Objective Extracellular recombinant protein fragment of PbTIP (rPbTIP) was expressed by prokaryotic vector in vitro and stimulated DC2.4 cells with rPbTIP, to study whether rPbTIP can exert immunomodulatory function on DC2.4 cells.
Methods E. coil BL-21 strains containing pET32a (+) -rPbTIP vector were used to express and purify rPbTIP protein; then different concentrations of rPbTIP protein were added to DC2.4 cells cultured in vitro. Cell proliferation assay was used to test whether rPbTIP could promote proliferation of DC2.4 cells. The DC2.4 cells was stimulated by rPbTIP, and the mRNA contents of MyD88, NF-κB, TLR9, and TLR4 on the surface molecules of DC2.4 cells were detected by RT-PCR. After the rPbTIP stimulation of DC2.4, the cytokine levels in the supernatant were detected by ELISA.
Results The rPbTIP protein was successfully expressed and purified at a concentration of 1 mg/mL; rPbTIP of 1 ng/mL could have effect on promoting proliferation of DC2.4 cells, and the level of TNF-α in the supernatant of cell culture was significantly increased, which was statistically significant (P<0.05). While, the changes of other cytokines were not statistically significant (P>0.05), 100 ng/L of rPbTIP may have certain inhibitory effect on DC2.4 cells, and can up-regulate the contents of MyD88, NF-κB and TLR9 mRNA on DC2.4 surface.
Conclusion In vitro, rPbTIP protein has a certain regulatory effect on the immune function of DC2.4 cells, and the effects of rPbTIP protein in different concentrations may be different, requiring further studies.
摘要： 目的 的应用原核载体表达PbTIP胞外重组蛋白片段(rPbTIP)并在体外刺激树突状细胞DC2.4, 研究在体外 PbTIP片段蛋白对DC2.4细胞是否有一定的免疫调节功能。 方法 用实验室保存的含有pET32a( + )-rPbTIP载体的 E. coil BL-21菌株表达并纯化rPbTIP蛋白；然后在体外培养的DC2.4细胞中加人不同浓度的rPbTIP蛋白刺激, 应用细胞 增殖实验检测rPbTIP对DC2.4细胞是否有促进增殖的作用；对rPbTIP刺激后的DC2.4细胞进行RT-PCR检测, 分析 DC2.4细胞表面分子MyD88、NF-KB、TLR9、TLR4的mRNA含量变化;rPbTIP刺激DC2.4后, 应用ELISA法检测细胞培养 上清中TNF-a、IL-10、TGF-p和IFN- 7细胞因子水平。 结果 成功表达出rPbTIP蛋白, 纯化后蛋白浓度为1 mg/mL; 1 ng/mL的rPbTIP对DC2.4细胞有一定促进增殖作用, 并且细胞培养上清中TNF-a水平明显升高, 有统计学意义(P< 0.05), 其他细胞因子水平变化无统计学意义(P>0.05); 100 ng/L的rPbTIP对DC2.4细胞可能有一定抑制作用, 并且可以 上调DC2.4表面MyD88、NF-KB、TLR9 mRNA含量。结论在体夕卜, rPbTIP蛋白对DC2.4细胞的免疫功能具有一定的调 节作用, 不同浓度rPbTIP蛋白的作用可能不同, 需要进一步研究。