Eukaryotic Expression, Purification And Preliminary Activity Assay Of Toll-like Receptor 5 Agonist CBLB502-Fc Fusion Protein

Toll-like receptors(TLRs) are a family of pattern recognition receptors, which play an important role in innate immunity and acquired immunity. They can specifically recognize the pathogen-associated molecular patterns of bacteria, viruses and other pathogens. Currently, 13 kinds of TLR have been found in mammalian cells and include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, though the latter two are not found in humans. TLR5, one of the TLRs family, can activate innate immune response by recognizing bacterial flagellin. TLR5 is widely distributed in the epithelial cells and dendritic cells of the trachea, spleen, lung, liver, kidney, urinary tract and so on. After recognition of flagellin, TLR5 forms a homodimer and induces proinflammatory signaling through the recruitment of adaptor molecules. TLR5 signaling has mostly been thought to be myeloid differentiation factor 88(My D88) –dependent. Finally, it activates the NF-κB, c-Jun Nterminal kinase(JNK) and p38, which induce the transcript of genes related with immunity and inflammation and promote the secretion of cytokines.CBLB502 is a recombinant protein and optimized TLR5 agonist derived from Salmonella enterica flagellin and developed by Cleveland Bio Labs. After optimized the structure, CBLB502 lacks the central globular domain with a high degree of immunogenicity and contains the N-terminal and C-terminal functional domain of flagellin. It has lower immunogenicity than flagellin, but retains the ability to activate TLR5 and the radioprotective effect. The study found that CBLB502 could prevent the the hematopoietic and gastrointestinal tissue damage from radiation or chemical agents. But its tissue protective effect would not extend to the tumor. In addition, it had direct cytotoxicity to the tumor cells of TLR5+ and produced antitumor effect on liver metastasis by mobilizing the immune cells to the liver. At present, CBLB502 used in the research is expressed and purified from Escherichia coli. Although the prokaryotic expression system can express the target protein efficiently, the target protein is usually expressed as inclusion bodies which need to be denatured, renatured, dialysed before purification. And the heat source or endotoxin is not easy to be removed. So the separation and purification proces of the protein expressed in Escherichia coli are relatively complicated. Therefore, in order to overcome the shortcomings of CBLB502 prokaryotic expression and bring the advantages of Fc fusion protein, this study intends to obtain the fusion protein of CBLB502 and Ig G1 Fc fragment by eukaryotic expression. The fusion of CBLB502 and Fc fragment can simplify the separation and purification process. In addition, the fusion of Fc can increase the protein molecular weight. And the protection of the Fc Rn can extend the plasma half-life of protein. Fc fusion protein can form a stable dipolymer, which can improve the stability of protein molecule.First, it was need to carry out the construction of CBLB502-Fc eukaryotic expression vector. The gene of CBLB502 in the literature was synthesized which was suitable for translating and expressing in Chinese hamster ovary cell(CHO-S). The CBLB502 gene was amplified from p UC57-CBLB502 plasmid by PCR and cloned into p UC57-Fc vector after double enzyme digestion with restriction enzyme Avr II and Bam H I. Then the gene of CBLB502-Fc was inserted into the pc DNA3.1 vector after double enzyme digestion with restriction enzyme Eco R V and Hind III. The positive clones were picked up and subjected to sequencing. The recombinant plasmid pc DNA3.1-CBLB502-Fc was transfected into CHO-S cells. Then the highly expressed cell lines were screened. The expression of fusion protein was detected by ELISA and western blot. The highly expressed cells were expanded with shake flask culture method. After culture supernatant was collected, the target protein was purified by Protein A affinity column and identified by SDS-PAGE. Finally, the eukaryotic expression vector of pc DNA-CBLB502-Fc was successfully constructed. The cell lines with high expression of CBLB502-Fc fusion protein was screened after the vector was transfected into CHO-S cells. And the fusion protein with high purity was purified by Protein A affinity column.Then the activity of CBLB502-Fc fusion protein in vivo and in vitro was identified primarily. The activity of CBLB502-Fc in vitro was initially identified by the detection of NF-κB and JNK. A549 cells treated with starvation were stimulated for 20 minutes by CBLB502-Fc(20 μg/m L), TNF-α(20 ng/m L) and CBLB502(10 μg/m L). The cells were collected for the separation and preparation of cytoplasmic and nuclear. And the extracts were detected by western blot. At the same time, the NF-κB translocation was observed by immunofluorescence assay. The results showed that NF-κB signaling pathway could not be activated by CBLB502-Fc under the success of the separation. And immunofluorescence results were consistent with the western blot method. Then after stimulating the A549 cells with the same experimental conditions, the cells were split by RIPA lysis solution. The change of JNK phosphorylation level was detected by western blot in cell lysis solution. The results showed that CBLB502-Fc which was the same as CBLB502 could activate JNK to increase its phosphorylation through TLR5 signaling pathway. In order to test the activity of fusion protein in vivo, C57BL/6 mice were used for the experiment of radiation survival. Before irradiation, the mice were injected intraperitoneally with saline, CBLB502(0.2 mg/kg body weight), CBLB502-Fc(low dose, 0.2 mg/kg body weight) and CBLB502-Fc(high dose, 0.4 mg/kg body weight) according to the group of the mice. The mice were subjected to whole body irradiation with the 8 Gy γ-rays of 60 Co. After irradiation, the survival of mice was observed for 30 days. The results showed that CBLB502-Fc could significantly improve the survival of mice with lethal irradiation and was no significant difference to CBLB502. Moreover, CBLB502-Fc and CBLB502 can significantly prolong the survival time of mice in the decitabine(DAC) acute toxicity experiment. The experiments preliminary proved that CBLB502-Fc had the radioprotective effect and the biological activity in vivo. And it also could reduce the toxic effects of chemical drugs. Combined with the results of cell experiment in vitro, JNK signaling pathway may play an important role in the biological function of CBLB502 and CBLB502-Fc.Finally, study the effects of CBLB502-Fc on bone marrow suppression and the cell phenotype in mice. The mouse myelosuppression model was made by the injection of low dose DAC(1.0 mg/kg body weight, i.p.). 48 BALB/c mice were randomly divided into group saline, group CBLB502, group CBLB502-Fc, group DAC, group DAC+CBLB502 and group DAC+CBLB502-Fc. The mice of DAC group, DAC+CBLB502 group and DAC+CBLB502-Fc group were injected with DAC once daily for 3 consecutive days. After the last injection of DAC for 1 hour, the mice were injected with normal saline, CBLB502(0.2 mg/kg body weight, i.p.) and CBLB502-Fc(0.4 mg/kg body weight, i.p.). After 3 days, the drug administration process was repeated. The blood taken from the mice tail vein were detected before mice were dissected. On the last day, the blood and femoral bone marrow were collected from the mice. The blood and bone marrow samples added the fluorescein labelled monoclonal antibodies were split the red blood cells by lysing solution. The treated samples were investigated by flow cytometry methods. And the results of the flow cytometry were analysis by statistics. The results of peripheral blood test showed that the peripheral blood parameters of the DAC treated mice were significantly lower than the mice injected with saline. It indicated that the mouse model of bone marrow suppression was successfully established. CBLB502 and CBLB502-Fc could not alleviate the inhibition red blood cells, white blood cells, lymphocytes and neutrophils induced by DAC. But CBLB502-Fc could significantly relieve the inhibition of peripheral blood platelet induced by DAC. It meant that CBLB502-Fc had a certain effect on bone marrow suppression. In addition, CBLB502 and CBLB502-Fc could significantly promote the proliferation of neutrophils.The flow cytometry results showed that compared to the control mice, CD4+, CD8a+, CD4+/CD8a+ T lymphocytes and CD3e+, NK, NKT cells in the peripheral blood and bone marrow of the mice treated with DAC were significantly increased, while CD11b+ cells were decreased significantly. CBLB502 and CBLB502-Fc could significantly increase CD11b+ cells in the blood and bone marrow of the normal mice and reduce CD4+, CD8a+ and CD4+/CD8a+T lymphocytes in the blood. In addition, NK and NKT cells were slightly increased in peripheral blood of the normal mice treated with CBLB502 and CBLB502-Fc. For myelosuppressive mice, CBLB502 and CBLB502-Fc could significantly increase CD4+ T lymphocytes and decreased NK and NKT cells in blood, but had no obvious effect on CD11b+ cells. Moreover, CBLB502 also significantly increased NK and NKT cells in bone marrow of myelosuppressive mice, while CBLB502-Fc can significantly reduce CD4+, CD4+/CD8a+ T lymphocytes. The results showed the effects of CBLB502-Fc on CD4+ and CD8a+ T lymphocytes, NK and NKT cells and CD11b+ cells were consistent with CBLB502 in mice. Both CBLB502 and CBLB502-Fc could promote the proliferation of NK cells, NKT cells and CD11b+ cells through the activation of the innate immune response.In summary, It is preliminarily proved that CBLB502-Fc fusion protein with the same bioactivity of CBLB502 has the radioprotective effect and bone marrow protection. The signaling pathway, the function and mechanism of CBLB502-Fc are worth further researching and discussing.