±Ï³à½ÍĸʵÑé²Ù×÷ÊÖ²á ÏÂÔØ±¾ÎÄ

±Ï³à½Íĸ±í´ïʵÑéÊÖ²á

´ó³¦¸Ë¾ú±í´ïϵͳ×îÍ»³öµÄÓŵãÊǹ¤ÒÕ¼òµ¥¡¢²úÁ¿¸ß¡¢Éú²ú³É±¾µÍ¡£È»¶ø£¬Ðí¶àµ°°×ÖÊÔÚ·­Òëºó£¬Ðè¾­¹ý·­ÒëºóµÄÐÞÊμӹ¤£¬ÈçÁ×Ëữ¡¢ÌÇ»ù»¯¡¢õ£°·»¯¼°µ°°×øˮ½âµÈ¹ý³Ì²ÅÄÜת»¯³É»îÐÔÐÎʽ¡£´ó³¦¸Ë¾úȱÉÙÉÏÊö¼Ó¹¤»úÖÆ£¬²»ÊʺÏÓÃÓÚ±í´ï½á¹¹¸´Ôӵĵ°°×ÖÊ¡£ÁíÍ⣬µ°°×ÖʵĻîÐÔ»¹ÒÀÀµÓÚÐγÉÕýÈ·µÄ¶þÁò¼ü²¢ÕÛµþ³É¸ß¼¶½á¹¹£¬Ôڴ󳦸˾úÖбí´ïµÄµ°°×ÖÊÍùÍù²»ÄܽøÐÐÕýÈ·µÄÕÛµþ£¬ÊÇÒÔ°üº¬Ìå״̬´æÔÚ¡£°üº¬ÌåµÄÐγÉËäÈ»¼ò»¯Á˲úÎïµÄ´¿»¯£¬µ«²»ÀûÓÚ²úÎïµÄ»îÐÔ£¬ÎªÁ˵õ½ÓлîÐԵĵ°°×£¬¾ÍÐèÒª½øÐбäÐÔÈܽ⼰¸´ÐԵȲÙ×÷£¬ÕâÒ»¹ý³Ì±È½Ï·±Ëö£¬Í¬Ê±Ôö¼ÓÁ˳ɱ¾¡£ Óë´ó³¦¸Ë¾úÏà±È£¬½ÍĸÊǵ͵ÈÕæºËÉúÎ¾ßÓÐϸ°ûÉú³¤¿ì£¬Ò×ÓÚÅàÑø£¬ÒÅ´«²Ù×÷¼òµ¥µÈÔ­ºËÉúÎïµÄÌØµã£¬ÓÖ¾ßÓÐÕæºËÉúÎïʱ±í´ïµÄµ°°×ÖʽøÐÐÕýÈ·¼Ó¹¤£¬ÐÞÊΣ¬ºÏÀíµÄ¿Õ¼äÕÛµþµÈ¹¦ÄÜ£¬·Ç³£ÓÐÀûÓÚÕæºË»ùÒòµÄ±í´ï£¬ÄÜÓÐЧ¿Ë·þ´ó³¦¸Ë¾úϵͳȱ·¦µ°°×·­Ôóºó¼Ó¹¤¡¢ÐÞÊεIJ»×ã¡£Òò´Ë½Íĸ±í´ïϵͳÊܵ½Ô½À´Ô½¶àµÄÖØÊÓºÍÀûÓᣠ´ó³¦¸Ë¾úÊÇÓõÃ×î¶à¡¢Ñо¿×î³ÉÊìµÄ»ùÒò¹¤³Ì±í´ïϵͳ£¬µ±Ç°ÒÑÉÌÒµ»¯µÄ»ùÒò¹¤³Ì²úÆ·´ó¶àÊÇͨ¹ý´ó³¦¸Ë¾ú±í´ïµÄ£¬ÆäÖ÷ÒªÓŵãÊdzɱ¾µÍ¡¢²úÁ¿¸ß¡¢Ò×ÓÚ²Ù×÷¡£µ«´ó³¦¸Ë¾úÊÇÔ­ºËÉúÎ²»¾ßÓÐÕæºËÉúÎïµÄ»ùÒò±í´ïµ÷¿Ø»úÖÆºÍµ°°×Öʵļӹ¤ÐÞÊÎÄÜÁ¦£¬Æä²úÎïÍùסÐγÉûÓлîÐԵİüº­Ì壬ÐèÒª¾­¹ý±äÐÔ¡¢¸´ÐԵȴ¦Àí£¬²ÅÄÜÓ¦Ó᣽üÄêÀ´£¬ÒÔ½Íĸ×÷Ϊ¹¤³Ì¾ú±í´ïÍâÔ´µ°°×ÈÕÒæÒýÆðÖØÊÓ£¬Ö÷¸üÊÇÒòΪ½ÍĸÊǵ¥Ï¸°ûÕæºËÉúÎ²»µ«¾ßÓд󳦸˾úÒײÙ×÷¡¢·±Ö³¿ì¡¢Ò×ÓÚ¹¤Òµ»¯Éú²úµÄÌØµã£¬»¹¾ßÓÐÕæºËÉúÎï±í´ïϵͳ»ùÒò±í´ïµ÷¿ØºÍµ°°×ÐÞÊι¦ÄÜ£¬±ÜÃâÁ˲úÎï»îÐԵͣ¬°üº­Ìå±äÐÔ¡¢¸´ÐԵȵȼäÌâ£Û1£Ý¡£

Óë´ó³¦¸Ë¾úÏà±È£¬½ÍĸÊǵ¥Ï¸°ûÕæºËÉúÎ¾ßÓбȽÏÍ걸µÄ»ùÒò±í´ïµ÷¿Ø»úÖÆºÍ¶Ô±í´ï²úÎïµÄ¼Ó¹¤ÐÞÊÎÄÜÁ¦£¬ÈËÃǶÔÄð¾Æ½Íĸ£¨Saccharomyces.Cerevisiae£©·Ö×ÓÒÅ´«Ñ§·½ÃæµÄÈÏʶ×îÔ磬Äð¾Æ½ÍĸҲ×îÏÈ×÷ΪÍâÔ´»ùÒò±í´ïµÄ½ÍĸËÞÖ÷£®1981Äê

Äð¾Æ½Íĸ±í´ïÁ˵ÚÒ»¸öÍâÔ´»ùÒòÒ»¸ÉÈÅËØ»ùÒò£¬ËæºóÓÖÓÐһϵÁÐÍâÔ´»ùÒòÔÚ¸ÃϵͳµÃµ½±í´ï¡£ËäÈ»¸ÉÈÅËØºÍÒȵºËØÒÑ´óÁ¿Éú²ú²¢ÔÚÈËȺÖй㷺ӦÓ㬵«ºÜ´ó²¿·Ö±í´ïÓÉʵÑéÊÒÀ©Õ¹µ½¹¤Òµ¹æÄ£Ê±£¬ÅàÑø»ùÖÐÎ¬ÌØÖÊÁ£¸ß¿½±´ÊýµÄÑ¡ÔñѹÁ¦Ïûʧ£¬ÖÊÁ£±äµÃ²»Îȶ¨£¬¿½±´ÊýϽµ£¬¶ø´ó¶àÊýÍâÔ´»ùÒòµÄ¸ßЧ±í´ïÐèÒª¸ß¿½±´ÊýµÄÎ¬ÌØ£¬Òò´ËÒýÆð²úÁ¿Ï½µ¡£Í¬Ê±£¬ÊµÑéÊÒÓÃÅàÑø»ù¸´ÔÓ¶ø°º¹ó£¬²ÉÓù¤Òµ¹æÄ£Äܹ»½ÓÊܵÄÅàÑø»ùʱ£¬ÍùÍùµ¼Ö²úÁ¿µÄϽµ¡£Îª¿Ë·þÄð¾Æ½ÍĸµÄ¾ÖÏÞ£¬ÈËÃÇ·¢Õ¹ÁËÒÔ¼×»ùÓªÑøÐͽÍĸ£¨methylotrophic yeast£©Îª´ú±íµÄµÚ¶þ´ú½Íĸ±í´ïϵͳ£Û2£Ý¡£

¼×»ùÓªÑøÐͽÍĸ°üÀ¨£ºPichia¡¢CandidaµÈ£®ÒÔPichia.pastoris£¨±Ï³à°Í˹µÂ½Íĸ£©ÎªËÞÖ÷µÄÍâÔ´»ùÒò±í´ïϵͳ½üÄêÀ´·¢Õ¹×îΪѸËÙ£¬Ó¦ÓÃÒ²×îΪ¹ã·º£¬ÒÑÀûÓôËϵͳ±í´ïÁËһϵÁÐÓÐÖØÒªÉúÎïѧ»îÐԵĵ°×ÔÖÊ¡£±Ï³à½ÍĸϵͳµÄ¹ã·ºÓ¦Óã¬Ô­ÒòÔÚÓÚ¸Ãϵͳ³ýÁ˾ßÓÐÒ»°ã½ÍĸËù¾ßÓеÄÌØµãÍ⣬»¹ÓÐÒÔϼ¸¸öÓŵã£Û1¡¢2¡¢3£Ý£» ¢Å ¾ßÓд¼Ñõ»¯Ã¸AOX1»ùÒòÆô¶¯×Ó£¬ÕâÊÇĿǰ×îÇ¿£¬µ÷¿Ø»úÀí×îÑϸñµÄÆô¶¯×ÓÖ®Ò»¡£ ¢Æ ±í´ïÖÊÁ£ÄÜÔÚ»ùÒò×éµÄÌØ¶¨Î»µãÒÔµ¥¿½±´»ò¶à¿½±´µÄÐÎʽÎȶ¨ÕûºÏ¡££¨¼´Í¬Ô´ÖØ×飩

¢Ç ¾úÖêÒ×ÓÚ½øÐиßÃܶȷ¢½Í£¬ÍâÔ´µ°°×±í´ïÁ¿¸ß¡£

¢È ±Ï³à½ÍĸÖдæÔÚ¹ýÑõ»¯ÎïøÌ壬±í´ïµÄµ°°×Öü´æÆäÖУ¬¿ÉÃâÊܵ°°×øµÄ½µ½â£¬¶øÇÒ¼õÉÙ¶Ôϸ°ûµÄ¶¾º¦×÷Óá£

Pichia.pastoris»ùÒò±í´ïϵͳ¾­¹ý½üÊ®Äê·¢Õ¹£¬ÒÑ»ù±¾³ÉΪ½ÏÍêÉÆµÄÍâÔ´»ùÒò±í´ïϵͳ£¬¾ßÓÐÒ×ÓÚ¸ßÃܶȷ¢½Í£¬±í´ï»ùÒòÎȶ¨ÕûºÏÔÚËÞÖ÷»ùÒò×éÖУ¬ÄÜʹ²úÎïÓÐЧ·ÖÃÚ²¢Êʵ±ÌÇ»ù»¯£¬ÅàÑø·½±ã¾­¼ÃµÈÌØµã¡£ÀûÓÃǿЧ¿Éµ÷¿ØÆô¶¯×ÓAOX1£¬ÒѸßЧ±í´ïÁËHBsAg¡¢TNF¡¢EGF¡¢ÆÆÉË·ç¶¾ËØ CƬ¶Î¡¢»ùÒò¹¤³Ì¿¹ÌåµÈ¶àÖÖÍâÔ´»ùÒò£¬Ö¤Êµ¸ÃϵͳΪ¸ßЧ¡¢ÊµÓᢼò±ã£¬ÒÔÌá¸ß±í´ïÁ¿²¢±£³Ö²úÎïÉúÎïѧ»îÐÔΪͻ³öÌØÕ÷µÄÍâÔ´»ùÒò±í´ïϵͳ£¬¶øÇҷdz£ÊÊÒË×ÓÀ©´óΪ¹¤Òµ¹æÄ££Û4£Ý¡£Ä¿Ç°ÃÀ¹úFDAÒÑÄÜÆÀ¼ÛÀ´×Ô¸ÃϵͳµÄ»ùÒò¹¤³Ì²úÆ·£¬×î½üÀ´×Ô¸ÃϵͳµÄCephelonÖÆ¼ÁÒÑ»ñµÃFDAÅú×¼£¬

ËùÒÔ¸Ãϵͳ±»ÈÏΪÊǰ²È«µÄ£® Pichia.pastoris±í´ïϵͳÔÚÉúÎ﹤³ÌÁìÓò½«·¢»ÓÔ½À´Ô½ÖØÒªµÄ×÷Ó㬴ٽø¸ü¶àÍâÔ´»ùÒòÔÚ¸ÃϵͳµÄ¸ßЧ±í´ï£¬Ìṩ¸üΪ¹ã·ºµÄ»ùÒò¹¤³Ì²úÆ·£Û2¡¢3£Ý¡£

½üÄêÀ´£¬Invitrogon¹«Ë¾¿ª·¢Á˱ϳà½Íĸ±í´ïϵͳµÄϵÁвúÆ·£¬¶Ì¶Ì¼¸ÄêÒѾ­ÓÐ300¶àÖÖÍâÔ´µ°×ÔÔÚ¸ÃϵͳµÃµ½ÓÐЧ±í´ï£¬±»ÈÏΪÊÇĿǰ×îÓÐЧµÄ½Íĸ±í´ïϵͳ¡£ ±Ï³à½ÍĸËÞÖ÷¾ú³£ÓõÄÓÐGS115ºÍKM71Á½ÖÖ£¬¶¼¾ßÓÐHIS4ÓªÑøÈ±Ïݱê¼Ç¡£ÆäÖУ¬GS115ÒðÖê¾ßÓÐAOX1»ùÒò£¬ÊÇMut£«£¬¼´¼×´¼ÀûÓÃÕý³£ÐÍ£»¶øKM71¾úÖêµÄAOX1λµã±»ARG4»ùÒò²åÈ룬±íÐÍΪMuts£¬¼´¼×´¼ÀûÓûºÂýÐÍ£¬Á½ÖÖ¾úÖê¶¼ÊÊÓÃÓÚÒ»°ãµÄ½Íĸת»¯·½·¨¡£

Pichia.pastoris½Íĸ¾úÌåÄÚÎÞÌìÈ»ÖÊÁ££¬ËùÒÔ±í´ïÔØÌåÐèÓëËÞÖ÷ȾɫÌå·¢ÉúÍ¬Ô´ÖØ×飬½«ÍâÔ´»ùÒò±í´ï¿ò¼ÜÕûºÏÓÚȾɫÌåÖÐÒÔʵÏÖÍâÔ´»ùÒòµÄ±í´ï£Û5£Ý£®°üÀ¨Æô¶¯×Ó¡¢ÍâÔ´»ùÒò¿Ë¡λµã¡¢ÖÕÖ¹ÐòÁС¢É¸Ñ¡±ê¼ÇµÈ¡£±í´ïÔØÌå¶¼ÊÇ´©ËóÖÊÁ££¬ÏÈÔڴ󳦸˾ú¸´ÖÆÀ©Ôö£¬È»ºó±»µ¼ÈëËÞÖ÷½Íĸϸ°û¡£ÎªÊ¹²úÎï·ÖÃÚ°ûÍ⣬±í´ïÔØÌ廹Ðè´øÓÐÐźÅëÄÐòÁС£

±Ï³à½Íĸ±í´ïϵͳÓжàÖÖ·ÖÃÚÐͱí´ïÖÊÁ££¬ÓÐÐí¶àµ°°×Ôڱϳà½ÍĸµÃµ½Á˸ßЧ·ÖÃÚ±í´ï¡£°ûÍâ±í´ïÐèÒªÔÚÍâÔ´µ°°×µÄNÄ©¶Ë¼ÓÉÏÒ»¶ÎÐźÅëÄÐòÁУ¬Òýµ¼ÖØ×éµ°°×½øÈë·ÖÃÚ;¾¶£¬¿Éʹµ°°×µ°°×ÖÊÔÚ·ÖÃÚµ½°ûÍâÖ®ºó»ñµÃ׼ȷµÄ¹¹ÐÍ¡£±Ï³à½Íĸ¶ÔÍâÔ´µ°°××ÔÉíµÄÐźÅÐòÁÐʶ±ðÄÜÁ¦²î£¬ÔÚ±¾ÊÔÑéÖÐËùʹÓÃpPICZ¦ÁAÖÊÁ££¬ÆäÐźÅëÄÀ´×ÔÄð¾Æ½ÍĸµÄ¦Á-½»ÅäÒò×Ó£¨¦Á-factor£©£¬ÄܺܺõĴﵽÒÔÉϵÄÒªÇó¡£²¢ÇÒ×÷ΪÐÂÒ»´úµÄ±Ï³à½Íĸ·ÖÃÚ±í´ïÖÊÁ££¬Ëü»¹ÓµÓÐÒ»¸öÌØµãÊÇÆä¾ßÓÐZeocin¿¹ÐÔ±ê¼Ç»ùÒò£¬¸øÎÒÃÇɸѡת»¯×ӵŤ×÷´øÀ´ºÜ´óµÄ±ãÀû£Û1¡¢2£Ý¡£

pPICZ¦ÁAÖÊÁ£ÊÇ×÷ΪÐÂÒ»´úµÄ±Ï³à½Íĸ·ÖÃÚ±í´ïÖÊÁ££¬ËüµÄÖ÷ÒªÌØµã¼ò½éÈçÏ£º

¢Å ¾ßÓÐǿЧ¿Éµ÷¿ØÆô¶¯×ÓAOX1£¨alcohol oxidase£¬´¼Ñõ»¯Ã¸£©£»

¢Æ ¾ßÓÐZeocin¿¹ÐÔɸѡ±ê¼Ç»ùÒò£¬ÖØ×éת»¯×Ó¿ÉÖ±½ÓÓÃZeocin½øÐÐɸѡ£¬¼´ÔÚYPDZƽ°åÉÏÉú³¤µÄת»¯×ÓÖУ¬100£¥¶¼ÓÐÍâÔ´»ùÒòµÄÕûºÏ£¬´ó´ó¼ò»¯ÁËÖØ×éת»¯½ÍĸµÄɸѡ¹ý³Ì£Û5£Ý¡£ÔÚ²Ù×÷¹ý³ÌÖУ¬ZeocinÒ²¿ÉÓÃÀ´É¸Ñ¡º¬±í´ïÔØÌåpPICZ¦ÁAµÄ´ó³¦¸Ë¾úת»¯×Ó£¬²»±ØÁíÍâʹÓÃAmp£¬¾­¼Ã¶øÓÖ¼ò±ã£»¡£

¢Ç ÔÚ±í´ïÔØÌåA0X1 5¡¯¶ËÆô¶¯×ÓÐòÁÐÏÂÓΣ¬Óй©ÍâÔ´»ùÒò²åÈëµÄ¶à¿Ë¡λµã£¬¶à¿Ë¡λµãÏÂÓÎÓÐA0X1 3¡¯¶ËÖÕÖ¹ÐòÁУ» ¢È ·ÖÃÚЧÂÊÇ¿µÄÐźÅëĦÁ-factor.

Invitrogen¹«Ë¾¿ª·¢µÄ±Ï³à½Íĸ±í´ïϵͳµÄϵÁвúÆ·×÷ΪĿǰ±»Ó¦ÓÃΪ×îΪ¹ã·ºµÄ½Íĸ±í´ïϵͳ£¬ÆäÖ÷ÒªµÄÓŵãÓУº´¼Ñõ»¯Ã¸¿Éµ÷¿ØµÄÇ¿Æô¶¯×Ó£¬ÄܸßÃܶȷ¢½Í£¬ÖØ×éµ°°×±í´ïÁ¿¸ß¡£ÍâÔ´»ùÒòÕûºÏÔÚ½Íĸ»ùÒò×éÉÏ£¬¿ÉÒÔÎȶ¨´æÔÚ¡£Í¬Ê±£¬¸ßЧ·ÖÃÚ±í´ïÖÊÁ£Äܽ«ÍâÔ´µ°°×±í´ïºó£¬½øÐз­Òëºó¼Ó¹¤´¦Àí£¬½«ÍâÔ´µ°°×·ÖÃÚµ½Ï¸°ûÍ⣬²»µ«Ìá¸ß±í´ïµ°°×µÄ»îÐÔ£¬¶øÇÒ£¬ÓÐÀûÓÚ²úÎïµÄ´¿»¯¡£

Ò»£®±Ï³à½Íĸ±í´ï³£ÓÃÈÜÒº¼°»º³åÒºµÄÅäÖÆ 1£®1 ¸÷ÖÖĸҺµÄÅäÖÆ

10*YNB £¨º¬ÓÐÁòËáï§¡¢ÎÞ°±»ùËáµÄ13.4%½Íĸ»ù´¡µªÔ´ÅàÑø»ù£© 4¡æ±£´æ¡£34g½Íĸ»ù´¡µªÔ´ÅàÑø»ù£¨ÎÞÁòËáï§£©+100gÁòËáï§£¬ÈÜÓÚ1000mlË®ÖУ¬¹ýÂ˳ý¾ú¡£

500*B £¨0.02%ÉúÎïËØ Biotin£© 4¡æ±£´æ ±£´æÆÚΪ1Äê¡£20mgµÄÉúÎïËØÈÜÓÚ100mlË®ÖУ¬¹ýÂ˳ý¾ú¡£

100*H £¨0.4%Histidine ×é°±Ëᣩ 4¡æ±£´æ ±£´æÆÚΪ1Äê¡£400mgµÄL-×é°±ËáÈÜÓÚ100mlË®ÖУ¬£¨¼ÓÈÈÖÁ50¡æÒÔ´Ù½øÈܽ⣩£¬¹ýÂ˳ý¾ú¡£

10*D £¨20Txtrose ÆÏÌÑÌÇ£© ±£´æÆÚΪ1Äê¡£200gÆÏÌÑÌÇÈÜÓÚ1000mlË®