SEB基因定位突变体构建及其重组表达产物生物学活性研究

Construction of the site-specific mutants of staphylococcal enterotoxin B gene and biological activities of their recombinant expression products

目的 构建葡萄球菌肠毒素B（SEB）基因及其突变体原核表达系统,了解突变前、后重组表达产物的细胞毒性、促淋巴细胞增殖和抑制肿瘤生长活性的变化.方法 采用突变引物PCR构建SEB基因定位突变体,建立SEB基因及其定位突变体原核表达系统;同时采用Ni-NTA亲和层析法提纯表达的目的 重组蛋白;采用TCID50法测定目的 重组蛋白对Vero细胞的毒性;采用MTT比色法分别检测不同浓度的目的 重组蛋白促使小鼠脾细胞增殖及对抑制KB和HL-60癌细胞株生长的作用.结果 所克隆的SEB基因核苷酸序列与各项研究报道的相似性为100%,3种突变体均在既定位置获得预期的密码子突变.rSEB和各突变体重组蛋白表达量约为细菌总蛋白的40%;rSEB对Vero细胞TCIC50为3.4μg,其突变体重组蛋白分别为3.2～16.8μg;1和5μg/ml的rSEB及突变体重组蛋白rSEB/D9N对小鼠脾细胞均有明显的促增殖作用（P＜0.05）,10和20μg/ml的rSEB及rSEB/D9N促进小鼠脾细胞增殖活性与100μg/ml植物血凝素PHA相似（P＞0.05）.5～20μg/ml的rSEB及rSEB/D9N作用的小鼠脾细胞上清液及其上清液与脾细胞混合物均能有效抑制KB和HL-60细胞生长（P＜0.05）.结论 本研究成功构建了SEB基因突变体及其高效原核表达系统;其中突变体重组蛋白rSEB/D9N细胞毒性较低,促脾细胞增殖和抑制肿瘤细胞生长活性较强,可作为研制升高白细胞、抗肿瘤的SEB相关药物的候选突变体.

Objective To construct prokaryotic expression systems of staphylococcal enterotoxin B （SEB） gene and its mutants, and to determine the activity changes of recombinant expression products. Methods The site-specific mutants of SEB gene by PCR using mutant primers as well as prokaryotic expression systems of the SEB gene and its mutants were constructed. Ni-NTA affinity chromatography was performed to extract the expressed target recombinant proteins. The cytotoxicity to Vero cells of the recombinant proteins was measured using TCID50 titration method. MTT colorimetry was applied to examine the effects of the recombinant proteins at different dosages on promoting proliferation of mouse splenocytes and inhibiting growth of tumor cell lines KB and HL-60. Results The similarity of nucleotide sequence of the cloned SEB gene was 100% compared to the reported SEB sequences and the expected mutation of codons in each the three mutants at the scheduled positions were present. All the recombinant protein expression outputs of SEB gene and its mutants were approximate 40% of the total bacterial proteins. The TCIC50 value of recombinant SEB to Vero cells was 3.4 μ g; while those of the recombinant SEB mutants were 3.2-16.8 μg, respectively. 1 and 5 μg/ml of rSEB and rSEB/D9N showed remarkable effects to promote proliferation of mouse splenocytes （P〈0.05）. The activities of promoting mouse splenocyte proliferation of 10 and 20 p g/ml rSEB and rSEB/D9N were similar to that of 100 μg/ml PHA （P 〉0.05）. The supernatants or the mixture of supernatant and splenocytes stimulated by 5N20 μ g/ml rSEB or rSEB/D9N efficiently inhibited the growth of KB and HL-60 cells （P〈0.05）. Conclusion The SEB gene mutants and their prokaryotic expression systems with high expression efficiency were successfully established in this study. Since rSEB/D9N displays lower cytotoxicity, stronger promoting effect on splenocyte proliferation and inhibiting effect on tumor cell growth, it might be a potential candidate for developing leukogenic and anti-tumor SEB associated drugs.