{"id":3447,"date":"2024-03-24T06:48:21","date_gmt":"2024-03-23T22:48:21","guid":{"rendered":"http:\/\/www.ccwifi.cc\/blogs\/?p=3447"},"modified":"2024-03-24T06:48:21","modified_gmt":"2024-03-23T22:48:21","slug":"%e7%97%85%e6%af%92%e5%8c%85%e8%a3%85%e6%8a%80%e6%9c%af%e8%85%ba%e7%9b%b8%e5%85%b3%e7%97%85%e6%af%92aav%e5%8c%85%e8%a3%857643","status":"publish","type":"post","link":"https:\/\/www.ccwifi.cc\/blogs\/2024\/03\/24\/%e7%97%85%e6%af%92%e5%8c%85%e8%a3%85%e6%8a%80%e6%9c%af%e8%85%ba%e7%9b%b8%e5%85%b3%e7%97%85%e6%af%92aav%e5%8c%85%e8%a3%857643\/","title":{"rendered":"\u75c5\u6bd2\u5305\u88c5\u6280\u672f\u2014\u2014\u817a\u76f8\u5173\u75c5\u6bd2(AAV)\u5305\u88c5\uff08\u4e00\uff09"},"content":{"rendered":"
\n

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\"cap\u5305\u88c5\"<\/p>\n

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    \n
  1. \u7b80\u4ecb
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    \"cap\u5305\u88c5\"<\/p>\n

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      \n
    1. \u6750\u6599\u548c\u65b9\u6cd5<\/li>\n<\/ol>\n

      \"cap\u5305\u88c5\"<\/p>\n

      2.1. \u8d28\u7c92\u3001\u7ec6\u80de\u548c\u75c5\u6bd2
      \n\u6211\u4eec\u4f7f\u7528pTR-UF5\u8d28\u7c92\u6765\u4f18\u5316\u6240\u6709\u5305\u88c5\u5b9e\u9a8c\uff0c\u8be5\u8d28\u7c92\u8868\u8fbe\u4eba\u7eff\u8272\u8367\u5149\u86cb\u767d\u3002pAVbgal\u8d28\u7c92\u542b\u6709AAV TR\uff0c\u4f4d\u4e8e\u5de8\u7ec6\u80de\u75c5\u6bd2\uff08CMV\uff09\u65e9\u671f\u8f6c\u5f55\u542f\u52a8\u5b50\u548cE.coli b\u534a\u4e73\u7cd6\u82f7\u9176\u57fa\u56e0\u7684\u4e24\u4fa7\u3002\u4f5c\u4e3a\u6bd4\u8f83\uff0c\u6211\u4eec\u4f7f\u7528pAAV\/Ad\u8d28\u7c92\u4f5c\u4e3a\u5305\u88c5\u8d28\u7c92\uff0c\u8be5\u8d28\u7c92\u542b\u6709AAV rep\u548ccap\u57fa\u56e0\u4ee5\u53caAd\u672b\u7aef\u91cd\u590d\u5143\u4ef6\u3002<\/p>\n

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      Fig.1. rAAV\u5305\u88c5\u6240\u7528\u7684\u8d28\u7c92\uff08A\uff09pTR-UF5\u542b\u6709CMV\u542f\u52a8\u5b50\uff08\u7bad\u5934\uff09\u9a71\u52a8\u7684\u4ebagfp\u57fa\u56e0\u548cHSV-tk\u542f\u52a8\u5b50\uff08\u7bad\u5934\uff09\u9a71\u52a8\u7684neoR\u57fa\u56e0\u3002\u8d28\u7c92\u4e24\u4fa7\u6709AAV TR\u5143\u4ef6\uff08\u586b\u5145\u7684\u65b9\u5757\uff09\u3002pAVbgal\u542b\u6709E.coli b-\u534a\u4e73\u7cd6\u82f7\u9176\u57fa\u56e0\uff0c\u53d7CMV\u542f\u52a8\u5b50\uff08\u7bad\u5934\uff09\u63a7\u5236\u3002\u8f6c\u5f55\u76d2\u4e24\u4fa7\u662fAAV TR\u5143\u4ef6\uff08\u586b\u5145\u7684\u65b9\u5757\uff09\u3002pAAV\/Ad\u542b\u6709AAV rep\u548ccap\u57fa\u56e0\uff0c\u4e24\u4fa7\u662fAd\u672b\u7aef\u91cd\u590d\u5143\u4ef6\uff08\u7a7a\u65b9\u5757\uff09\uff08B\uff09pSH3\u548cpSH5\u542b\u6709Ad E4\u3001E2a\u548cVA RNA\u57fa\u56e0\u3002\u4e24\u4e2a\u8d28\u7c92\u4e5f\u542b\u6709AAV rep\u548ccap\u57fa\u56e0\u3002\u56fe\u4e0b\u7684\u6570\u5b57\u6307\u7684\u662fAd\u548cAAV\u57fa\u56e0\u7ec4\u4e2d\u7684\u6838\u82f7\u9178\u6570\u76ee\u3002pSH3\u548cpSH5\u7684\u533a\u522b\u662f\u5728AAV DNA\u5e8f\u5217\u4e2d\u7684\u65b9\u5411\u3002\u4e3a\u4e86\u6e05\u695a\u8d77\u89c1\uff0c\u8d28\u7c92\u56fe\u8c31\u7684\u5176\u4f59\u90e8\u5206\u672a\u5217\u51fa\u3002<\/p>\n

      2.2. rAAV\u8f7d\u4f53\u4ea7\u751f\u548c\u6ef4\u5b9a
      \n\u5bf9\u4e8e\u5c0f\u89c4\u6a21\u7684\u5305\u88c5\u5b9e\u9a8c\uff0c\u5c068\u00d710^5\u4e2a293\u7ec6\u80de\u79cd\u690d\u572835mm\u76846\u5b54\u57f9\u517b\u677f\u4e2d\u300224\u5c0f\u65f6\u540e\uff0c\u4f7f\u7528lipofectamin\u8fdb\u884c\u8f6c\u67d3\uff0c\u8f6c\u67d3\u57f9\u517b\u7269\u7684\u8f7d\u4f53\u548c\u8f85\u52a9\u8d28\u7c92\u6bd4\u4f8b\u5982\u88681\u6240\u793a\u3002\u6839\u636e\u8f7d\u4f53\u548c\u8f85\u52a9\u8d28\u7c92\u7684\u5927\u5c0f\uff0c1mg:3mg\u7684\u6bd4\u4f8b\u76f8\u5f53\u4e8epTR-UF5\u548cpSH3\/5\u7684\u6469\u5c14\u6bd4\u4e3a1:1\u3002pAAV\/Ad\u8d28\u7c92\u4e0eAd\u5171\u611f\u67d3\uff0c\u4f7f\u75283\u62165mg\u7684pAAV\/Ad\u4e0e1mg\u7684pTR-UF5\u5171\u611f\u67d3\u3002\u6dfb\u52a0DNA\u5230\u7ec6\u80de\u4e4b\u524d\uff0c\u5c06\u75c5\u6bd2\u5438\u9644\u5230\u65e0\u8840\u6e05\u57f9\u517b\u57fa\u4e2d\u7684\u7ec6\u80de\u5355\u5c42\u4e0a1\u5c0f\u65f6\u3002\u8f6c\u67d3\u540e72\u5c0f\u65f6\uff0c\u5c06\u7ec6\u80de\u522e\u4e0b\u5e76\u6536\u96c6\u52302ml\u7684\u57f9\u517b\u57fa\u4e2d\u3002\u968f\u540e\u75281ml\u7684\u65e0\u83ccPBS\u6d17\u6da4\u7ec6\u80de\u3002\u5c06PBS\u52a0\u5165\u7ec6\u80de\u60ac\u6d6e\u6db2\u4e2d\uff0c\u7ecf\u8fc75\u6b21\u51bb\u878d\u8d85\u58f0\u7834\u788e\uff083,36mm 45s\uff09\u3002\u79bb\u5fc3\u63d0\u53d6\u726910\u5206\u949f\u4ee5\u53bb\u9664\u7ec6\u80de\u788e\u7247\uff0c\u4e0a\u6e05\u6db2\u4e2d\u542b\u6709\u91cd\u7ec4\u8f7d\u4f53vAVgfp\uff0c\u5c06\u7528\u4e8e\u968f\u540e\u7684\u8f6c\u5bfc\u3002\u5728\u8f6c\u5bfc\u4e4b\u524d\uff0c\u5bf9\u8fd9\u4e9b\u8f6c\u67d3\u7684Ad\u8fdb\u884c56\u00b0C\u70ed\u5904\u740630\u5206\u949f\u3002\u5bf9\u4e8e\u5927\u89c4\u6a21\u7684\u8f7d\u4f53\u5305\u88c5\uff0c\u5c065\u00d710^6\u4e2a293\u7ec6\u80de\u79cd\u690d\u572820\u00d71 125px\u7684\u57f9\u517b\u677f\u4e2d\uff0c\u5e76\u4f7f\u7528\u9499\u78f7\u9178\u76d0\u6c89\u6dc0\u6cd5\u8fdb\u884c\u8f6c\u67d3\u3002<\/p>\n

      72\u5c0f\u65f6\u540e\uff0c\u6536\u96c6\u57f9\u517b\u7269\uff0c\u4f4e\u901f\u79bb\u5fc3\u6c89\u6dc0\u7ec6\u80de\uff0c\u51bb\u878d4\u6b21\uff0c\u7136\u540e\u8fdb\u884c\u8d85\u58f0\u7834\u788e\u3002\u5c06CsCl2\u52a0\u5165\u88c2\u89e3\u7269\u4e2d\uff0c\u6700\u7ec8\u6d53\u5ea6\u4e3a1.41g\/ml\uff0c\u4f7f\u7528\u6298\u5c04\u6307\u6570\u786e\u5b9a\u3002\u5c06\u60ac\u6d6e\u6db2\u5728SW41\u8f6c\u5934\u4e0a\u4ee540,000rpm\u79bb\u5fc348\u5c0f\u65f6\u3002\u4ece\u68af\u5ea6\u4e2d\u53bb\u9664\u542b\u6709\u53ef\u89c1\u8f7d\u4f53\u7684\u6761\u5e26\uff0c\u5176\u5bc6\u5ea6\u53ef\u901a\u8fc7\u6d4b\u91cf\u6298\u5c04\u6307\u6570\u6765\u786e\u5b9a\u3002\u91cd\u590dCsCl2\u79bb\u5fc3\u6b65\u9aa4\uff0c\u83b7\u5f97\u7684\u8f7d\u4f53\u7ecf\u8fc7\u900f\u6790\uff0c\u66f4\u63624\u6b21PBS\uff0c\u4fdd\u5b58\u5728-80\u00b0C\u3002\u4f7f\u7528\u4ee5\u4e0b\u65b9\u6cd5\u5bf9\u4ea7\u751f\u7684\u8f7d\u4f53vAVgal\u8fdb\u884c\u6ef4\u5b9a\u3002\u4e3a\u4e86\u786e\u5b9arAAV\u7684\u6ef4\u5ea6\uff0c\u5c064\u00d710^4\u4e2aHela\u7ec6\u80de\u79cd\u690d\u572824\u5b54\u57f9\u517b\u677f\u4e2d\uff0c\u611f\u67d3m.o.i\u4e3a10\u7684Ad\uff0c\u4f7f\u7528\u4e00\u7cfb\u5217\u7a00\u91ca\u7684\u8f6c\u67d3\u7ec6\u80de\u88c2\u89e3\u6db2\u8fdb\u884c\u8f6c\u5bfc\u30022-3\u5929\u540e\uff0c\u5728\u89c2\u5bdf\u5230\u5e7f\u6cdb\u7684\u7ec6\u80de\u75c5\u7406\u6548\u5e94\u4e4b\u524d\uff0c\u4f7f\u7528\u5e26\u6709EPI-\u8367\u5149\u8bbe\u5907\u7684DIAPHOT-Nikon\u5012\u7f6e\u663e\u5fae\u955c\u572820\u500d\u653e\u5927\u4e0b\u89c2\u5bdf\u3002\u8ba1\u6570\u4ea7\u751f\u826f\u597d\u5206\u79bb\u7684\u8367\u5149\u7ec6\u80de\u7684\u7a00\u91ca\u500d\u6570\uff0c\u6bcf\u4e2a\u9633\u6027\u7ec6\u80de\u4ee3\u8868\u4e00\u4e2a\u8f7d\u4f53\u8f6c\u5bfc\u5355\u4f4d\uff08T.U.\uff09\u3002\u4e3a\u4e86\u786e\u5b9arAAVgal\u8f7d\u4f53\u7684\u6ef4\u5ea6\uff0c\u4f7f\u7528\u7eaf\u5316\u8f7d\u4f53\u7684\u7a00\u91ca\u6db2\u611f\u67d3\u7ec6\u80de\u3002\u7ec6\u80de\u7528PBS\u6eb6\u89e3\u768495%\u7532\u9187\u56fa\u5b9a\uff0c\u7528X-gal\u8fdb\u884c\u67d3\u8272\u3002\u6bcf\u4e2a\u84dd\u8272\u67d3\u8272\u7684\u7ec6\u80de\u4ee3\u8868\u4e00\u4e2a\u8f7d\u4f53\u8f6c\u5bfc\u5355\u4f4d\uff08T.U.\uff09\u3002\u4f7f\u7528\u70b9\u5370\u8ff9\u6742\u4ea4\u5206\u6790\u786e\u5b9a\u8f7d\u4f53\u57fa\u56e0\u7ec4\u7684\u62f7\u8d1d\u6570\uff0c\u5982\u524d\u8ff0\u65b9\u6cd5\u6d4b\u5b9aA260\u3002 <\/p>\n

      \u75c5\u6bd2\u5305\u88c5
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