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Construction, Expression, and Activity of a Genetic Engineered Anti-ErbB-2 scFv-Fc-IL-2 Fusion Protein

Author: ShiMing
Tutor: GuoNing
School: PLA Military Academy of Medical Sciences
Course: Immunology
Keywords: ErbB-2 IL-2 Immune cytokines Antibody Expression
CLC: R392
Type: Master's thesis
Year: 2006
Downloads: 75
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Abstract


Recombinant antibodies and cytokine fusion protein combines unique antibody targeting function and multifunctional cytokine activity, known as immune cells the factor (Immunocytokines ICK), pre-clinical study results show good prospects. This study using genetic engineering methods to build a scFv-Fc-IL-2 fusion protein (HFI) and their antigen-binding properties of IL-2 biological activity in vivo and anti-ErbB-2 overexpressing tumor cells of the anti-ErbB-2 function of a preliminary study. This study is divided into three parts. 1 anti-human ErbB-2 scFv-Fc-IL-2 fusion gene and expression of mouse anti-human ErbB-2 single-chain antibody (scFv) gene using genetic engineering techniques, the IgGl Fc gene and the human IL-2 gene fusion inserted into plasmid pCI / dhfr, recombinant plasmid pCI / HFI. This vector transformed CHO cells, the activity of the fusion protein in the culture supernatant of transfected CHO cells was detected using sandwich ELISA, cell ELISA, flow cytometry and MTT method. Construction of the fusion protein was named as HFI, HFI can be identified by anti-human IgG and anti-human IL-2 antibody, specific binding with high expression of ErbB-2 in SKBR3 cells, and can promote the IL-2-dependent cell line CTLL- The 2 cells identify the capacity of the ErbB-2 antigen, and the biological activity of IL-2, i.e. HFI retained. HFI actual protein sequence design consistent with the theoretical mass spectrometry to prove. 2 using a two-stage culture method to improve the HFI expression in CHO cells using genetic engineering techniques to delete the 72 bp enhancer sequences in the SV40 promoter in the vector, in order to weaken the dihydrofolate reductase (the dihydrofolate reductase dhfr). Reconstructive expression vector transfected CHO cells, pressure screening, engineering cell lines stably expressing HFI. The level of expression of the HFI from the initial 100 ng / ml to 25μg/ml. Incubation temperature and two-stage culture strategy by optimizing the engineered cells, HFI expression levels get further improve (60-80μg/ml). At the same time, a systematic study reduce cultivation temperature of the the engineered cell proliferation, cell viability, apoptosis, cell cycle and impact HFI expression levels and biological activity. The results show that reducing the culture temperature may enhance cell viability, reduced apoptosis, without affecting the biological activity of the HFI. This part of the study by optimizing expression vector and engineering cell culture programs to improve the expression level of the HFI, for HFI enter pilot production and pre-clinical experiments laid a good foundation. Preliminary study 3 HFI in vivo anti-tumor activity in this part of the study proved through in vivo experiments in vitro HFI can be effective in mediating ADCC, and shows a good anti-tumor activity of the ErbB-2 expression in tumor animal model . The inhibitory activity was significantly better than free IL-2 and scFv. The proof of this section, HFI may have important applications for the next ErbB-2 high expression of tumor treatment and postoperative residual tumor, metastases Clear.

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