大肠杆菌Ⅰ型菌毛基因的克隆﹑表达及呈现表达载体的构建(部

1、 大肠杆菌大肠杆菌型菌毛基因的型菌毛基因的 克隆表达及呈现表达载体的构建克隆表达及呈现表达载体的构建 （部分内容）（部分内容）学生：王国相学生：王国相导师：戴鼎震导师：戴鼎震 秦爱建秦爱建型菌毛基因结构图型菌毛基因结构图 B E A I C D F G HP1P2P3P4P5P6 P7P8 P9引物引物P1,P2P1,P2扩增片段扩增片段BEABEA引物引物P3,P4P3,P4扩增片段扩增片段ICIC引物引物P5,P6P5,P6扩增片段扩增片段D D引物引物P5,P7P5,P7扩增片段扩增片段DD引物引物P8,P9P8,P9扩增片段扩增片段FGHFGH引物引物P5,P9P5,P9用于用于SOE

2、 PCRSOE PCR，扩增片段，扩增片段DFGH DFGH RegulationMajor subunit ExportChaperoneUsherMinor subunitsNumber regulatorLength regulatorAdhesin型菌毛呈现表达载体结构模式图型菌毛呈现表达载体结构模式图ABCA, Fimbriated E.coli. B,C, Model for high- and low-valency display of heterologousBy type fimbriae . Black knobs represent the passenger epit

3、opes.片段片段BEA PCR扩增图扩增图 1 2 3 1 2 3 23130231309416941665576557436143613300330023202320202720271，3 片段片段BEA PCR产物产物2 分子量标准分子量标准Hind digest digest片段片段IC,D PCR扩增扩增 1 2 323130941665574361232020271 分子量标准分子量标准Hind digest2 片段片段IC PCR扩增产物约扩增产物约2050bp3 片段片段D PCR扩增产物约扩增产物约2150bp片段片段D ，FGH PCR扩增扩增 1 2 3 23130941

4、665574361232020271 分子量标准分子量标准Hind digest2 片段片段D PCR扩增产物约扩增产物约2200bp3 片段片段FGH PCR扩增产物约扩增产物约2300bpSOE PCR 扩增片段扩增片段DFGH1 2 bp500040003000200010001片段片段DFGH PCR扩增产物约扩增产物约4300bp2 1Kbp DNA Landder 质粒质粒pBEA的构建的构建1pUC182686AmprColE1orilacZMCSEcoR1Kpn1fimB fimE fimAEcoR1Kpn11pBEA6016bpAmprColEorifimBfimEfimAL

5、acZEcoR1Kpn1EcoR1+Kpn1双酶切双酶切 质粒质粒pBEA酶切鉴定酶切鉴定 1 2 3 4 5 20272322436165579416231302670330060001,2为为pBEA EcoR+Kpn双酶切图双酶切图3,43,4为为pBEA EcoR单酶切图单酶切图5为为-Hind digest marker 片段片段IC，D的的T/A克隆克隆 1 2 3 4 1 2 3bp2313094166557436123202027bp6000500040003000200010001 分子量标准分子量标准Hind digest2,3,4 IC T/A 克隆克隆Kpn1+BamH

6、1双酶切双酶切1 1Kbp DNA Landder2,3 D T/A克隆克隆BamH1+Sal1双酶切双酶切 质粒质粒pBC的构建的构建1pBEA6016bpAmprColEorifimBfimEfimALacZEcoR1Kpn1BamH1Kpn1+BamH1双酶切双酶切1pUC-IC4680bpAmprColE1orifimIfimCLacZKpn1BamH11pBC8000bpAmprColEorifimBfimEfimALacZfimIfimCEcoR1Kpn1BamH1Sal1 质粒质粒pBD的构建的构建BamH1+Sal1双酶切双酶切1pBC8000bpAmprColEorifimB

7、fimEfimALacZfimIfimCEcoR1Kpn1BamH1Sal11pUCm-D4880bpAmprColE1 oriLacZfimDBamH1Sal11pBD10100bpAmprColEorifimBfimEfimALacZfimIfimCfimDEcoR1Kpn1Sal1BamH1质粒质粒pBC,pBD酶切鉴定酶切鉴定 1 2 3 4 519002100102008000610080001，pBC BamH单酶切单酶切2 2，pBC BamH+Kpn双酶切双酶切3， -Hind digest marker 4， pBD BamH单酶切单酶切5， pBD BamH+Sal双酶切双

8、酶切pBH的构建的构建1pBD10100bpAmprColEorifimBfimEfimALacZfimIfimCfimDEcoR1Kpn1Sal1BamH11pBH12400bpAmprColEorifimBfimEfimALacZfimIfimCfimDfimFfimGfimHEcoR1Kpn1BamH1Sal1BamH1 fimD fimF fimG fimH Sal1 BamH1+Sal1双酶切双酶切pBH酶切鉴定酶切鉴定1 2 4bp2313094166557436123202027bp12400800043001 分子量标准分子量标准Hind digest2 pBH BamH1+Sal1双酶切双酶切3 pBH Sal1单酶切单酶切 SDS-PAGE电泳电泳 1 2 3 4