ÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎïÃâÒßÖƱ¸¿¹ÒȵºËص¥¿Ë¡¿¹Ìå_ҩѧרҵ±ÏÒµÂÛÎÄ
·¢²¼Ê±¼ä£º
2014-11-09
À´Ô´£º
ÈË´ó¾¼ÃÂÛ̳
¡¾ÕªÒª¡¿ Ä¿µÄ ÖƱ¸¿¹ÒȵºËص¥¿Ë¡¿¹Ì壨mAb£©¡£·½·¨ ÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎïÃâÒßBALB/cСÊóÖƱ¸ÃâÒßƢϸ°û£¬ÔËÓÃÁÜ°Íϸ°ûÔÓ½»Áö¼¼Êõ½«ÆäÓëСÊó¹ÇËèÁöϸ°ûSP2/0Èںϡ£½á¹û ½¨Á¢ÁË7Öê·ÖÃÚ¿¹ÒȵºËØmAbµÄÔÓ½»Áöϸ°ûϵ£¬ÃüÃûΪA¡¢B¡¢C¡¢D¡¢E¡¢FºÍG£»7ÖêmAbµÄIgÑÇÀà·Ö±ðΪIgM¡¢IgM¡¢IgM¡¢IgG2b¡¢IgG1¡¢IgG1ºÍIgG1£»ÔÚ¼ÐÐÄELISAÊÔÑéÖУ¬7ÖêmAb¾ùÄÜÓëÈË¡¢Å£ºÍÖíÒȵºËØ·¢ÉúÌØÒìÐÔÃâÒß·´Ó¦£»ÔÚÃâÒßÓ¡¼£ÊÔÑéÖУ¬7ÖêmAb¾ùÄÜÓëÖØ×éÈËÒȵºËØÔÀàËÆÎï½áºÏ³ÊÏÖÒ»Ìõµ°°×Ó¡¼£´ø¡£½áÂÛ 7ÖêmAb¶ÔÈË¡¢Å£ºÍÖíÒȵºËؾù¾ßÓÐÃâÒß·´Ó¦ÐÔ£¬¿ÉÓÃÓÚÈ˺Ͷ¯ÎïÒȵºËصIJⶨºÍ´¿»¯¡£
¡¾¹Ø¼ü´Ê¡¿ µ¥¿Ë¡¿¹Ì壨mAb£©£»ÒȵºËØ£»ÖØ×éÈËÒȵºËØÔÀàËÆÎï
Development of monoclonal antibodies against insulin by immunization with recombinant human proinsulin analog
¡¾Abstract¡¿ Objective To prepare monoclonal antibody (mAb) against insulinMethods Spleen cells from BALB/c mice immunized with recombinant human proinsulin analog were fused with SP2/0 myeloma cells by using hybridoma techniques.Results Seven hybridomas secreting mAb against insulin were established and designated A, B, C, D, E, F and G. The subclass of the seven mAb was IgM, IgM, IgM, IgG2b, IgG1, IgG1 and IgG1 respectively. The seven mAb could specifically react with insulin derived from human, cattle or pig in sandwich ELISA. The specific reaction of each mAb with recombinant human proinsulin analog was confermed by immunoblotting assay, only one stained protein band appeared on the transferred film.Conclusion The seven mAb are immunological specific to insulin derived from human, cattle or swine, and are available to the detection and purification of insulin.
¡¾Key words¡¿ monoclonal antibody (mAb); insulin; recombinant human proinsulin analog
ÒȵºËØÊǾßÓÐÖØÒªÉúÀí¹¦ÄܵÄÒ»ÖÖ¶àëÄÀ༤ËØ£¬ÔÚÖÎÁÆÌÇÄò²¡·½Ãæ¾ßÓÐÖØÒªÓÃ;¡£ÒȵºËØ¿¹ÌåÊÇÒȵºËزⶨºÍ´¿»¯Ëù±ØÐèµÄÃâÒßѧÊÔ¼Á¡£Îª´Ë½øÐÐÁ˱¾Ñо¿¡£
1 Óë·½·¨
1.1 ¾úÖÖ¡¢ÖÊÁ£ºÍϸ°û ´ó³¦¸Ë¾úJM109¡¢Ð¡Êó¹ÇËèÁöSP2/0ϸ°ûϵ¡¢º¬ÓЦËÊɾúÌåPRPL´®ÁªÆô¶¯×ÓµÄÈËÒȵºËØÔÀàËÆÎBKRA£©»ùÒò±í´ïÖÊÁ£pBV- BKRAÓɱ¾¿ÎÌâ×é¹¹½¨ºÍ±£´æ£Û1£Ý¡£
1.2 ÃâÒßѪÇåµÄÖƱ¸ ÊÕ¼¯´ó³¦¸Ë¾úJM109ÅàÑøÎÀëÐijÁµí¾úÌ壬Óó¬Éù²¨ÆÆË飬¼Ó¸£ÊÏ×ô¼ÁÈ黯ºó£¬¾Æ¤ÏÂ×¢ÉäÃâÒßÍã¬ÖƱ¸JM109ÃâÒßѪÇå¡£
1.3 ÖØ×éÈËÒȵºËØÔÀàËÆÎïµÄÖƱ¸ ÓÃpBV-
BKRAת»¯JM109£Û1¡½¡£È¡µ¥¸ö¾úÂ䣬30¡æÅàÑø14h£¬ÒÔ20%½ÓÖÖÔÚº¬¿¨ÄÇùËØ£¨Kan£©µÄÐÂÏÊLBÅàÑøÒºÖУ¬42¡æÕñµ´ÅàÑø4h£¬¸ßËÙÀëÐijÁµí¾úÌå¡£²ÎÕÕÎÄÏ×£Û2¡½·ÖÀë°üºÌ壬ÓöþÏõ»ù±½»ÇËáÄÆ´¦Àí£Û3¡½ºó×÷ΪBALB/cСÊóÃâÒß¿¹Ô¡£
1.4 ÃâÒßÊó ½«ÖƱ¸µÄÖØ×éÈËÒȵºËØÔÀàËÆÎïÓëµÈÁ¿¸£ÊÏÍêÈ«×ô¼Á»ìºÏÈ黯£¬¾¸¹Ç»×¢ÉäÃâÒß6ÖÜÁäµÄBALB/cСÊó£¬20mg/£¨´Î¡¤Ö»£©£¬ÃâÒß2´Î£¬¼ä¸ô2ÖÜ¡£ÔÚϸ°ûÈÚºÏÇ°3¡«4Ìì, ¼ÓÇ¿ÃâÒß1´Î¡£
1.5 ϸ°ûÈÚºÏ ²ÎÕÕÎÄÏ×£Û4¡½£¬ÓÃÃâÒßµÄBALB/cСÊóÖƱ¸Æ¢Ï¸°û£¬ÓëСÊó¹ÇËèÁöϸ°ûSP2/0Èںϡ£
1.6 ÔÓ½»Áöϸ°ûµÄɸѡÓë¿Ë¡»¯ ²ÉÓüä½ÓÒÖÖÆELISA·½·¨¡£ÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎï°ü±»ELISA·´Ó¦°å£¬10%µÄJM109ÃâÒßѪÇå·â±Õ£¬¼ÓÈëÔÓ½»Áöϸ°ûÅàÑøÉÏÇåÒºÓëÖ®·´Ó¦£¬ÔÙÓëÀ±¸ù¹ýÑõ»¯Îïø£¨HRP£©±ê¼ÇµÄÍÿ¹ÊóIgG·´Ó¦£¬ÏÔÉ«¡£´ÓÁ½´Î¼ì²â¾ùΪÑôÐÔµÄÔÓ½»Áöϸ°û¿×ÖÐÑ¡È¡7¸öÑôÐÔ¿×£¬ÓÃÓÐÏÞÏ¡ÊÍ·¨½øÐпË¡»¯¡£
1.7 µ¥¿Ë¡¿¹ÌåµÄÖƱ¸ ½«µ¥¿Ë¡»¯µÄÑôÐÔÔÓ½»Áöϸ°ûÀ©´óÅàÑø£¬°´5¡Á106/Ö»µÄϸ°ûÁ¿×¢ÉäÓÚ7¡«10ÌìÇ°Ô¤Ïȸ¹Ç»×¢ÉäÎÞ¾úҺ״ʯÀ¯µÄBALB/cСÊó¸¹Ç»ÄÚ£¬ÓÕÉú¸¹Ë®£¬ÖƱ¸mAb¡£
1.8 µ¥¿Ë¡¿¹ÌåµÄЧ¼Û ÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎï°ü±»ELISA·´Ó¦°å£¬ÒÔ³£¹æ¼ä½ÓELISA·Ö±ð²â¶¨¸÷µ¥¿Ë¡»¯µÄÔÓ½»Áöϸ°ûÅàÑøÉÏÇåÒººÍÓÕÉú¸¹Ë®ÖеÄmAbЧ¼Û¡£
1.9 µ¥¿Ë¡¿¹ÌåµÄ¼ø¶¨
1.9.1 ÑÇÀà ½«Å¨Ëõ10±¶ÒÔÉϵÄÔÓ½»Áöϸ°ûÅàÑøÉÏÇåÒº»òÔÓ½»Áöϸ°ûÓÕÉú¸¹Ë®£¬Óÿ¹ÊóIgÑÇÀàѪÇ壨Sigma²úÆ·£©£¬ÒÔÇí֬˫À©É¢ÊÔÑé¼ø¶¨mAbµÄÑÇÀà¡£
1.9.2 ÌØÒìÐÔ
1.9.2.1 ¼ÐÐÄELISA ÓÃ7ÖêmAb×÷Ϊ²¶×½¿¹Ìå·Ö±ð°ü±»ELISA·´Ó¦°å£¬ÒÔÈËÒȵºËØ£¨Sigma²úÆ·£©¡¢Å£ÒȵºËØ£¨Sigma²úÆ·£©¡¢ÖíÒȵºËØ£¨ÄϾ©Éú»¯ÖƳ§²úÆ·£©×÷Ϊ±»²¶×½Îï·Ö±ðÓëÖ®·´Ó¦£¬ÔÙ¼ÓÈëÀ±¸ù¹ýÑõ»¯Îïø£¨HRP£©±ê¼ÇµÄ¿¹ÒȵºËص¥¿Ë¡¿¹Ì壨Sigma²úÆ·£©·´Ó¦ÏÔÉ«£¬7ÖêmAbÄÜ·ñ²¶×½ÒȵºËØ¡£
1.9.2.2 ÃâÒßÓ¡¼£ÊÔÑé ²ÉÓÃÃâÒßÓ¡¼£·¨£Û5¡½·ÖÎö¡£ÖØ×é¾úÓÕµ¼±í´ïºó£¬½«¾úÌå½øÐÐSDS-PAGE£¬µçӾתÒƵ½ÏõËáÏËάËØĤÉÏ£¬ÓÃ7ÖêmAb·Ö±ðÓëתÒÆĤ·´Ó¦ºó£¬ÔÙ¼Ó¼îÐÔÁ×Ëáø£¨AP£©±ê¼ÇµÄɽÑò¿¹ÊóIgGÓëתÒÆĤ·´Ó¦£¬ÏÔÉ«¡£ÊµÑéͬ²½ÉèÁ¢JM109¾úÓëpBV220ת»¯¾ú¶ÔÕÕ¡£
2 ½á¹û
2.1 ÖØ×éÈËÒȵºËØÔÀàËÆÎïµÄÖƱ¸ ÓÃSDS-PAGEºÍÄý½ºÉ¨Ãè·ÖÎö£¬°üºÌåÖÐÖØ×éÈËÒȵºËØÔÀàËÆÎïµÄº¬Á¿´ïµ½80%£¨Í¼1£©¡£ ͼ1 pBV-BKRAµÄÓÕµ¼±í´ïÓëmAb±í´ï£¨ÂÔ£©
2.2 ϸ°ûÈÚºÏÓë¿Ë¡»¯ ÈÚºÏϸ°ûµÄÑôÐÔÂÊΪ2.4%£¨7/288£©¡£´ÓÖÐÑ¡È¡7¸öÑôÐÔ¿×½øÐпË¡»¯£¬¾3´Î¿Ë¡ºó£¬ËùÓÐϸ°ûÉú³¤¿×¾ù³ÊÑôÐÔ·´Ó¦¡£
2.3 µ¥¿Ë¡¿¹ÌåµÄЧ¼ÛºÍÑÇÀà ¼ä½ÓELISA·¨²â¶¨7ÖêmAbϸ°ûÅàÑøÉÏÇåÒºÖеĿ¹ÌåЧ¼ÛΪ102£¬¶øÓÕÉú¸¹Ë®ÖеĿ¹ÌåЧ¼Û´ïµ½105¡«106¡£IgÑÇÀà¼ø¶¨½á¹û¼û±í1¡£±í1 7Ö꿹ÒȵºËØmAbµÄЧ¼ÛºÍIgÑÇÀࣨÂÔ£©
2.4 µ¥¿Ë¡¿¹ÌåµÄÌØÒìÐÔ ÔÚ¼ÐÐÄELISAÊÔÑéÖУ¬7ÖêmAb¾ùÄܲ¶×½µ½ÈË¡¢Å£ºÍÖíÒȵºËØ£»ÔÚÃâÒßÓ¡¼£ÊÔÑéÖУ¬7ÖêmAb¾ùÄÜÓëÖØ×éÈËÒȵºËØÔÀàËÆÎ·Ö×ÓÁ¿12KD£©½áºÏ³ÊÏÖÒ»Ìõµ°°×Ó¡¼£´ø£¨Í¼1-A¡«G£©¡£±íÃ÷ÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎïÃâÒßÖƱ¸µÄ7ÖêmAb¶ÔÈË¡¢Å£ºÍÖíÒȵºËؾù¾ßÓÐÌØÒìµÄÃâÒß·´Ó¦ÐÔ¡£
3 ÌÖÂÛ
±¾ÊµÑéÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎïÃâÒßBALB/cÊ󣬳ɹ¦µØ»ñµÃÁË7Ö꿹ÒȵºËØmAb£¬¿ÉÓëÈË¡¢Å£¡¢ÖíÒȵºËØ·¢ÉúÌØÒìÐÔÃâÒß·´Ó¦£¬ÎªÒȵºËصIJⶨºÍ´¿»¯Ôö¼ÓÁËеĿ¹ÌåÑ¡Ôñ¡£
ÖƱ¸ÌìÈ»ÒȵºËØ´¿Æ·±È½ÏÀ§ÄÑ¡£Òò´Ë£¬±¾ÊµÑéÓÃÖØ×éÈËÒȵºËØÔÀàËÆÎï×÷ΪÃâÒß¿¹Ô£¬ÖƱ¸ºÍ´¿»¯¹ý³Ì¼ò±ã£¬ÔÚÃâÒßÇ°£¬¶ÔÆäÓöþÏõ»ù±½»ÇËáÄƽøÐÐÁË´¦Àí£¬Ä¿µÄÊÇÌá¸ßÃâÒßÔÐÔ£Û3¡½¡¢ÔöÇ¿BALB/cÊóµÄÃâÒß·´Ó¦£»ÔÚɸѡ¼ì²âʱ²ÉÓÃÁËͬԴËÞÖ÷¾úÃâÒßѪÇåÒÖÖÆELISA£¬ÒÔ½µµÍ»òÏû³ýɸѡ¼ì²â¹ý³ÌÖеķÇÌØÒìÐÔ·´Ó¦¡£½á¹û±íÃ÷£¬ÒÔÉÏ´ëÊ©¶ÔÌá¸ßʵÑéЧÂÊÊÇÓÐñÔÒæµÄ¡£
¡¾²Î¿¼ÎÄÏס¿
1 ·¿µÂÐË£¬ÍõÓÀɽ£¬ÖÜ×Ú°²£¬µÈ. ÈËÒȵºËØÔÀàËÆÎBKRA£©»ùÒòµÄºÏ³ÉÓë±í´ï. ÖйúÉúÎﻯѧÓë·Ö×ÓÉúÎïѧѧ±¨£¬1998£¬14£¨5£©£º518-524.
2 ¬ʥ¶°. ÏÖ´ú·Ö×ÓÉúÎïѧʵÑé¼¼Êõ. ±±¾©£º¸ßµÈ³ö°æÉ磬1993£¬380.
3 Öìƽ£¬·ëÊéÕÂ. ¿¹ÌåʵÑé¼¼Êõ. ³¤´º£º³¤´º³ö°æÉ磬1994£¬117-118.
4 ÁõÐãèó. µ¥¿Ë¡¿¹ÌåÔÚÉϵÄÓ¦ÓÃ. ºÏ·Ê£º°²»Õ¿Æѧ¼¼Êõ³ö°æÉ磬1994£¬30.
5 ½ð¶¬Ñã, ÀèÃÏ·ã, ºîÔƵÂ,Òë. ·Ö×Ó¿Ë¡ʵÑéÖ¸ÄÏ,µÚ2°æ.±±¾©£º¿Æѧ³ö°æÉ磬1992£¬889-897.