论文部分内容阅读
目的表达、纯化重组葡萄球菌肠毒素B突变体蛋白D9N(rSEB-D9N),了解表达产物rSEB-D9N的细胞毒性、促淋巴细胞增殖和肿瘤生长抑制作用。方法不同浓度IPTG诱导SEB-D9N突变体原核表达系统pET32a-SEB-D9N-E.coliBL21DE3表达rSEB-D9N,经Ni-NTA亲和层析法纯化rSEB-D9N,10%SDS-PAGE和高效液相色谱检测其纯度。以Vero细胞为靶细胞,进行rSEB-D9N的细胞毒性作用,并计算TCID50值。采用MTT法分别检测不同浓度的rSEB-D9N促人淋巴细胞增殖及抑制KB及MCF-7癌细胞株生长的作用。流式细胞术检测rSEB-D9N刺激人淋巴细胞后CD3、CD4、CD8的表达。结果rSEB-D9N表达产量约占细菌总蛋白30%,纯化后获得纯度为90.48%的rSEB-D9N。rSEB和rSEB-D9N对Vero细胞的TCID50值分别为3.12,8.85μg,10.0,20.0mg·L-1的rSEB-D9N具有显著的促PBMC增殖的作用(P<0.05),且主要上调CD3,CD4和CD8的表达。5.0～20.0mg·L-1rSEB-D9N作用的PBMC上清对KB和MCF-7细胞生长均可产生明显抑制作用(P<0.05)。结论获得并纯化了重组葡萄球菌肠毒素B突变体蛋白(rSEB-D9N),其细胞毒性作用有所降低,但促人淋巴细胞增殖和抑制肿瘤细胞生长作用仍较强,可作为研制升白细胞、抗肿瘤相关药物的候选突变体。
Objective To express and purify the recombinant staphylococcal enterotoxin B mutant protein D9N (rSEB-D9N) and investigate the cytotoxicity of the expressed product rSEB-D9N and the inhibition of lymphocyte proliferation and tumor growth inhibition. Methods The rSEB-D9N was expressed in pET32a-SEB-D9N-E.coli BL21DE3 by different concentrations of IPTG. The rSEB-D9N and 10% SDS-PAGE were purified by Ni-NTA affinity chromatography. Chromatography to determine its purity. Cytotoxicity of rSEB-D9N was performed using Vero cells as target cells and TCID50 values were calculated. The effect of different concentrations of rSEB-D9N on proliferation of human lymphocytes and inhibition of the growth of KB and MCF-7 cancer cell lines were detected by MTT assay. Flow cytometry was used to detect the expression of CD3, CD4 and CD8 after rSEB-D9N stimulated human lymphocytes. Results rSEB-D9N expression accounted for about 30% of bacterial total protein, purified to obtain a purity of 90.48% rSEB-D9N. The TCID50 values of rSEB and rSEB-D9N in Vero cells were 3.12,8.85μg, 10.0,20.0mg · L-1, respectively. rSEB-D9N had significant effect on promoting PBMC proliferation (P <0.05) And CD8 expression. The supernatant of PBMC of 5.0-20.0 mg · L-1 rSEB-D9N could significantly inhibit the growth of KB and MCF-7 cells (P <0.05). Conclusions The recombinant protein of staphylococcal enterotoxin B (rSEB-D9N) was obtained and purified and its cytotoxicity was reduced. However, the effect of promoting the proliferation of human lymphocytes and inhibiting the growth of tumor cells was still strong. Candidate mutants of anti-tumor related drugs.