| 178 | 4 | 47 |
| 下载次数 | 被引频次 | 阅读次数 |
目的通过基因重组方式获得高纯度、高含量重组金黄色葡萄球菌肠毒素A(rSEA)。方法将金黄色葡萄球菌肠毒素A(SEA)基因片段插入pET28a载体中,测序并正确鉴定后,将重组质粒转化到大肠杆菌BL21(DE3)中,经IPTG诱导,表达目的蛋白。重组蛋白用Ni-NTA His蛋白亲和柱纯化。结果 SDS-PAGE结果表明成功表达了分子量约为30 kD的重组蛋白,经Western blot和小鼠生物学鉴定,表达产物为重组金黄色葡萄球菌肠毒素A,并且具有较好的免疫原性,为制备抗金黄色葡萄球菌肠毒素A单克隆抗体及建立相应的免疫学检测方法奠定了基础。结论构建的rSEA表达载体稳定,可高效表达目的 rSEA蛋白。
Abstract：Objective To clone the sequence of Staphylococcus aureus enterotoxin A( SEA) gene,construct its expression vector,and obtain high levels of recombinant Staphylococcus aureus enterotoxin A( rSEA) with high purity.Methods The enterotoxin A gene was synthesized and inserted into the prokaryotic expressed vector pET28a possessing histiding-tag.The recombinant vector was transfected into E.Coli BL21( DE3) after it had been verified by DNA sequencing.The strain can express the recombinant protein after the induction by the IPTG.The recombinant protein was purified by Ni-NTA His Bind affinity purification column.Results The recombinant protein was obtained.SDS-PAGE showed that its molecular mass was about 30kD.The western bolt showed that it can specifically bind rabbit anti-SEA antibody.The BALB/c mouse was immunized by the recombinant protein followed by detection of anti-SEA antibody by ELISA.The results revealed that the protein has reactogenicity and immunogenicity,which provides the foundation for either the preparation of monoclonal antibodies or the further study of the immunological detection of SEA.Conclusion The rSEA expression vector was constructed successfully and highly expressed the rSEA protein.
[1]SCHLIEVERT P M,JABLONSKI L M,ROGGIANI M,et al.Pyrogenic toxin superantigen site specificity in toxic shock syndrome and food poisoning in animals[J].Infect Immun,2000,68(6):3630-3634.
[2]JANTRA J,KANATHARANA P,ASAWATRERATANAKUL P,et al.Detection of staphylococcal enterotoxin A(SEA)at pictogram level by a capacitive immunosensor[J].J Environ Sci Health A Tox Hazard Subst Environ Eng,2011,46:560-568.
[3]REN K,BANNAN J D,PANCHOLI V,et al.Characterization and biological properties of new staphylococcal exotoxin[J].J Exp Med,1994,180(5):1675-1683.
[4]SHAFER V M,LANDOLO J J.Staphylococcal enterotoxin A:a chromosomal gene product[J].Appl Environ Microbiol,1978,36(2):389-391.
[5]TSUTSUURA S,SHIMAMURA Y,MURATA M.Temperature dependence of staphylococcal enterotoxin A production by Staphylococcus aureus[J].Biosci Biotechnol Biochem,2013,77(1):30-37.
[6]LANGFORD M P,STANTON G J,JOHNSON H M.Biological effects of staphylococcal enterotoxin A on human peripheral lymphocytes[J].Infect Immun,1978,22(1):62-68.
[7]U.S.department of health and human services.Surveillance for Foodborne-Disease Outbreaks-United States,1988-1992[J].MMWR,1996,45:SS-5.
[8]Snow Brand coverup suspected in food poisonings[N/OL].Japan Times,2000-08-25.http://www.japantimes.co.jp/text/nn20000825a4.html.
[9]HORSMON J R,CAO C J,KHAN A S,et al.Realtime fluorogenic PCR assays for the detection of entA,the gene encoding staphylococcal enterotoxin A[J].Biotechnol Lett,2006,28(11):823-829.
[10]KUANG H,WANG W,XU L,et al.Monoclonal antibody-based sandwich ELISA for the detection of staphylococcal enterotoxin A[J].Int J Environ Res,2013,10(4):1598-1608.
[11]VARSHNEY A K,MEDIAVILLA J R,ROBIOUS N,et al.Diverse enterotoxin gene profiles among clonal complexes of Staphylococcus aureus isolates from the Bronx,New York[J].Appl Environ Microbiol,2009,75(21):6839-6849.
[12]丁军颖,伊瑶,毕胜利,等.不同温度、时间、IPTG和菌种浓度对丁肝抗原蛋白表达量的影响[J].医学研究杂志,2012,41(5):31-34.
[13]王丽婵,张庶民,杨晓明,等.金黄色葡萄球菌肠毒素A基因克隆、表达、纯化及鉴定[J].中国生物制品学杂志,2006,19(2):120-123.
基本信息:
DOI：10.19813/j.cnki.weishengyanjiu.2013.06.006
中图分类号:R378
引用信息:
[1]王佳慧,李楠,沈青,等.重组金黄色葡萄球菌肠毒素A的表达及鉴定[J].卫生研究,2013,42(06):920-924.DOI:10.19813/j.cnki.weishengyanjiu.2013.06.006.
2013-08-28
2013
2013-08-29
2013
1
2013-11-30
2013-11-30