Preparation, In Vitro And In Vivo Immunomodulatory And Synergistic Anti-Tumor Activities Of Thymosinα1-Thymopentin Fusion Peptide And Its Binding Character To TLR2

Malignant tumors, which have high mortality, are one kind of the major diseases that seriously affect human health and threaten human life. Currently, the commenly used methods in the treatment of tumors in clinic are chemotherapy and radiotherapy, but, these remedies result in the severe damage on the immune function of patients. Therefore, searching for immunomodulators which can improve the immune function in cancer therapy has significant social and economic significance. Thymopentin (TP5) and thymosin alpha1(Tα1) are two thymic peptides with similar immunoregulatory activities and are being used in clinic for the treatment of some malignancies. They can improve the immune response and tolerance of patients to chemotherapy or radiotherapy, inhibit or reduce the progression of the diseases and improve the patient’s quality of life. However, the in vivo half-life (t1/2) of TP5is very short, which is only30s. So. frequent administration is required to maintain its efficacy, which brings great inconvenience to patients. Tα1is mainly synthesized and purified by the method of solid phase synthesis, which is very expensive. The high price of Tal is a burdern for the majority of patients.In order to prolong the t1/2and enhance the activity of TP5. a Tα1-TP5fusion peptide was designed and expressed in Pichia pastor is by our research team. Previous study showed that Tα1-TP5had higher activity than TP5in inducing T-lymphocyte proliferation in vitro. In in vivo carbon clearance test, the abilities of Tα1-TP5in promoting phagocytic capability of macrophages as well as the secretion of IL-2in peripheral blood of mice were also higher than those of TP5and Tα1. Besides. Tal-TP5had longer in vitro plasma t1/2than TP5and Tα1. However, the amount of Tα1-TP5secreted from Pichia pastor is was small and could not meet the demand of further in vivo study. Besides, the thrombin was needed to remove the fusion tag to obtain Tα1-TP5, which resulted in the high purification costs and complex purification steps. After thrombin cutting, two extra amino acids existed in the N-terminal of Tal-TP5and four extra amino acids existed in the C-terminal of Tα1-TP5, which might increase immunogenicity.To obtain large quantity of active Tα1-TP5with low cost, a new intein-fused expression and purification strategy was used for the production of Tα1-TP5fusion peptide from the soluble fraction and inclusion bodies. The in vivo immune regulating activity and synergistic anti-tumor activity of Tα1-TP5were also investigated. Besides, the potential receptor of Tα1-TP5was investigated by surface plasmon resonance (SPR) binding studies. In sum, the research may provide important basis for developing Tα1-TP5as a novel immunomodulatory agent.The main contents and results obtained were summarized as follows:1Expression and purification of Tα1-TP5in Escherichia coliTα1-TP5was expressed in Escherichia coli using pTYB2as the expression vector. The gene of Tα1-TP5was inserted into the N-terminal of the gene of Sce intein-Chitin binding domain (CBD). Then the recombinant plasmid pTYB2-Tα1-TP5-Sce intein-CBD was transformed into Escherichia coli ER2566and recombinant protein Tα1-TP5-Sce intein-CBD was expressed in ER2566. After sonication, SDS-PAGE was used to confirm whether the recombinant protein was soluble. The influences of different induction temperature and the concentration of isopropyl-β-D-galactoside (IPTG) on the amount of soluble recombinant protein were examined. Affinity chromatography was used to purify the recombinant protein. The target peptide was released from the chitin beads via self-cleaving of intein induced by dithiothreitol (DTT). In order to increase production, Tal-TP5was also obtained from inclusion bodies. The condition of washing inclusion body was investated. Subsequently, the inclusion body was renatured by direct dilution method. Besides, the effect of redox ratios, L-Arg and final urea concentration on refolding efficiency was evaluated. The refolded protein was applied to affinity chromatography columns, and Tα1-TP5was released from the chitin beads via self-cleaving of intein induced by DTT. Further, Tα1-TP5was purified and desalted by Superdex30. The correctness of its molecular weight was identified by ESI-MS and the purity of Tα1-TP5was evaluated by Tricine-SDS-PAGE.Tα1-TP5was successfully expressed in E.coli ER2566. After sonication, the fusion protein was partially in the form of inclusion bodies. As the induction temperature decreased from37℃to16℃, the amount of soluble protein increased from15.9%to18.9%of total bacterial protein. Change of IPTG concentration from1mmol/L to0.lmmol/L showed no effect on soluble expression level. When redox ratio (reduced:oxidized) was1:22.5and the final urea concentration was0.8mol/L, the fusion protein has the highest refolding efficiency. L-Arg showed no effect on the refolding efficiency. After further purified and desalted by Superdex30,4.6mg Tal-TP5was obtained from the supernatant and3.0mg Tal-TP5was obtained from inclusion bodies per liter medium. The purity was up to95%determined by16.5%Tricine-SDS-PAGE. Finally, this approach reported here allowed the production of approximately7.6mg of Tα1-TP5from1L shaking flask culture of E. coli.2Study on the in vitro and in vivo immunomodulatory activity of Tal-TP5The ability of Tal-TP5on promoting mouse spleen lymphocytes proliferation was detected by CCK-8assay. The effect of Tα1-TP5on promoting the expression of CD69on mouse spleen lymphocytes was investigated by flow cytometry. Then the effect of Tal-TP5on promoting the expression of IFN-γ was detected by RT-PCR. The result showed that Tal-TP5purified from the supernatant and inclusion bodies could promote the proliferation of mouse spleen lymphocytes in a concentration-dependent manner. When the concentration of Tα1-TP5was40μg/mL. the proliferation rate was26.21%and21.84%, respectively. Compared to negetive control, the difference was significant (p<0.01, p<0.01). Tα1-TP5could promote the expression of CD69and compared to negative control, the difference was significant (21.51%vs.9.5%, p<0.05). The ability of Tα1-TP5in promoting CD69expression was also higher than that of TP5(16.06%) and Tα1(16.65%). RT-PCR results showed that Tα1-TP5could promote IFN-γ expression at the gene level, and the ability was higher than that of TP5and Tα1. These all indicated that purified Tal-TP5possessed stronger immune regulating activity than TP5and Tal.The immunosuppressive mice model was established by intraperitoneal injection of hydrocortisone (HC) at a dose of250mg/kg for two consecutive days. Tα1-TP5was subcutaneously injected for two consecutive weeks to evaluate the resistance of Tal-TP5to immunosuppression induced by HC. The results showed that, Tal-TP5could significantly enhance the thymus index of immunesuppressed mice, and compared with the model group, the difference was significant (2.253±0.427vs0.444±0.407,p<0.05). Compared with the TP5and Tal alone group, the difference was also significant (p<0.05). However, the effect of Tα1-TP5on spleen index was little. HE staining indicated that Tal-TP5could antagonize thymic atrophy induced by HC and increase the number of thymocytes in the thymus tissue. In addition, Tal-TP5made CD4+CD8+thymocytes returned to normal levels. Compared with model group, Tal-TP5significantly increased the concentration of IFN-γ in the peripheral blood. In sum, Tal-TP5has the ability to alleviate the immunosuppression induced by HC, and the activity was stronger than that of TP5and Tα1.3Study on the synergistic anti-tumor activity of Tal-TP5The inhibitory effect of Tal-TP5on breast cancer cell MDA-MB-231, lung cancer cell A549, hepatoma carcinoma cell HepG2and melanoma cell B16was evaluated by CCK-8assay. Tal-TP5could inhibit the proliferation of these four tumor cells in a time-dependent manner and Tal-TP5showed the highest inhibitory effect on MDA-MB-231. After72h incubation, cell viability was only64.6%of that of the negetive control, and compared to TP5group, the difference was significant (64.6%vs.90.8%, p<0.05), but compared to Tal group, there was no significant difference (64.6%vs.70.2%, p>0.05). The inhibitory effect of Tal-TP5on HepG2cell and A549cell was weaker than on MDA-MB-231cell, and cell viabilities were73.9%and69.9%of that of the negative group after72h incubation, which was stronger than that of TP5but had no significant difference from that of Tα1. Tα1-TP5also could inhibit the proliferation of B16cells, and after72h incubation, the cell viability was82.5%of that of the negative group. Compared to TP5and Tal group. there were no significant differences.C57BL/6mice were inoculated subcutaneously with2×105of B16melanoma cells/mouse in the forelimb armpit. The effect of Tα1-TP5in combination with cyclophosphamide (CY) on inhibition of tumor growth was investigated. Compared to CY alone, Tα1-TP5combined with CY significantly reduced tumor volume and tumor weight, and the tumor weight was reduced by nearly88.1%after two weeks treatment. Tal-TP5also increased leukocyte number lowered by CY treatment, and leukocyte number in peripheral blood increased to normal levels after two weeks treatment of Tα1-TP5. In CY group, MHC I expression rate was only11.65%detected by flow cytometry, however, Tal-TP5significantly increased MHC I expression in the tumor tissue, and compared to CY group, the difference was statistically significant (28.71%vs.11.65%, p<0.05). Immunohistochemistry results showed that Tal-TP5promoted more CD8+T cell infiltration and CD86expression in tumor tissue than TP5and Tα1. From all the results above it was concluded that Tal-TP5alone had weak killing effect on tumor cells and mainly activated the immune system in combination therapy.4Study on the binding character of Tal-TP5to TLR2Confocal microscope was used to observe the binding character of Tα1-TP5to bone marrow-derived macrophages (BMDMs). Tα1-TP5. TP5and Tα1were labeled with fluorescein isothiocyanate (FITC) and incubated with cultured BMDMs for2h. Then, the cell nucleus and cell membrane were stained by Hoechst33342and Dil. respectively. The results showed that all the three peptides were located on the cell membrane of BMDMs.SPR was used to detect the binding of Tα1-TP5to TLR2. which exist on the cell membrane of BMDMs. TLR2was immobilized by standard amine coupling using an amine coupling kit. The peptides were dissolved in running buffer, and a flow rate of30μL/min was employed for association and dissociation at a constant. SPR technology could accurately reflect the level of the binding of the three polypeptides with TLR2. The KD of Tal-TP5, TP5and Tal were6.84×10-6mol/L,1.57×10-6mol/L and35.4×10-6mol/L, respectively. The KD of Tal-TP5was much smaller than that of Tα1, indicating that Tα1-TP5had higher affinity to TLR2than Tα1. Compared with TP5, the affinity of Tal-TP5to TLR2was slightly low but there was no statistical significance. Thus, the potential receptor of the three peptides was first confirmed as TLR2.The main results and conclusions are as follows:(1) The expression strain of Tal-TP5was firstly constructed. The fusion protein was successfully expressed in E.coli and Tal-TP5was purified from the supernatant and inclusion bodes with intein-mediated purification systems. High purity of Tal-TP5was obtained by further purification with Superdex30. There was only a Gly residue at the end of Tal-TP5, which might greatly reduce the immunogenicity. The approach described here is a low-cost, convenient and potential way for obtaining a large amount of Tα1-TP5.(2) Purified Tal-TP5can significantly promote the proliferation of mouse spleen lymphocytes, as well as the expression of CD69and IFN-y. Tal-TP5showed good in vitro immunoregulatory activity.(3) The in vivo immunomodulatory activity of Tal-TP5was evaluated for the first time. Tα1-TP5can antagonize immune suppression induced by HC, and the effect was stronger than TP5and Tα1.(4) The synergistic anti-tumor activity of Tal-TP5was evaluated for the first time. Tα1-TP5combined with CY exhibited good anti-tumor activity. Moreover, Tα1-TP5activated the immune system, which was responsible for killing tumor cells. The synergistic anti-tumor activity of Tα1-TP5was stronger than that of TP5and Tal.(5) The receptor of Tα1-TP5was first studied. Tα1-TP5could bind to TLR2, suggesting that Ta-TP5may mediate immune response through TLR2signaling pathway.