大肠杆菌肠毒素ST_1—LT_B融合基因的高效表达

用限制性核酸内切酶ＥｃｏＲＩ和ＨｉｎｄⅢ双酶切含大肠杆菌肠毒素ＳＴ１—ＬＴＢ融合基因的质粒ｐＸＳＬＴ１，回收０．８４ｋｂ的ＳＴ１—ＬＴＢ融合基因，再将载体ｐＥＴ—２８ｂ（＋）用ＥｃｏＲⅠ和ＨｉｎｄⅢ双酶切，然后将其与ＳＴ１—ＬＴＢ融合基因连接，转化至受体菌ＢＬ２１（ＤＥ３）中。经ＥｃｏＲⅠ、ＨｉｎｄⅢ、ＢａｍＨⅠ酶切反应鉴定重组子质粒，得到了理想重组质粒ｐＸＥＴ－ＳＬＴ１。重组菌株ＢＬ２１（ＤＥ３）（ｐＸＥＴＳＬＴ１）经ＩＰＴＧ诱导后，其表达产物经ＳＤＳ—ＰＡＧＥ和ＥＬＩＳＡ检测，结果表明重组菌株可以高效表达ＳＴ１—ＬＴＢ融合蛋白，该融合蛋白占菌体总蛋白的３３．２１％，而且无天然ＳＴ１生物毒性

High—Level Expression of Fusion Gene of the Heat—Stable Enterotoxin Ⅰ(ST 1) and Heat-Labile Enterotoxin B Subuint(LT B) of Escherichia Coli

おhe authors used restriction endonucleases EcoR Ⅰ and Hind Ⅲ to cleave 084kb E.coli ST 1—LT B fusion gene from plasmid pXSLT1,isolated by 15% agarose gel electrophoresis,recovered 084kb gene fragment,then inserted it into an expression vector pET—28b(+) which cleaved with EcoR Ⅰ and Hind Ⅲ by blunt—end ligation.The recombinant plasmid pXET—SLT1 was studied in detail by restriction endonuclease analysis.The results showed that the recombinant plasmid carried ST1—LTB fusion gene.By transformation of E.coli BL21(DE3),we obtained E.coli BL21(DE3)(pXETSLT1).The recombinant strain can produce pre-ST1—LTB fusion protein by ELISA and SDS-PAGE,and the fusion protein was nontoxic.After recombinat strain was induced by IPTG,is expressed product was about 3321% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.