葡萄球菌肠毒素A基因原核表达系统的构建及其表达产物的鉴定

 目的：构建葡萄球菌肠毒素A（SEA）基因原核表达系统，了解重组表达产物rSEA促淋巴细胞增殖和抑制肿瘤生长的作用。方法：采用高保真PCR从金黄色葡萄球菌ATCC13565株DNA中扩增全长SEA基因片段，T-A克隆后测序，构建SEA基因原核表达系统pET32a-SEA-E.coliBL21DE3。采用SDS-PAGE检测rSEA表达量，Ni-NTA亲和层析法提纯rSEA。采用TCID₅₀法测定rSEA对Vero细胞的细胞毒性并计算TCIC₅₀值。采用MTT比色法分别检测不同浓度rSEA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（人肝癌细胞）、HeLa细胞（人宫颈癌上皮细胞）的生长抑制作用。结果: 与公布的相关序列比较，所克隆的SEA基因核苷酸序列相似性为100%。rSEA表达量约为细菌总蛋白的25%。rSEA对Vero细胞的TCIC₅₀为3.14 μg。1.0-20.0 mg/L的rSEA对小鼠脾细胞和人PBMC均有明显的促增殖作用（P<0.05）。5.0-20.0 mg/L rSEA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）。结论:成功地构建了rSEA原核表达系统。rSEA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rSEA突变体的筛选奠定了基础。

Construction and identification of prokaryotic expression system of staphylococcal enterotoxin A gene and expressed product

AIM: To construct a prokaryotic expression system of staphylococcal enterotoxin A (SEA) gene and determine the effects of the recombinant expression product rSEA in promoting lymphocyte proliferation and inhibiting tumor cell growth. METHODS: PCR was used to amplify entire SEA gene of S. aureus strain ATCC13565. The cloned SEA gene was sequenced after T-A cloning. SDS-PAGE was applied to measure the output of rSEA expressed by pET32a-SEA- E. coli BL21DE3. Ni-NTA affinity chromatography was performed to extract rSEA. Cytotoxicity of rSEA to Vero cells was detected using TCID₅₀ titration method and then the value of TCIC₅₀ was determined. MTT colorimetry was established to examine the effects of rSEA at different dosages on proliferation of mouse splenocytes and human peripheral blood mononuclear cells (PBMC) as well as on growth of HepG2 cells and HeLa cells in vitro. RESULTS: In comparison with the published corresponding sequences, similarities of the nucleotide and putative amino acid sequences of the cloned SEA gene were 100%. The output of rSEA was approximate 25% of the total bacterial proteins. rSEA had a cytotoxicity with TCIC₅₀ of 3.14 μg to Vero cells. 1.0-20.0 mg/L rSEA showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC (P<0.05). The supernatants of human PBMCs treated with 5.0-20.0 mg/L rSEA efficiently inhibited the growth of HepG2 and HeLa cells (P<0.05). CONCLUSION: A prokaryotic expression system of rSEA was successfully established. rSEA still retains 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 rSEA mutants.