sea基因定位突变体构建及其重组表达产物生物学活性的研究  

Construction of site-specific mutants of Staphylococcus enterotoxin A gene and biological activities of recombinant expression products

作　　者：范芳华[1] 严杰[2] 毛亚飞[1] 

机构地区：[1]浙江大学医学院病原生物学系,杭州310031 [2]浙江大学附属第一医院卫生部传染病重点实验室,杭州310031

出　　处：《中华微生物学和免疫学杂志》2007年第6期525-531,共7页Chinese Journal of Microbiology and Immunology

基　　金：浙江省科技计划项目(2003C34010)

摘　　要：目的构建葡萄球菌肠毒素A(SEA)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。方法采用高保真PCR和T- A克隆法,从金黄葡萄球菌ATCC13565株DNA中扩增并克隆了不含信号肽序列的sea基因。采用突变引物PCR构建sea基因定位突变体。建立sea基因及其定位突变体原核表达系统。Ni-NTA亲和层析法提纯表达的目的重组蛋白。采用TCID_(50)法测定目的重组蛋白对Vero细胞的细胞毒性。采用MTT比色法分别检测不同浓度的目的重组蛋白促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sea基因核苷酸序列与报道序列的相似性为100%,7种突变体均在既定位置获得预期的密码子突变。rSEA和各突变体重组蛋白表达量约为细菌总蛋白的52%。rSEA对Vero细胞TCID_(50)为4.1μg,其突变体重组蛋白分别为5.8～23.5μg。1和5μg/ml的rSEA及其5种突变体重组蛋白rSEA/H187A、rSEA/H225A、rSEA/D227A、rSEA/H187A/D227A和rSEA/H225A/D227A对小鼠脾细胞均有明显的促增殖作用(P<0.05),10和20μg/ml的rSEA及上述5种突变体重组蛋白促小鼠脾细胞增殖活性与100μg/ml PHA相似(P>0.05)。5～20μg/ml的rSEA及上述5种突变体重组蛋白作用的小鼠脾细胞上清及其上清与脾细胞混合物均能有效地抑制KB和HL-60细胞生长(P<0.05)。结论本研究成功地构建了sea基因突变体及其高效原核表达系统,其表达产物的细胞毒性均有所降低。由于rSEA/H225A、rSEA/D227A、rSEA/H225A/D227A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,可作为研制升白细胞、抗肿瘤SEA相关药物的候选突变体。Objective To construct prokaryotic expression systems of Staphylococcus enterotoxin A （SEA） gene and its mutants, and to determine the activity changes of recombinant expression products before and after mutation shown by cytotoxicity, promoting lymphocyte proliferation and inhibiting tumor cell growth. Methods sea gene with no signal peptide sequence of S. aureus strain ATCC13565 was amplified and then cloned by using high fidelity PCR and T-A cloning method. The site-specific mutants of sea gene using mutant primers as well as prokaryotic expression systems of the sea 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 sea gene was 100% as compared to the reported SEA sequences and the expected mutation of codons in each seven mutants at the scheduled positions was proved. All the recombiant protein expression outputs of sea gene and its mutants took approximate 52% of the total bacterial proteins. The TCID50 of rSEA＇s cytotoxicity to Veto cells was 4.1μg while the TCID50 values of the recombinant of sea gene mutants was 5.8-23.5μg, respectively. One and fiveμg/ml of rSEA and its five mutant proteins rSEA/H187A, rSEA/H225A, rSEA/D227A, rSEA/H187A/D227A and rSEA/H2.25A/D227A showed remarkable effects to promote proliferation of mouse splenocytes （P 〈 0.05）. The activities of promoting mouse splenocyte proliferation of 10 and 20 μg/ml rSEA and the five mutant proteins were similar to that of 100μg/ml PHA （ P 〉 0.05）. The supernatants or the mixture of supernatant and splenocytes stimulated by 5-20μg/ml rSEA or each five mutant proteins efficient

关 键 词：葡萄球菌肠毒素A sea基因/定位突变 原核表达系统/构建 重组蛋白 细胞毒性 脾细胞/增殖 肿瘤细胞/抑制 

分 类 号：R346[医药卫生—基础医学]