葡萄球菌肠毒素B基因原核表达系统的构建及其表达产物促淋巴细胞增殖和抑制肿瘤细胞生长作用的研究

Construction of prokaryotic expression system of staphylococcal enterotoxin B gene and effects of its expressed product on promoting lymphocyte proliferation and inhibiting the growth of tumor cell.

构建葡萄球菌肠毒素B（staphylococcal enterotoxins B，SEB）基因原核表达系统，了解重组表达产物rSEB促淋巴细胞增殖和抑制肿瘤生长的作用．采用高保真PCR从金黄色葡萄球菌FRIS6B株DNA中扩增全长SEB基因片段，T—A克隆后测序，构建SEB基因原核表达系统pET32α-SEB—E．coliBL2IDE3．采用SDS-PAGE检测rSEB表达产量，Ni—NTA亲和层析法提纯rSEB．采用TCID50法测定rSEB对Vero细胞的细胞毒性作用并计算TCID50值．采用MTT比色法分别检测不同浓度rSEB体外对小鼠脾细胞、人外周血单个核细胞（PBMC）的促增殖作用以及对HepG2细胞（人肝腺癌细胞）、HeLa细胞（人宫颈癌上皮细胞）的生长抑制作用．与公布的相关序列比较，所克隆的SEB基因核苷酸序列相似性为100％．rSEB表达量约为细菌总蛋白的40％．rSEB对Vero细胞的TCIC50为3．02μg．5．0～20．0μg／ml的rSEB对小鼠脾细胞和人PBMC均有明显的促增殖作用（P〈0．05）．5．0～20．0μg／ml rSEB作用的人PBMC上清均能有效地抑制HepG2细胞和HeLa细胞生长（P〈0．05）．本研究成功地构建了rSEB高效原核表达系统．rSEB仍然具有生物学活性．所建立的细胞毒性、促淋巴细胞增殖和抑制肿瘤细胞生长作用的检测方法，为以后减毒rSEB突变体的筛选奠定了基础．

To construct a prokaryotic expression system of staphylococcal enterotoxin B gene and determine the effects of the recombinant expression product rSEB on promoting lymphocyte proliferation and inhibiting tumor cell growth. A high fidelity PCR was used to amplify entire SEB gene from DNA of S. aureus strain FRIS6B. The cloned SEB gene was sequenced after T-A cloning, pET32α-SEB-E, coliBL21DE3, a prokaryotic expression system of the SEB gene, was then constructed. SDS-PAGE was applied to measure the output of rSEB under inducement. Ni-NTA affinity chromatography was performed to extract and purify rSEB. Cytotoxicity of rSEB to Vero cells was detected by using TCID50 titration method and then the value of TCIC50 was determined. MTT colorimetry was established to examine the effects of rSEB at different dosages on promoting proliferation of mouse splenocytes and hu- man peripheral blood mononuclear cells （PBMC） as well as on inhibiting the growth of HepG2 cells and HeLa cells in vitro. In comparison with the published corresponding sequences, similarities of the nucleotide and putative amino acid sequences of the cloned SEB gene were 100%.Output of rSEB was approximate 40% of the total bacterial proteins, rSEB had a cytotoxicity with TCIC50 of 3.02μg to Vero cells. 5.0-20.0μg/ml rSEB showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC （P〈0.05）. The supernatants of human PBMCs treated with 5.0-20.0μg/ml rSEB could efficiently inhibit the growth of HepG2 and HeLa cells （P〈0.05）. A prokaryotic expression system of rSEB with high efficiency was successfully established in this study, rSEB still maintain the original biological activities. The established methods to detect cytotoxicity, promoting proliferation of lymphocytes and inhibiting growth of tumor cells lay a foundation for further screening of attenuated rSEB mutants.