µ¥Á´¿¹Ì壨single-chain variable fragment£¬ scFv£©ÊÇÓÉ¿¹ÌåÖØÁ´µÄ¿É±äÇøÓëÇáÁ´µÄ¿É±äÇøÔÚÒ»¶ÎëÄÁ´µÄÁ¬½ÓϹ¹³ÉµÄС·Ö×Ó£¬ÊǾßÓп¹Ìå»îÐÔµÄ×îС¹¦Äܽṹµ¥Î»¡£µ¥Á´¿¹Ìå¿ÉÒÔÓɱí´ïϵͳ½øÐбí´ï£¬Ò²¿ÉÒÔÓÃÊɾúÌåչʾ¼¼Êõ½øÐÐÖƱ¸¡£ÓÉÓÚÆä·Ö×ÓÖÊÁ¿Ð¡£¬´©Í¸Á¦Ç¿£¬°ëË¥Æڶ̣¬ÃâÒßÔÐԵ͵ÈÌص㣬ÔÚ¼²²¡ÁÙ´²Õï¶Ï¡¢ÖÎÁÆ¡¢Ô¤·ÀµÈ·½Ãæ¾ßÓÐÖØÒª×÷Óú͹ãÀ«µÄÓ¦ÓÃÇ°¾°¡£±¾Îľ͵¥Á´¿¹ÌåµÄ½á¹¹¡¢ÖƱ¸¡¢Ó¦Óõȷ½Ãæ×öÒ»¸ö¸ÅÊö¡£
ÍêÕûµÄ¿¹ÌåÓÉÁ½ÌõÖØÁ´£¨heavy chain£¬H£©ºÍÁ½ÌõÇáÁ´£¨light chain£¬L£©¹¹³É£¬Í¸¹ýÈ˹¤¸ÄÔ죬¿ÉʹÆäÖ»±í´ï¿É±äÇø£¬Í¨¹ýÈ˹¤ºÏ³ÉµÄÁ¬½ÓëÄ£¨Linker£©»ùÒò½«¿¹ÌåÖØÁ´¿É±äÇø£¨VH£©ºÍÇáÁ´¿É±äÇø£¨VL£©Á¬½Ó³ÉÖØ×é»ùÒò£¬ÓɸÃÖØ×é»ùÒò±í´ïµÄ¿¹Ìå¾Í³ÆΪµ¥Á´¿¹Ì壨scFv£©¡£ÔڽṹÉÏ£¬¿ÉÒÔ½«ÖØÁ´µÄN¶ËÓëÇáÁ´µÄC¶ËÏàÁ¬£¬Ò²¿ÉÒÔ½«ÇáÁ´µÄN¶ËÓëÖØÁ´µÄC¶ËÏàÁ¬¡£Linker³¤¶Èͨ³£Îª15~25¸ö°±»ùËᣬͨ³£Óɸʰ±ËᣨGly£©ºÍË¿°±ËᣨSer£©¹¹³É£¬¾ßÓÐÒ»¶¨µ¯ÐÔ¼°µ°°×ø¿¹ÐÔ£¬LinkerµÄ×÷ÓÃÊÇÁ¬½ÓVH£¬VL£¬²¢±£³ÖÒ»¶¨µÄµ¯ÐÔ£¬Ê¹VH£¬VLµÄ¹¦ÄÜÇøÕÛµþºóÈÔ¿ÉÅä¶Ô£¬¹¹³Éµ¥¼Û¿¹Ô½áºÏλµã¡£
scFvƬ¶ÎΪһ¼Û£¬¿ÉÒÔͨ¹ýÁ¬½ÓÁ½¸öscFvƬ¶ÎÖÆ×÷³ö¶þ¼ÛµÄscFv¡£¶þ¼ÛscFvÒ»°ãÓÐÁ½Öֽṹ£¬Ò»ÖÖÊÇÐγÉÒ»Ìõº¬ÓÐÁ½¸öÖØÁ´¿É±äÇøÓëÁ½¸öÇáÁ´¿É±äÇøµÄµ¥ëÄÁ´£¬½Ð×ö´®Áª¿¹Ì壨tandem di-scFvs£©£»ÁíÒ»ÖÖÊǽ«LinkerµÄ³¤¶È´Ó15Ëõ¶Ìµ½3~12¸ö£¬Ê¹À´×ÔÁ½¸ö²»Í¬·Ö×ÓµÄVHºÍVL¹¦ÄÜÇøÏ໥Åä¶Ô£¬ÐγÉÒ»¸ö¶þ¾ÛÌå½á¹¹£¬½Ð×öDiabodies£¨Èç¹ûDiabodiesÊÇÓÉÀ´×ÔÁ½¸ö²»Í¬¿¹ÔµÄ¿É±äÇø×é³É£¬ÄÇô¾Í»áÐγÉË«ÌØÒìÐÔ¿¹Ì壩¡£Óë´ËÀàËÆ£¬¿ÉÒÔÉú²úÒ»Ìõ¾ßÓÐÈý¸öÖØÁ´¿É±äÇøÓëÈý¸öÇáÁ´¿É±äÇøµÄ´®Áª¿¹Ì壨tandem di-scFvs£©£¬Ò²¿ÉÒÔͨ¹ý½øÒ»²½Ëõ¶ÌLinker³¤¶È£¬´Ó¶øʹÀ´×ÔÈý¸ö²»Í¬·Ö×ÓµÄVHºÍVL¹¦ÄÜÇøÏ໥Åä¶Ô£¬ÐγÉÒ»¸öÈý¾ÛÌ壨triabodies£©¡£ÓëÒ»°ãµÄscFvƬ¶ÎÏà±È£¬scFv¶à¾ÛÌ忹ԽáºÏ¼ÛÔö¼Ó£¬¾ßÓиü¸ßµÄÇ׺ÍÁ¦¡£
scFv¿ÉÔÚ¶à¸ö±í´ïϵͳÖнøÐбí´ï£¬Ä¿Ç°±È½Ï³£Óõģ¬ÊǴ󳦸˾ú±í´ïϵͳºÍ²¸È鶯Îï±í´ïϵͳ¡£Í¨¹ýÊɾúÌåչʾ¼¼ÊõÒ²¿ÉÉú²úµÃµ½scFvƬ¶Î¡£Í¨¹ýÃâÒ߶¯ÎïʵÑéµÃµ½ÔÓ½»Áöϸ°û£¬´ÓÖÐÌáÈ¡cDNA£¬ÀûÓÃÄæת¼PCR£¨RT-PCR£©µÃµ½È«Ì׿¹ÌåµÄÖØÁ´¿É±äÇøVHÓëÇáÁ´¿É±äÇøVL£¬ÀûÓÃÖصþPCR£¨SOE-PCR£©½«VH£¬VLƬ¶ÎÆ´½ÓÀ©ÔöµÃµ½µ¥Á´¿¹Ìå¿É±äÇø»ùÒòƬ¶Î£¨scFv£©¡£½«scFv»ùÒò¿Ë¡µ½ºÏÊʵÄÊɾúÌåÔØÌåÖУ¬µçת¸ÐÊÜ̬´ó³¦¸Ë¾ú£¬¸¨ÖúÊɾúÌ峬Ⱦ£¬µÃµ½µÄÉÏÇåÒº¼´Îªµ¥Á´¿¹Ìå¿â¡£ÒÔÌØÒìÐÔ¿¹ÔΪ¹ÌÏ࣬¾3~5Âֵġ°Îü¸½-Ï´ÍÑ-À©Ôö¡±É¸Ñ¡£¬¼´¿ÉµÃµ½¸»¼¯µÄÄÜÓ뿹ÔÌØÒìÐÔ½áºÏµÄµ¥Á´¿¹Ìå¡££¨ÊɾúÌåչʾ¼¼ÊõÖƱ¸scFvÏêϸʵÑé²½Ö裩
µ¥Á´¿¹Ìåȱ·¦ÔÚÍêÕû¿¹ÌåÖк¬ÓеÄFcƬ¶Î£¬Òò´ËÆÕͨµÄ½áºÏλµã£¨Protein G£©²»ÄÜÓÃÀ´´¿»¯¿¹Ì塣ͨ³££¬ÔÚscFvƬ¶ÎµÄC¶Ë¼ÓÉÏ6¸ö×é°±ËᣬȻºóÀûÓýðÊôòüºÏÇ׺ͲãÎö£¨IMAC£©¿ÉÒÔ¶Ôµ¥Á´¿¹Ìå½øÐд¿»¯¡£³ý´ËÖ®Í⣬µ°°×L¿ÉÒÔºÍÇáÁ´µÄ¿É±äÇøÏ໥×÷Óã¬Òò´ËÒ²¿ÉÒÔÓõ°°×L½øÐп¹ÌåµÄ´¿»¯¡£
ijЩÌØÊâµÄScFvƬ¶ÎÒ²¿ÉÒÔ±»Protein A²¶»ñ²¢´¿»¯£¬µ«ÆäÖеÄÔÀí´¦ÓÚδ֪״̬¡£
scFv¾ßÓзÖ×ÓÖÊÁ¿Ð¡¡¢´©Í¸Á¦½«Ç¿¡¢ÌØÒìÐԽϸߵÈÓŵ㣬ÔÚ°ÐÏòÖÎÁÆ¡¢Ó°ÏñÕï¶Ï¡¢Ï¸°ûÄÚÃâÒß¡¢ÉúÎï¼ì²âµÈ·½ÃæÓÐ×ÅÖØÒªµÄÓ¦Óá£ÔÚ°ÐÏòÖÎÁÆ·½Ã棬¿ÉÒÔ½«Ò©ÎïºÍ¶¾ËØÓëscFvÁ´½Ó£¬ÐγÉÃâÒßÒ©Îï»òÕßÃâÒ߶¾Ëصȣ¬ÀûÓÿ¹ÔÓ뿹ÌåÌØÒìÐÔ½áºÏµÄÌص㣬¶¨Î»ÓÚ°Ðϸ°û£¬¶Ô°Ðϸ°û½øÐÐÌØÒìÐÔÃâÒßɱÉË£»ÔÚÓ°ÏñÕï¶Ï·½Ã棬ÓÉÓÚscFv´©Í¸Á¦Ç¿£¬ÔÚÖ×Áö×éÖ¯Öеķֲ¼Ö¸Êý½ÏÍêÕû¿¹Ìå·Ö×Ó·Ö×Ӹߣ¬ÔÚ·ÅÉäÏÔÏñʱ£¬·ÅÉäÐÔºËËØÅųö½Ï¿ì£¬¶ÔÉíÌåΣº¦Ð¡£¬¿ÉÓÃÓÚÖ×ÁöµÄÏÔÏñ¶¨Î»Õï¶Ï£»ÔÚϸ°ûÄÚÃâÒß·½Ã棬²¡¶¾¸ÐȾÉúÎïÌåºó£¬Ï¸°ûÄÚ±í´ïÄÜʶ±ðijÖÖ²¡¶¾±àÂëµ°°×µÄ¿¹Ì壬´Ó¶ø×èÖ¹²¡¶¾ÔÚϸ°û¼ä´«µÝ£¬¿¹²¡¶¾µ°°×µÄµ¥Á´¿¹Ìå¿ÉÒÔÔÚϸ°ûÄÚ¿Ë¡Óë±í´ï£¬´Ó¶ø¿ÉÓÃÓÚ²¡¶¾¸ÐȾÐÔ¼²²¡µÄÕï¶ÏºÍÖÎÁÆ£»ÔÚÉúÎï¼à²â·½Ã棬scFvÇ׺ÍÁ¦ºÃ£¬Ò×ÓÚÖƱ¸£¬Òò´Ë±»ÓÃÀ´¿ìËÙ׼ȷʳÎïºÍË®µÈÎïÖÊÖеÄÓк¦ÎïÖÊ¡£
Ä¿Ç°£¬scFvÒѾ³ÉΪ»ùÒò¹¤³ÌÑо¿µÄÈȵ㡣Ëæ×ŶÔscFvÑо¿µÄ²»¶ÏÉîÈ룬ÏàÐÅÔÚ²»¾ÃµÄ½«À´£¬scFv½«Æ¾½è×ÔÉíµÄÓÅÊÆ£¬ÔÚҽѧ¡¢Ê³Æ·µÈÁìÓò·¢»Ó¸ü¾Þ´óµÄÓ¦Óá£
¿¹ÌåƬ¶ÎFab¡¢Fab’¼°F£¨ab’£©2½éÉÜ