Human thymus stromal lymphopoietin monoclonal antibody and application thereof
A lymphocyte and progenitor technology, applied in the direction of antibodies, anti-cytokine/lymphokine/interferon immunoglobulin, microorganisms, etc., can solve the problems affecting the quality of life, health care burden, etc.
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Embodiment 1
[0160] The preparation of embodiment 1 immunogen
[0161] The eukaryotic expression vector of human TSLP was constructed, transfected into 293F cells, and the culture supernatant was purified by Protein A column to prepare human TSLP protein, which was used for mouse immunization, clone screening and functional identification.
[0162] (1) Construction of human TSLP eukaryotic expression vector
[0163] The human source TSLP sequence starts from Tyr 29 of natural human TSLP to Gln 159, a total of 131 amino acids (sequence shown in SEQ ID NO.102), and a mouse Fc fragment (mFc) (Accession number: AJ487681, Allele or Gene:IGHG1*01), can be combined with the recombinant protein A in the protein A column, thereby can be purified by affinity chromatography, therefore, construct rhTSLP-mFc fusion protein (amino acid sequence such as SEQ ID NO.103 shown).
[0164] The above rhTSLP-mFc fusion gene was amplified by PCR, and the recombinant plasmid was constructed by homologous recombi...
Embodiment 2
[0170] Example 2 Mouse immunization and antibody titer determination in serum
[0171] Use rhTSLP-His (rhTSLP coupled with a His tag, the sequence is shown in SEQ ID NO.104) or rhTSLP-mFc prepared in Example 1 as an immunogen for Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Ltd) for immunization. A total of 10 mice were immunized, among which 6 mice were immunogened with rhTSLP-His and immunized with complete Freund's adjuvant; the other 4 mice were immunogened with rhTSLP-mFc and quickly immunized with water-soluble adjuvant.
[0172] When using complete Freund's adjuvant for immunization, multi-point subcutaneous injection was used on the back, and the immunization dose was 50 μg / 200 μl / mouse. Mix 50 μg rhTSLP-His with 100 μl Freund’s complete adjuvant (Sigma, F5881) for the first immunization, mix 50 μg rhTSLP-His with 100 μl Freund’s adjuvant for the second immunization, third immunization, fourth immunization and fifth immunization Co...
Embodiment 3
[0179] The preparation of embodiment 3 hybridomas
[0180] After the last immunization in Example 2, the spleens of the two mice (No. 1 and No. 6 immunized with complete Freund's adjuvant; No. 2 and No. 4 rapidly immunized) with the highest titers obtained by each immunization method were mixed respectively, After milling in normal saline, the suspension rich in B cells was taken, and cell fusion was carried out with myeloma cell SP2 / 0 under the action of fusion agent PEG (Sigma, P7181). The fused cells were divided into 15 96-well cell culture plates, placed in 5% CO in 20% fetal bovine serum RPMI-1640 full medium (Thermo, 31800089) containing HAT (Sigma, H0262) 2 After one week of culture at 37°C, HT medium (Sigma, H0137) was used to continue the culture. Screening and subcloning of embodiment 4 hybridomas
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