Anti CD52 monoclonal antibody, coding sequence and use thereof
A monoclonal antibody, CDR2 technology, applied in the medical field, can solve the problem of unsatisfactory characteristics such as monoclonal antibody specificity
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Embodiment 1
[0048] Screening the Variable Region Genes of Anti-CD52 Antibody from Antibody Library
[0049] 1) Construction of human antibody library
[0050] According to Marks et al. J. Mol. Biol., 222, 581-597; Hoogenboom and Winte, J. Mol. Biol., 227, 381-388; Haidaris CG et al., J Immunol Methods. 2001 Nov 1; 257 (1- 2): 185-202; Griffiths, A.D. et al. EMBO J., 13, 3245-3260 (1994); Nissim, A. et al. The method described in EMBO J., 13, 692-698 (1994) to construct a human antibody library . In short, the genes of heavy and light chains of immunoglobulins were prepared from peripheral blood lymphocytes, amplified in vitro by PCR, and cloned into phage vectors. CD52 protein (purchased from Shenzhen Jingmei Company) was used as the antigen, and the corresponding specific antibody was screened.
[0051] 2) screening
[0052] Add 1 ml of the revived antibody library strain to 14 ml of fresh LB medium, and culture in a 50 ml Erlenmeyer flask at 37°C for 16 hours.
[0053] Centrifuge a...
Embodiment 2
[0061] Cloning of Expression Vectors for Antibody Variable Region Coding Sequences
[0062] The cloned bacterial strain obtained in Example 1 was amplified in 100 ml of LB medium, and the plasmid DNA was purified with a plasmid DNA extraction and purification kit from Promega Company.
[0063] After digesting the above plasmid DNA with XbaI and NheI, the digested fragments were separated on 1.5% agarose gel electrophoresis, and a band of about 350 bp was taken for gel recovery, and the resulting fragment was the heavy chain variable region coding sequence.
[0064] The above plasmid DNA was digested with HindIII and Bsi WI, and the digested fragments were separated on 1.5% agarose gel electrophoresis, and a band of about 320 bp was taken for gel recovery, and the resulting fragment was the light chain variable region coding sequence.
[0065] Then first insert the above heavy chain variable region coding sequence into the expression vector pMG18-3K (see book DEVELOPMENT OF TOO...
Embodiment 3
[0067] Transfection of CHO cells and screening of recombinant clones
[0068] The expression vector with the antibody gene constructed in the above-mentioned Example 2 was transferred into the Escherichia coli DH5α strain, and then inoculated in 100 milliliters of LB medium for amplification, and the ultrapure plasmid DNA purification kit (Ultrapure Plasmid DNA) from Qiagen Company was used to Purification Kit) to extract and purify plasmid DNA. The above-mentioned purified plasmid DNA was transfected into CHO cells using the liposome method kit of Invitrogen Company, and the operation was performed according to the manufacturer's instructions.
[0069] The transformed CHO cells were continuously selected on the selection medium for 9 weeks, and finally cultured in extreme dilution on a 96-well plate for 3 consecutive times for monoclonalization.
[0070] The selected monoclonal cell lines were cultured on RPM1641 medium, and Western Blotting experiments were performed on the...
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