombinant expression vector system and host cell
A CHI3L1, host cell technology, applied in nucleic acid, recombinant expression vector system and host cells, CHI3L1 antibody and its application fields, can solve the problems of poor specificity, specificity and affinity of polyclonal antibodies, etc.
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[0053] In addition, one embodiment of the present invention also provides a method for preparing the above-mentioned CHI3L1 antibody, which includes the following steps: converting the light chain variable region of the CHI3L1 antibody encoding the amino acid sequence shown in SEQ ID No. 7 to After the nucleic acid fragment encoding the nucleic acid fragment of the heavy chain variable region of the CHI3L1 antibody whose amino acid sequence is shown in SEQ ID No. 8 is introduced into the host cell, the host cell is cultured to prepare the CHI3L1 antibody.
[0054] In one of the embodiments, the recombinant expression vector comprising the nucleic acid fragment of the light chain variable region of the CHI3L1 antibody encoding the above-mentioned amino acid sequence such as SEQ ID No.7 and the recombinant expression vector comprising the above-mentioned encoding amino acid sequence such as SEQ ID No.8 After the recombinant expression vector of the nucleic acid fragment of the he...
Embodiment 1
[0066] (1) Immunization: Recombinant CHI3L1 protein was mixed with Freund's adjuvant in equal amounts, fully emulsified, injected into 3 Balb / c mice, 25 μg per mouse, and immunized 3 times with an interval of 15 days.
[0067] (2) Cell fusion, selective culture, and screening: 3 days before fusion, the mice were intraperitoneally injected with 25 μg of immune protein, and the spleen cells of the immunized mice were taken out, and 2.0×10 7 2.0×10 SP2 / 0 myeloma cells 8 Splenocytes of 1 immunized Balb / c mouse were mixed, centrifuged, discarded supernatant, slightly oscillated and mixed, in 37°C water bath, 1 mL of 50% PEG-1500 aqueous solution was added dropwise within 90 seconds, Then add 20 mL of 1640 medium dropwise, centrifuge, discard the supernatant, wash once more, centrifuge, discard the supernatant to obtain hybridoma cells. Hybridoma cells were selected on a 96-well cell culture plate using HAT selection medium, and the total fusion rate was >95% as detected under a mi...
Embodiment 2
[0090] Affinity detection
[0091]Use Biacore T200 to detect the affinity of the CHI3L1 recombinant antibody prepared in step (11) of Example 1: immobilize the recombinant CHI3L1 protein on the CM5 chip, use different concentrations of the CHI3L1 recombinant antibody to be tested, and react with the CHI3L1 protein on the chip according to the appropriate time, The kinetic curve obtained from the reaction was fitted by software, and the affinity test results were as follows image 3 shown. According to the kinetic curve, the affinity KD of the CHI3L1 recombinant antibody was 2.656×10 -10 , where Kd is 2.893×10 -4 , Ka is 1.089×10 6 .
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