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\u51b3\u5b9a\u4f7f\u7528\u4e24\u79cd\u4e0d\u540c\u7684\u6db2\u4f53\u5904\u7406\u7cfb\u7edf\u662f\u51fa\u4e8e\u6700\u9ad8\u6548\u5229\u7528\u5b9e\u9a8c\u5ba4\u8bbe\u5907\u7684\u9700\u6c42\uff1a\u53ea\u8981\u6709\u53ef\u80fd\uff0c\u5c31\u4f7f\u7528 OT-2 \u8fdb\u884c\u5355\u5143\u64cd\u4f5c\u3002\u4e0e\u66f4\u6602\u8d35\u7684 EVO 200 \u76f8\u6bd4\uff0c\u8fd9\u8282\u7701\u4e86\u65f6\u95f4\uff0c\u56e0\u6b64\u53ef\u7528\u4e8e\u8981\u6c42\u66f4\u9ad8\u7684\u5b9e\u9a8c\u3002\u5728\u672c\u7814\u7a76\u4e2d\uff0c\u5355\u5143\u64cd\u4f5c\u201c\u83cc\u843d\u91c7\u6458\u201d\u662f\u624b\u52a8\u5b8c\u6210\u7684\u3002\u5b83\u53ef\u4ee5\u901a\u8fc7\u4e13\u7528\u8bbe\u5907\u81ea\u52a8\u5b8c\u6210\uff0c\u4f46\u5f53\u65f6\u6ca1\u6709\u8fd9\u4e9b\u8bbe\u5907\u3002
\u57fa\u4e8e\u8f6c\u5f55\u56e0\u5b50\u7684\u751f\u7269\u4f20\u611f\u5668\u7684\u5408\u7406\u7ec4\u5408\u8bbe\u8ba1\u4e3a\u4e86\u8bc1\u660e\u6211\u4eec\u7684\u5de5\u4f5c\u6d41\u7a0b\u7684\u9002\u7528\u6027\uff0c\u8bbe\u8ba1\u3001\u6784\u5efa\u5e76\u5728\u8c37\u6c28\u9178\u68d2\u6746\u83cc\u4e2d\u8868\u8fbe\u4e86\u4e0d\u540c\u7248\u672c\u7684 Lrp \u751f\u7269\u4f20\u611f\u5668\u3002Lrp \u751f\u7269\u4f20\u611f\u5668\u5c06\u7ec6\u80de\u5185l -\u86cb\u6c28\u9178\u3001l -\u4eae\u6c28\u9178\u3001l -\u5f02\u4eae\u6c28\u9178\u548cl -\u7f2c\u6c28\u9178\u6d53\u5ea6\u4e0e\u8367\u5149\u62a5\u544a\u86cb\u767d eYFP \u7684\u8868\u8fbe\u76f8\u7ed3\u5408\u3002\u4e3a\u4e86\u6784\u5efa\u4e0d\u540c\u7248\u672c\u7684 Lrp \u751f\u7269\u4f20\u611f\u5668\uff0c\u5bf9\u4f20\u611f\u5668\u7684\u4e0d\u540c\u90e8\u5206\u8fdb\u884c\u4e86\u4fee\u6539\u3002Lrp \u751f\u7269\u4f20\u611f\u5668\u7531lrp\u57fa\u56e0\uff08\u7f16\u7801 Lrp \u8f6c\u5f55\u56e0\u5b50\uff09\u3001\u53d1\u6563\u8868\u8fbe\u7684eyfp\u57fa\u56e0\uff08\u7f16\u7801\u8367\u5149\u62a5\u544a\u57fa\u56e0\uff09\u548c\u57fa\u56e0\u95f4\u533a\uff08\u5305\u542blrp\u542f\u52a8\u5b50\u548ceyfp\u4e0a\u6e38\u7684brnF\u542f\u52a8\u5b50\u4ee5\u53caeyfp RBS\uff09\u7ec4\u6210(19)\uff08\u56fe2 \uff09\u9488\u5bf9lrp\u8d77\u59cb\u5bc6\u7801\u5b50\u548c RBS\u3001brnF\u542f\u52a8\u5b50\u548ceyfp RBS\u8bbe\u8ba1\u4e86\u4e0d\u540c\u7684\u53d8\u4f53\u3002<\/p>\n\n\n\n

\"\u8c37\u6c28\u9178\u68d2\u6746\u83cc\u4e2d\u6784\u5efa\u548c\u8868\u8fbe\"
\u56fe2.\u4e3a\u5728\u8c37\u6c28\u9178\u68d2\u6746\u83cc\u4e2d\u6784\u5efa\u548c\u8868\u8fbe\u800c\u8bbe\u8ba1\u7684\u4e0d\u540c Lrp \u751f\u7269\u4f20\u611f\u5668\u53d8\u4f53\u7684\u56fe\u5f62\u6982\u89c8\u3002\u5de6\u4fa7\u8bbe\u8ba1\u4e86 9 \u79cd\u4e0d\u540c\u7684\u5f15\u7269\uff0c\u5305\u542b\u4e0d\u540clrp\u8d77\u59cb\u5bc6\u7801\u5b50\u548clrp RBS \u7684\u7ec4\u5408\u3002\u53f3\u4fa7\u8bbe\u8ba1\u4e86 12 \u79cd\u4e0d\u540c\u7684\u5f15\u7269\uff0c\u5305\u542b\u4e0d\u540cbrnF\u542f\u52a8\u5b50\u5e8f\u5217\u548ceyfp RBS \u7684\u7ec4\u5408\u3002<\/figcaption><\/figure>\n\n\n\n

\u5bf9\u4e8elrp\u8d77\u59cb\u5bc6\u7801\u5b50\uff0c\u9009\u62e9\u4e86\u4e09\u79cd\u53d8\u4f53\uff1a\u5929\u7136\u8d77\u59cb\u5bc6\u7801\u5b50 ATG\uff08\u5e94\u5bfc\u81f4\u6700\u9ad8\u8868\u8fbe\uff09\u3001\u8d77\u59cb\u5bc6\u7801\u5b50 GTG\uff08\u5e94\u5bfc\u81f4\u8f83\u4f4e\u8868\u8fbe\uff09\u548c TGT\uff08\u5e94\u5bfc\u81f4\u975e\u5e38\u4f4e\u6216\u65e0\u8868\u8fbe\uff09\u3002\u8fd9\u4e9b\u8d77\u59cb\u5bc6\u7801\u5b50\u53d8\u4f53\u4e2d\u7684\u6bcf\u4e00\u4e2a\u90fd\u4e0e\u4e09\u79cd\u4e0d\u540c\u7684lrp RBS \u7ec4\u5408\uff0c\u540c\u6837\u4ece\u6700\u9ad8\u8868\u8fbe\u5230\u6700\u4f4e\u8868\u8fbe\uff1aGCTAAAATGG\uff08\u6700\u5f3a\uff09\u3001GCTATTGTGC\uff08\u5929\u7136\uff0c\u8f83\u5f31\uff09\u548c CAATCCTACC\uff08\u6700\u5f31\uff09\u3002lrp\u4fa7\u7684\u4e09\u4e2a\u8d77\u59cb\u5bc6\u7801\u5b50\u548c\u4e09\u4e2a RBS \u7684\u7ec4\u5408\u4ea7\u751f\u4e86 3 \u00d7 3 = 9 \u79cd\u4e0d\u540c\u7684\u5f15\u7269\u3002
eyfp\u57fa\u56e0\u5728brnF\u542f\u52a8\u5b50\u7684\u63a7\u5236\u4e0b\u8868\u8fbe\uff0c\u8be5\u542f\u52a8\u5b50\u542b\u6709 Lrp \u86cb\u767d\u7684\u7ed3\u5408\u4f4d\u70b9\u3002(25)\u56e0\u6b64\uff0c\u5f53\u542f\u52a8\u5b50\u5e8f\u5217\u7f29\u77ed\u6216\u5ef6\u957f\u65f6\uff0c\u53ef\u4ee5\u6dfb\u52a0\u6216\u79fb\u9664\u7ed3\u5408\u4f4d\u70b9\u3002\u7531\u4e8ebrnF\u8f6c\u5f55\u4e3a\u65e0\u524d\u5bfc\u8f6c\u5f55\u672c\uff0c\u5e76\u4e14brnF\u7684\u7b2c\u4e00\u90e8\u5206\u53ef\u80fd\u5177\u6709\u8c03\u63a7\u529f\u80fd\uff0c(26)\u6807\u51c6Lrp\u4f20\u611f\u5668\u7684eyfp\u542f\u52a8\u5b50\u5305\u542bbrnF\u7684\u524d30bp \u3002\u672c\u7814\u7a76\u8bbe\u8ba1\u4e86\u56db\u79cd\u4e0d\u540c\u7684\u53d8\u4f53\uff0c\u4ece+30\uff08\u6807\u51c6Lrp\u751f\u7269\u4f20\u611f\u5668\u542f\u52a8\u5b50\uff09\u5f00\u59cb\uff0c\u4ee515\u4e3a\u6b65\u957f\u8bbe\u8ba1\u7f29\u77ed\u7248\u672c\uff0c\u5373+15\u30010\u548c\u221215\u3002\u5bf9\u4e8eeyfp\u7684RBS\uff0c\u9009\u62e9\u4e86\u4e09\u79cd\u53d8\u4f53\uff1aAAAAGGAGAT\uff08\u6700\u5f3a\uff09\u3001AGAAGGAGAT\uff08\u5929\u7136\uff0c\u5f3a\u5ea6\u8f83\u4f4e\uff09\u548cATCCGACCAT\uff08\u6700\u5f31\uff09\u3002\u56db\u79cd\u542f\u52a8\u5b50\u957f\u5ea6\u548ceyfp\u4fa7\u4e09\u79cdRBS\u7684\u7ec4\u5408\u4ea7\u751f\u4e864\u00d73=12\u79cd\u4e0d\u540c\u7684\u5f15\u7269\u3002\u8fd9\u79cd\u8bbe\u8ba1\u7b56\u7565\u603b\u5171\u5305\u62ec9\u00d712=108\u79cd\u4e0d\u540c\u7684Lrp\u751f\u7269\u4f20\u611f\u5668\u53d8\u4f53\u3002
\u6bcf\u79cd\u53d8\u4f53\u90fd\u53ef\u4ee5\u901a\u8fc7\u5355\u4e2a PCR \u53cd\u5e94\u6784\u5efa\uff0c\u5e76\u4e14\u6240\u6709 PCR \u53cd\u5e94\u90fd\u53ef\u4ee5\u4f7f\u7528\u76f8\u540c\u7684 PCR \u6a21\u677f\uff0c\u5373\u8d28\u7c92 pJC1- lrp - brnF\u2032 - eyfp\u3002\u4e3a\u4e86\u7ec4\u88c5\u751f\u7269\u4f20\u611f\u5668\u8d28\u7c92\uff0c\u6bcf\u4e2a PCR \u53cd\u5e94\u4e4b\u540e\u90fd\u4f1a\u8fdb\u884c\u53cc\u7247\u6bb5 Gibson \u7ec4\u88c5\uff0c\u5176\u4e2d PCR \u4ea7\u7269\u88ab\u7ec4\u88c5\u5230 pEC-Tmob18- lrp - eyfp\u4e3b\u94fe\u4e2d\uff1b\u6240\u6709 PCR \u4ea7\u7269\u90fd\u7ecf\u8fc7\u8bbe\u8ba1\uff0c\u56e0\u6b64\u5b83\u4eec\u53ef\u4ee5\u7ec4\u88c5\u5230\u76f8\u540c\u7684\u4e3b\u94fe\u4e2d\u3002<\/p>\n\n\n\n

\u5927\u80a0\u6746\u83cc\u7684DNA\u7ec4\u88c5\u4e0e\u8f6c\u5316
\u5728\u6784\u5efa Lrp \u751f\u7269\u4f20\u611f\u5668\u53d8\u4f53\u4e4b\u524d\uff0c\u901a\u8fc7\u5c06lrp\u548ceyfp\u63d2\u5165\u53ef\u79fb\u52a8\u7684 pEC-Tmob18 \u9aa8\u67b6\u6765\u6784\u5efa\u9aa8\u67b6\u8d28\u7c92\u3002\u5728\u4e24\u4e2a\u57fa\u56e0\u4e4b\u95f4\u63d2\u5165\u4e00\u4e2a\u5305\u542b\u4e24\u4e2a NcoI \u9650\u5236\u4f4d\u70b9\u7684\u77ed\u95f4\u9694\u533a\u3002\u6b64\u5e8f\u5217\u5141\u8bb8\u8d28\u7c92\u7ebf\u6027\u5316\uff0c\u5e76\u5728\u4e0b\u4e00\u6b65\u4e2d\u63d2\u5165\u5305\u542b\u4e0d\u540clrp\u8d77\u59cb\u5bc6\u7801\u5b50\u3001lrp RBS\u3001brnF\u542f\u52a8\u5b50\u957f\u5ea6\u548ceyfp RBS \u7684\u4e0d\u540c\u7248\u672c\u7684\u5e8f\u5217\u53d8\u4f53\u3002
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