大肠杆菌肠毒素ST_1-LT_B融合基因的高效表达

HIGHLEVELEXRESSIONOFFUSIONGENEOF ESCHERICHIACOLI HEATSTABLEENTEROTOXIN(ST1)ANDHEATLABILEENTEROTOXINBSUBUINT(LTB)

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）（ｐＸＥＴＳＬＴ１）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。

WeusedrestrictionendonucleasesEcoRandHindtocleave08kb Ecoli ST1LTBfusiongenefromplasmidpXSLT1,isolatedby15%agarosegelelectrophoresis,recovered084kbgenefragment,theninserteditintoanexpressionvectorpET28b(+)whichcleavedwithEcoRandHindbybluntendligationTherecombinantplasmidpXETSLT1wasstudiedindetailbyrestrictionendonucleaseanalysisTheresultshaveshownthattherecombinantplasmidcarriedST1LTBfusiongeneBytransformationof Ecoli BL21(DE3),wegot Ecoli BL21(DE3)(pXETSLT1)TherecombinantstraincouldproducepreST1LTBfusionproteinbyELISAandSDSPAGE,andthefusionproteinwasnontoxicAfterrecombinatstrainwasinducedbyIPTG,itsexpressedproductwasabout3321%oftotalcellularproteinbySDSPAGEandthinlayergelscanninganalysis