רÀûÃû³Æ£ºÓÃÓÚí¦³æ²¡Õï¶ÏµÄÖØ×鿹Լ°Æä¿ìËÙÕï¶ÏµÄÊÔÖ½µÄÖÆ×÷·½·¨
¼¼ÊõÁìÓò£º
±¾·¢Ã÷Éæ¼°¼²²¡Õï¶ÏµÄ¿¹Ô£¬¾ßÌå˵ÊÇÒ»ÖÖÓÃÓÚí¦³æ²¡Õï¶ÏµÄÖØ×鿹Լ°Æä¿ìËÙÕï¶ÏµÄÊÔÖ½¡£
í¦³æ²¡µÄÁÙ´²Ö¢×´ÖУ¬³æÒ§À£ÑñÊÇÆäÌØÒìÐÔµÄÌåÕ÷£¬µ«ÓÉÓÚ³æÒ§À£Ññ´ó¶¼²¿Î»Òþ±Î£¬ÊýÁ¿ÉÙ£¬¶ÔÆä¼ì³öÂʵı¨µÀ¸÷µØ²îÒì½Ï´ó£¬Òò´Ë±ØÐ븨ÒÔʵÑéÊÒ¼ì²éÒԱ㼰ʱȷÕï¡£í¦³æ²¡¶«·½ÌåÊÇÑϸñµÄרÐÔϸ°ûÄÚ¼ÄÉúµÄ΢ÉúÎ·±Ö³Âý£¬´¿»¯·±Ëö£¬Òò¶øÄÑÒÔ»ñµÃ´óÁ¿´¿»¯µÄí¦³æ²¡¶«·½Ì忹ԣ¬ÏÞÖÆÁ˼ìÑé·½·¨µÄ·¢Õ¹¡£´«Í³µÄÕï¶Ï·½·¨£¬ÈçÍâì³ÊÏÊÔÑé(Weil-Felix test)¡¢ÃâÒß¹ýÑõ»¯ÎïøÊÔÑé(IIP)¡¢¼ä½ÓÃâÒßÓ«¹â¿¹Ìå¼ì²â(IFAT)µÈ£¬¾ùÒÔѪÇåѧ·´Ó¦Îª»ù´¡£¬µ«Òò¸÷×ԵľÖÏÞÐÔ¶øÏÞÖÆÁËËüÃǵÄʹÓá£Íâì³ÊÏÊÔÑéËäÈ»²Ù×÷¼òµ¥£¬µ«¶Ôí¦³æ²¡Õï¶Ïȱ·¦Ãô¸ÐÐÔºÍÌØÒìÐÔ£»IFAT¾ßÓкܺõÄÃô¸ÐÐÔºÍÌØÒìÐÔ£¬µ«ÐèÒªÓ«¹âÏÔ΢¾µºÍѵÁ·ÓÐËØµÄרҵÈËÔ±£¬ÄÑÒÔÔÚÅ©´å¡¢»ù²ãÒ½Ôº¼°ÏÖ³¡Ê¹Óã»IIPËäÈ»ÒÔÆÕͨ¹âѧÏÔ΢¾µ´úÌæÁËÓ«¹âÏÔ΢¾µ»òÉõÖÁ²»ÐèÒªÏÔ΢¾µ£¬µ«ÈÔÈ»ÐèÒªÅàÑøºÍ´¿»¯í¦³æ²¡¶«·½Ì壬ÐèÒª°º¹óµÄ×éÖ¯ÅàÑøÉ豸¡£Òò´Ë£¬¼±Ð轨Á¢Ò»ÖÖ¼ò±ã¡¢¿ìËÙ²¢Äܿ˷þÉÏÊö¾ÖÏÞµÄѪÇåѧÕï¶Ï·½·¨£¬ÒÔÂú×ãí¦³æ²¡¶«·½ÌåÁÙ´²¼ì²âµÄÐèÒª¡£
í¦³æ²¡¶«·½Ìå56kD¿¹Ô(Scrub typhus antigen£¬Sta56)×÷Îªí¦³æ²¡¶«·½Ìå±íÃæº¬Á¿×î·á¸»µÄÍâĤµ°°×£¬¿ÉÕ¼¾úÌå×ܵ°°×µÄ10¡«15£¥£»ÃâÒßÔÐÔÇ¿£¬×î³£±»ËÞÖ÷ÃâÒßϵͳËùʶ±ð£»Sta56¿ÉÓëÖêÌØÒìÐÔµ¥¿¹ºÍÈºÌØÒìÐÔµ¥¿¹·´Ó¦£¬±íÃ÷Sta56´æÔÚÖêÌØÒìÐÔ±íλºÍÈºÌØÒìÐÔ±í룬Òò¶øSta56ÊÇí¦³æ²¡Õï¶Ï¿¹ÔµÄÖ÷ÒªºòÑ¡ÕßÖ®Ò»¡£
½ºÌå½ðÊÇÒ»ÖÖ´¦Óڰ뻹Ô״̬µÄÐü¸¡¿ÅÁ££¬ÄÜѸËÙÎü¸½µ°°×Öʵȴó·Ö×Ó¶ø²»Ó°ÏìÆä»îÐÔ£¬½ºÌå½ð¾ßÓÐÒ×·Ö±æµÄºìÖÁ×ϺìÉ«£¬ÔÚÓë´ó·Ö×Ó½áºÏºó£¬Ò×ÓÚÓÃÈâÑÛʶ±ð£¬²»Ðè¶þ¼¶·Å´ó¼´¿ÉÖ±½Ó¹Û²ì½á¹û£¬½ºÌå½ðÕï¶Ï²úÆ·Òò¶ø¿ÉÖ±½Ó½øÈëûÓи´ÔÓ¼ì²âÉ豸µÄÕïËùÉõÖÁ¼ÒÍ¥Àï¡£½üÄêÀ´£¬ÒÔÔçÔмì²âÊÔֽΪ´ú±íµÄ½ºÌå½ðÃâÒßÕï¶ÏÊÔ¼ÁÒѽøÈëǧ¼ÒÍò»§£¬¸øÈËÃǵÄÉú»î´øÀ´ºÜ´óµÄ·½±ã¡£
´Ó°üÀ¨ÖйúרÀûµÈÓйØ×ÊÁϼìË÷±íÃ÷£¬Ä¿Ç°ÉÐδÓÐÀûÓûùÒò¹¤³Ì·½·¨»ñµÃµÄÖØ×鿹ÔÓÃÓÚí¦³æ²¡µÄÕï¶Ï¼°í¦³æ²¡¿ìËÙÕï¶ÏÊÔÖ½µÄÏà¹Ø±¨µÀ¡£
±¾·¢Ã÷½â¾öÆä¼¼ÊõÎÊÌâËù²ÉÓõļ¼Êõ·½°¸ÊÇÒ».±¾·¢Ã÷Éæ¼°µÄÈÜÒºÅ䷽Ϊ(1)ÖØ×鿹ÔÈܽâÒºSDS 0.1£¥¸Ê°±Ëá 250mmol/LTris.Cl 25mmol/L(2)½ºÌå½ðÈÜÒº3¡«5/ÍòµÄËÄÂȽðËᣬ¼Ó1¡«2£¥ÄûÃÊËáÈýÄÆÖÆ±¸³É½ºÌå½ð¿ÅÁ££»(3)¶þ¿¹¿¹ÈËIgG¡¢¿¹ÈËIgM¶à¿¹£¬¹º×ÔSigma¹«Ë¾£»(4)°ü±»»º³åÒº1¡«3£¥ÕáÌÇin PBS£»(5)»ù´¡»º³åÒº¹º×ÔÏÃÃŲ¨ÉúÉúÎï¼¼ÊõÓÐÏÞ¹«Ë¾£»(6)·´Ó¦»º³åÒº0.01¡«0.05£¥NaN3in PBS£»(7)PBSÔÚ800mlÕôÁóË®ÖÐÈܽâ8g NaCl¡¢0.2g KCl¡¢1.44g Na2HPO4ºÍ0.24gKH2PO4£¬ÓÃÑÎËáµ÷½ÚÈÜÒºµÄpHÖµÖÁ7.2¡«7.6£¬¼ÓË®¶¨ÈÝÖÁ1L¡£
¶þ.±¾·¢Ã÷ÖØ×鿹ԵŤÒÕÁ÷³Ì(1)´Óí¦³æ²¡¶«·½ÌåKarpÖê»ùÒò×éÖÐÀ©Ôö³östa56»ùÒòµÄORF»òÆäÖÐµÄ´óÆ¬¶Î£¬ÓÃTA¿Ë¡¼¼Êõ½«´Ë´óƬ¶Î¿Ë¡£»(2)ÒÔ¸ÃÖØ×éÖÊÁ£ÎªÄ£°å£¬À©Ôö³ö½Ø¶ÌµÄsta56Ƭ¶Î£¬¶¨Ïò²åÈëpET30aÔØÌ壬ת»¯´ó³¦¸Ë¾úBL21(DE3)£¬IPTGÓÕµ¼±í´ï£»Western blot֤ʵ¸ÃÖØ×éµ°°×Äܱ»í¦³æ²¡»¼ÕßÑôÐÔѪÇåËùʶ±ð£»(3)²ÉÓõçÏ´ÍÑ·½·¨´¿»¯ÖØ×éµ°°×¶Ô´óÁ¿ÓÕµ¼Ê±ÊÕ¼¯µÄ¾úÌå½øÐг¬Éù·ÛËéºó£¬½«³¬ÉùÉÏÇå½øÐÐÖÆ±¸µçÓ¾£¬È·¶¨Ä¿±êÌõ´øÔÚÄý½ºÖеÄλÖã¬ÇÐϺ¬Ä¿±êµ°°×µÄÄý½º½ºÖÃÓÚ͸Îö´üÖнøÐеçÏ´ÍÑ£»´¿»¯ÑùÆ·½øÐÐSDS-PAGE£¬È·¶¨´¿¶È²¢½øÐе°°×¶¨Á¿¡£
Èý.±¾·¢Ã÷µÄÊÔÖ½ÖÆ×÷¹¤ÒÕ(1)ÒÔ½ºÌå½ð±ê¼ÇÖØ×鿹ԺÍÊóIgG£»(2)Á½ÖÖ½ð±ê¿¹ÔÈÜÒº»ìºÏºó³ä·Ö½þÈó²£Á§ÏËάֽ£¬Öö³¸É»ú¶³¸É£»(3)Ñò¿¹È˦ÌÁ´1.0¡«5.0mg/ml¡¢Ñò¿¹ÈËIgG 1.0¡«5.0mg/mlÓëÑò¿¹ÊóIgGµ¥¿¹1.0¡«5.0mg/ml°ü±»NCĤ£¬×ÔÈ»ÁÀ¸É£»(4)½«Îüˮֽ¡¢NCĤºÍÎü¸½Á˽ð±ê¿¹ÔµÄ²£Á§ÏËά×é×°³É¼ì²âIgM¡¢IgGºÍ×ÜIgÊÔÖ½Ìõ¡£
Óñ¾·¢Ã÷ÖÆ×÷µÄÖØ×鿹Լ°ÀûÓÃÖØ×鿹ÔÖÆ×÷³ÉµÄÊÔÖ½Ìõ¾Í¿ÉÒÔÓÃÓÚí¦³æ²¡µÄÁÙ´²Õï¶ÏÁË¡£
±¾·¢Ã÷µÄÓÐÒæÐ§¹ûÊDZ¾·¢Ã÷ÀûÓûùÒò¹¤³Ì¼¼Êõ£¬¿ËÂ¡í¦³æ²¡¶«·½ÌåµÄÃâÒßÓÅÊÆ¿¹ÔµÄ½á¹¹»ùÒò£¬²¢Ôڴ󳦸˾úÄÚ±í´ï£¬ÒÔ·½±ãµØ»ñµÃ´óÁ¿Á®¼ÛµÄÖØ×鿹ԣ¬´¿»¯ºó¼´¿ÉÓÃÓÚÃâÒßѧÕï¶Ï¡£ÓÉ´Ë·½·¨µÃµ½µÄÖØ×鿹Լ°¿ìËÙÕï¶ÏÊÔÖ½¿Ë·þÁËÏÖÓÐí¦³æ²¡Õï¶Ï·½·¨µÄ¾ÖÏÞÐÔ£¬¿ÉÒÔ¿ìËÙ¡¢¼ò±ãµØÕï¶Ïí¦³æ²¡£¬ÎÞÐèÌØÊâÒÇÆ÷£¬ÎÞÐèѵÁ·ÓÐËØµÄרҵÈËÔ±£¬¿ÉÒÔÂú×ã»ù²ãÒ½Ôº¡¢Å©´åÎÀÉúÔºµÈÁÙ´²¼ì²âµÄÐèÒª£¬¶øÇÒ´ó´óµÄ½µµÍÁ˳ɱ¾£¬·½±ãÁË»¼Õß¡£Í¬Ê±±¾·¢Ã÷Õï¶Ï¾«È·¶È¸ß£¬´Ó¶øÊ¹»¼ÕßÄܹ»µÃµ½¼°Ê±µÄÖÎÁÆ£¬´ïµ½ÁË·ÀÖεÄÄ¿µÄ¡£
ʵʩÀý2¿ìËÙÕï¶ÏÊÔÖ½µÄÖÆ×÷(1)È¡3/ÍòËÄÂȽðËá1000ml£¬¼ÓÈÈÖÁ·ÐÌÚ£¬¼ÓÈë1£¥ÄûÃÊËáÈýÄÆÈÜÒº25ml£¬¼ÌÐøÖó·Ð5·ÖÖÓ£¬´ý½ºÌå½ðÈÜÒºÑÕÉ«ÓÉÓÐÀ¶¾×ϱäºìºó£¬×ÔÈ»ÀäÈ´ºó±¸Óã»(2)È¡½ºÌå½ðÈÜÒº1ml£¬¼ÓÈë0.2M K2CO310¦Ìl¡¢50¦Ìg´¿»¯¿¹Ô£¬½ÁÔȺó¾²ÖÃ1h£»(3)¼ÓÈë20£¥Å£ÑªÇå°×µ°°×25¦Ìl¡¢20£¥ PEG 20,000 15¦Ìl£¬8,000r/min£¬ÀëÐÄ8min£¬ÆúÉÏÇ壻(4)³Áµí¼ÓÈë1ml»ù´¡»º³åÒºÐü¸¡£¬¼´Îª½ð±ê¿¹ÔÈÜÒº£»(5)ͬ·¨±ê¼ÇÊóIgG£»(6)Á½ÖÖ½ð±ê¿¹ÔÈÜÒº»ìºÏºó³ä·Ö½þÈó²£Á§ÏËάֽ£¬Öö³¸É»ú¶³¸É£»(7)Ñò¿¹ÊóIgGµ¥¿¹3.0mg/ml¡¢Ñò¿¹ÈËIgG 3.0mg/ml¡¢Ñò¿¹È˦ÌÁ´1.0mg/ml·Ö±ðÔÚNCĤµÄÉÏ¡¢ÖС¢ÏÂλÖðü±»NCĤ£¬×ÔÈ»ÁÀ¸É£»»òÑò¿¹ÊóIgGµ¥¿¹3.0mg/ml¡¢ÖØ×鿹ÔÈÜÒº2mg/mlÔÚNCĤµÄÉÏ¡¢ÏÂÈý·ÖÖ®Ò»´¦°ü±»NCĤ£¬×ÔÈ»ÁÀ¸É£»(8)ÔÚÔ¼10cm¿íµÄµ×°åÉÏ£¬½«Îüˮֽ¡¢NCĤ¡¢Îü¸½Á˽ð±ê¿¹ÔµÄ²£Á§ÏËάºÍδÎü¸½¿¹ÔµÄ²£Á§ÏËά×é×°³É¼ì²âIgMÓëIgGºÍ¼ì²â×ÜIgµÄÊÔÖ½Ìõ¡£
ÆÀ¼ÛÔÚÊÔÖ½ÌõµÄ²£Á§ÏËάÉϼÓ3¦Ìl(¼ì²âIgMºÍIgG)»ò5¦Ìl´ý¼ìѪÇå(¼ì²â×ÜIg)£¬ÔٵμÓ1¡«2µÎ·´Ó¦Òº¡£¹Û²ì½ð±ê¿¹ÔÊͷźóNCĤÉϼì²âÏߺͶÔÕÕÏߵķ´Ó¦Çé¿ö£¬¼ì²âÏߺͶÔÕÕÏßͬʱ³öÏÖºìÉ«Ìõ´øÅÐΪÑôÐÔ£¬½ö¶ÔÕÕÏß³öÏÖºìÉ«Ìõ´øÅÐΪÒõÐÔ£¬¶ÔÕÕÏßδ³öÏÖÌõ´øÎªÎÞЧÊÔÑé¡£½ð±êÃâÒß²ãÎö·¨¼ì²âIgM¡¢IgG¡¢×ÜIgµÄÃô¸ÐÐÔ·Ö±ðΪ92.3£¥£¬90.9£¥ºÍ98.6£¥£»ÌØÒìÐÔ·Ö±ðΪ94.7£¥£¬94.7£¥ºÍ93.3£¥¡£
ȨÀûÒªÇó
1.Ò»ÖÖÓÃÓÚí¦³æ²¡Õï¶ÏµÄÖØ×鿹Լ°Æä¿ìËÙÕï¶ÏÊÔÖ½µÄÈÜÒºÖÆ±¸£¬ÆäÌØÕ÷ÊÇÈÜÒºÅ䷽Ϊ(1)ÖØ×鿹ÔÈܽâÒº SDS 0.1£¥¸Ê°±Ëá250mmol/LTris.Cl 25mmol/L(2)½ºÌå½ðÈÜÒº2¡«5/ÍòµÄËÄÂȽðËᣬ¼Ó1¡«2£¥ÄûÃÊËáÈýÄÆ»¹Ô³ÉÖеȴóСµÄ½ºÌå½ð¿ÅÁ££»(3)¶þ¿¹¿¹ÈËIgG¡¢¿¹ÈËIgM¶à¿¹£¬¹º×ÔSigma¹«Ë¾£»(4)°ü±»»º³åÒº1¡«3£¥ÕáÌÇin PBS£»(5)»ù´¡»º³åÒº¹º×ÔÏÃÃŲ¨ÉúÉúÎï¼¼ÊõÓÐÏÞ¹«Ë¾£»(6)·´Ó¦»º³åÒº0.01¡«0.05£¥NaN3in PBS£»(7)PBSÔÚ800mlÕôÁóË®ÖÐÈܽâ8g NaCl¡¢0.2g KCl¡¢1.44g Na2HPO4ºÍ0.24g KH2PO4£¬ÓÃÑÎËáµ÷½ÚÈÜÒºµÄpHÖµÖÁ7.2¡«7.6£¬¼ÓË®¶¨ÈÝÖÁ1L¡£
2.Ò»ÖÖÓÃÓÚí¦³æ²¡Õï¶ÏµÄÖØ×鿹ԵÄÖÆ±¸¹¤ÒÕÁ÷³Ì£¬ÆäÌØÕ÷ÊÇÖØ×鿹ԵŤÒÕÁ÷³ÌΪ(1)´Óí¦³æ²¡¶«·½ÌåKarpÖê»ùÒò×éÖÐÀ©Ôö³östa56»ùÒòµÄORF»òÆäÖÐµÄ´óÆ¬¶Î£¬ÓÃTA¿Ë¡¼¼Êõ½«´Ë´óƬ¶Î¿Ë¡£»(2)ÒÔ¸ÃÖØ×éÖÊÁ£ÎªÄ£°å£¬À©Ôö³ö½Ø¶ÌµÄsta56Ƭ¶Î£¬¶¨Ïò²åÈëpET30aÔØÌ壬ת»¯´ó³¦¸Ë¾úBL21(DE3)£¬IPTGÓÕµ¼±í´ï£»Western blot֤ʵ¸ÃÖØ×éµ°°×Äܱ»í¦³æ²¡»¼ÕßÑôÐÔѪÇåËùʶ±ð£»(3)²ÉÓõçÏ´ÍÑ·½·¨´¿»¯ÖØ×éµ°°×¶Ô´óÁ¿ÓÕµ¼Ê±ÊÕ¼¯µÄ¾úÌå½øÐг¬Éù·ÛËéºó£¬½«³¬ÉùÉÏÇå½øÐÐÖÆ±¸µçÓ¾£¬È·¶¨Ä¿±êÌõ´øÔÚÄý½ºÖеÄλÖã¬ÇÐϺ¬Ä¿±êµ°°×µÄÄý½º½ºÖÃÓÚ͸Îö´üÖнøÐеçÏ´ÍÑ£»´¿»¯ÑùÆ·½øÐÐSDS-PAGE£¬È·¶¨´¿¶È²¢½øÐе°°×¶¨Á¿¡£
3.Ò»ÖÖÓÃÓÚí¦³æ²¡Õï¶ÏµÄÖØ×鿹ԵĿìËÙÕï¶ÏÊÔÖ½£¬ÆäÌØÕ÷ÊÇÊÔÖ½ÖÆ×÷¹¤ÒÕΪ(1)ÒÔ½ºÌå½ð±ê¼ÇÖØ×鿹ԺÍÊóIgG£»(2)Á½ÖÖ½ð±ê¿¹ÔÈÜÒº»ìºÏºó³ä·Ö½þÈó²£Á§ÏËάֽ£¬Öö³¸É»ú¶³¸É£»(3)Ñò¿¹È˦ÌÁ´1.0¡«5.0mg/ml¡¢Ñò¿¹ÈËIgG 1.0¡«5.0mg/mlÓëÑò¿¹ÊóIgGµ¥¿¹1.0¡«5.0mg/ml°ü±»NCĤ£¬×ÔÈ»ÁÀ¸É£»(4)½«Îüˮֽ¡¢NCĤ¡¢Îü¸½ºÍδÎü¸½½ð±ê¿¹ÔµÄ²£Á§ÏËά×é×°³É¼ì²âIgM¡¢IgGºÍ×ÜIgÊÔÖ½Ìõ¡£
È«ÎÄÕªÒª
±¾·¢Ã÷¹«¿ªÁËÒ»ÖÖÓÃÓÚí¦³æ²¡Õï¶ÏµÄÖØ×鿹Լ°Æä¿ìËÙÕï¶ÏµÄÊÔÖ½¡£í¦³æ²¡£¬ÓֳƴÔÁÖ°ßÕîÉ˺®£¬ÊÇÓÉí¦³æ²¡¶«·½ÌåÒýÆðµÄÒÔ·¢ÈÈ¡¢Æ¤Õî¡¢³æÒ§À£Ññ(½¹ðè)ºÍdz±íÁܰͽáÖ×´óΪÖ÷ÒªÌØÕ÷µÄ·¢ÈÈÐÔ¼²²¡£¬ÑÏÖØÕß³öÏÖ¸ÎÆ¢Ö״󡢸¹Ë®£¬¾ÈÖβ»¼°Ê±¿Éµ¼ÖÂËÀÍö¡£±¾·¢Ã÷ÀûÓûùÒò¹¤³Ì¼¼Êõ£¬¿ËÂ¡í¦³æ²¡¶«·½ÌåµÄÃâÒßÓÅÊÆ¿¹ÔµÄ½á¹¹»ùÒò£¬²¢Ôڴ󳦸˾úÄÚ±í´ï£¬ÒÔ·½±ãµØ»ñµÃ´óÁ¿Á®¼ÛµÄÖØ×鿹ԣ¬´¿»¯ºó¼´¿ÉÓÃÓÚÃâÒßѧÕï¶Ï¡£ÓÉ´Ë·½·¨µÃµ½µÄÖØ×鿹Լ°¿ìËÙÕï¶ÏÊÔÖ½¿Ë·þÁËÏÖÓÐí¦³æ²¡Õï¶Ï·½·¨µÄ¾ÖÏÞÐÔ£¬¿ÉÒÔ¿ìËÙ¡¢¼ò±ãµØÕï¶Ïí¦³æ²¡£¬ÎÞÐèÌØÊâÒÇÆ÷£¬ÎÞÐèѵÁ·ÓÐËØµÄרҵÈËÔ±£¬¿ÉÒÔÂú×ã»ù²ãÒ½Ôº¡¢Å©´åÎÀÉúÔºµÈÁÙ´²¼ì²âµÄÐèÒª£¬¶øÇÒ´ó´óµÄ½µµÍÁ˳ɱ¾£¬·½±ãÁË»¼Õß¡£
Îĵµ±àºÅG01N33/558GK1479099SQ0314731
¹«¿ªÈÕ2004Äê3ÔÂ3ÈÕ ÉêÇëÈÕÆÚ2003Äê7ÔÂ5ÈÕ ÓÅÏÈȨÈÕ2003Äê7ÔÂ5ÈÕ
·¢Ã÷ÕßÑÏÑÓÉú, µËÑÞÇÙ ÉêÇëÈË:¸£½¨Ê¡ÎÀÉú·ÀÒßÕ¾