Construction and identification of prokaryotic expression system of staphylococcal enterotoxin A gene and expressed product
- VernacularTitle: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
- Author:
Shuiling XU
;
Yafei MAO
;
Meiguang ZHANG
;
Dongjiao LUO
;
Jie YAN
- Publication Type:Journal Article
- Keywords:
Staphylococcal enterotoxin A;
Gene expression;
Prokaryotic expression;
HepG2 cells;
HeLa cells;
Vero cells
- From:
Chinese Journal of Pathophysiology
1986;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
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 TCID_ 50 titration method and then the value of TCIC_ 50 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 TCIC_ 50 of 3.14 ?g to Vero cells.1.0-20.0 mg/L rSEA showed the significant effects of promoting proliferation of mouse splenocytes and human PBMC(P
