Single chain antibody-lidamycin fusion protein ER (Fv-LDP) targeting EGFR
A fusion protein and single-chain antibody technology, applied in the field of fusion protein ER, to promote tumor cell apoptosis, good clinical application prospects, and inhibit tumor growth
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Embodiment 1
[0129] . Construction, screening and identification of EGFR phage antibody library
[0130] 1.1 Immunization of mice
[0131] The cultured A431 cells were collected, and each mouse was intraperitoneally injected with 1×10 cells 7 indivual. Booster immunization 2 weeks later. On the 7th day after the last immunization, the mouse eye vein blood was taken to measure the titer by ELISA, and the titer reached 10 5 The spleens of the above mice were used for later use.
[0132]1.2 Phage antibody library construction, screening and identification
[0133] According to the instructions of the Expression Module / Recombinant Phage Antibody system kit from Pharmacia, the construction of the phage library and the screening of anti-EGFR antibodies were carried out. The brief steps are as follows:
[0134] Spleen RNA extraction from the above immunized mice→mRNA purification+OligdT→cDNA synthesis+library construction primer mixture→VH, VL→scFv→scFv+pCANTAB 5E (phagemid Pharmacia produc...
Embodiment 2
[0137] . Construction of fusion protein ER (Fv-LDP) recombinant expression plasmid
[0138] The recombinant plasmid pET-30a(+)-LDP contains the LDP gene and is preserved by our laboratory. The recombinant phagemid pCANTAB 5E contains the scFv gene, and two restriction sites, NdeI and EcoRI, were introduced by PCR (primers were synthesized by Invitrogen).
[0139] scFv 5' end primer (PH1): 5'GGAATTC CATATG GCCCAGGTCCAGCTGCAG 3′
[0140] Nde I
[0141] scFv 3' end primer (PL2): 5'CG GAATTC GGATCCGCCACCGCC CCGTTTTATTTTCCAAC
[0142] TTTGT 3′ EcoR I spacer
[0143] The recombinant phagemid pCANTAB 5E-scFv was used as a template, PH1 was used as the 5' end primer, and PL2 was used as the 3' end primer for PCR amplification to obtain a single-chain antibody gene fragment with a small peptide spacer at the C-terminus. The PCR reaction system was pre-denaturation at 94°C for 2 minutes, followed by 30 cycles of PCR: denaturation at 94°C for 1 minut...
Embodiment 3
[0144] . Fusion protein ER (Fv-LDP) in Escherichia coli BL21 (DE3) star TM inducible expression
[0145] The identified recombinant plasmid was transformed into Escherichia coli BL21(DE3)star TM (Invitrogen company product), randomly pick recombinant transformants and inoculate into 3 ml of LB medium containing 50 μg / ml kanamycin, and culture overnight at 37° C. with shaking. The next day they were inoculated at a ratio of 1:50, cultured with shaking at 37°C until the OD600 was 0.8, added IPTG to a final concentration of 0.8mM, and induced for 4-6 hours. Take an appropriate amount of bacterial liquid, and perform localization analysis of expression products on whole bacteria, medium supernatant, periplasmic cavity components, soluble cytoplasmic components and inclusion bodies. 10% SDS-PAGE electrophoresis analysis of exogenous protein expression The fusion protein was expressed in the inclusion body in an insoluble form ( Figure 4 ). Quantitative analysis of the gel imag...
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