full2010.pdf - page 1121

1083
‡ÎµœÎµ
„µ¦«¹„¬µÂ¨³„µ¦ª·‹´¥šµŠ—oµœ¡´œ›»ª·«ª„¦¦¤ (genetic engineering) šÎµÄ®oÁ„·—„µ¦¡´ •œµ°¥nµŠ¦ª—Á¦Èª
šµŠ—oµœ¡´œ›»«µ˜¦r¦³—´Ã¤Á¨„»¨Â¨³œÎµÅž¼n„µ¦Áž¨¸É ¥œÂž¨Š°¥nµŠ¤µ„šµŠ—oµœ¸ªª·š¥µÂ¨³Áš‡ÃœÃ¨¥¸¸ª£µ¡ —´Š
‹³Á®ÈœÅ—o‹µ„„µ¦œÎ µÁ°µ‡ªµ¤¦¼o—oµœÁš‡ÃœÃ¨¥¸¡´ œ›»ª·«ª„¦¦¤Ž¹ÉŠ¤¸‡ªµ¤Á‹¦· „oµª®œoµÁž}œ°´œ¤µ„¤µž¦³¥» „˜rčo°¥nµŠ
„ªoµŠ ªµŠšµŠ„µ¦Â¡š¥r¨³—o µœ°ºÉœÇ Ž¹ÉŠ„µ¦œÎµÁ°µÁš‡ÃœÃ¨¥¸ ¡´œ›»ª·«ª„¦¦¤ÅžÄo šµŠ„µ¦Â¡š¥rÁnœ„µ¦Á˜¦¸¥¤
ª´‡Ž¸œÂ¨³µ¦š¸É čoĜ„µ¦ª·œ·‹Œ´¥Ã¦‡ „µ¦ª·œ· ‹Œ´¥Ã¦‡˜·—ÁºÊ° „µ¦ª·œ·‹Œ´¥Ã¦‡šµŠ¡´œ›»„¦¦¤ „µ¦Ÿ¨·˜ÁžžÅš˜r
±°¦r䜝µŠœ·—ץčo DNA ´ŠÁ‡¦µ³®rš¸É¤¸¨Îµ—´ nucleotides ´¤¡´ œ›r °—‡¨o°Š„´¨Îµ—´ °Š„¦—°³¤·Ãœ °Š
±°¦r䜗´Š„¨nµªÂ¨oªœÎµÁ oµ¼nÁŽ¨¨rÁ‹oµo µœ (host) Á¡ºÉ°´Š‡´Ä®o¤¸„µ¦Â—Š°°„ °Š¥¸œÁ®¨nµœ´Êœ ץčo ¡¨µ¤·—Áž}œ
¡µ®³Ánœ „µ¦Än¥¸œ°·œŽ¼¨·œ (insulin gene) °Š¤œ»¬¥rÁ oµ¼n‡š¸Á¦¸¥šÎµÄ®o‡š¸Á¦¸¥œ·—œ´Êœµ¤µ¦™Ÿ¨·˜°·œŽ¼¨·œ
ŗo (ž¦µÃ¤š¥r, 2529; ·¬–» ¦¦, 2529) ˜nž{®µš¸É¤´„¡Å—on°¥Â¨³nŠŸ¨˜n°‡ªµ¤ÎµÁ¦È‹ °ŠŠµœª· ‹´¥šµŠ—oµœ¡´œ›»-
ª·«ª„¦¦¤‡º°¡¨µ¤·—¨¼„Ÿ¤ (recombinant plasmid) Ťn µ¤µ¦™‡Š°¥¼nŗo ĜÁŽ¨¨rÁ‹o µoµœ (structural instability)
®¦º°Å¤n µ¤µ¦™™nµ¥š°—Åž¥´ŠÁŽ¨¨r¦»nœ¨¼„Å—o (segregrational instability) (Uhlen et al., 1981; Ostroff and Pene,
1984) šÎµÄ®oÁ„·—‡ªµ¤¨nµoµÂ¨³¼Á¸¥‡nµÄo‹nµ¥¼Š ¹Êœ ץŗo¤¸¦µ¥Šµœ„µ¦ª·‹´¥®¨µ¥Œ´Á„¸É¥ª„´ž{‹‹´¥š¸É¤¸Ÿ¨˜n°
„µ¦‡Š°¥¼n°Š¡¨µ¤·—ÄœÁŽ¨¨rÁ‹oµoµœªn µ ¹Êœ°¥¼n„´°»–®£¼¤·š¸ÉčoĜ„µ¦Á¡µ³Á¨¸Ê¥Š µ¥¡´œ›»r°ŠÁºÊ°š¸ÉœÎµ¤µÁž} œÁŽ¨¨r
Á‹oµoµœ œµ—¨³‡» –¤´˜·°Š¡¨µ¤·— ‡»–¤´˜·°Š°µ®µ¦Á¨¸Ê¥ŠÁºÊ ° Áž}œ˜oœ (Gamel and Piot, 1992; Brigidi
et al., 1996) Ž¹ÉŠž{‹‹´¥¡ºÊœ“µœš¸É œnµ‹³Á„¸É¥ª o°Š„´„µ¦‡Š°¥¼n°Š¡¨µ¤·— (segregrational stability) ‡º° œµ— °Š
¡¨µ¤·—¨¼„Ÿ¤„´‡ªµ¤´¤¡´ œ›r„´ÁŽ¨¨rÁ‹oµoµœš¸ÉœÎ µ¤µÄo —´Šœ´Êœ‹¹ŠœÄ‹ž{‹‹´¥—´ Š„¨nµªš¸É¤¸Ÿ¨˜n °„µ¦‡Š°¥¼n°Š
¡¨µ¤·—ÄœÁŽ¨¨rÁ‹oµoµœÃ—¥Á¨º°„čo¡¨µ¤·—¡µ®³ 2 œ·—„¨nµª‡º ° pBluescript II KS (2.96 „·Ã¨Á) ¨³
pHY300PLK (4.87 „·Ã¨Á) ¨³¡¨µ¤·—¨¼„Ÿ¤¥¸œÅ‡˜·Áœš¸Éŗo ¤µ‹µ„¡¨µ¤·—¡µ®³—´Š„¨nµª¤¸ºÉ°ªn µ pCHIB1
(5.54 „·Ã¨Á) ¨³ pHYB43 (7.46 „·Ã¨Á) 宦´ÁŽ¨¨rÁ‹oµoµœš¸ÉœÎµ¤µÄoĜ„µ¦«¹ „¬µ‡º° Escherichia coli µ¥
¡´œ›»r DH5 v ¨³ INVvFc Á¡ºÉ°œÎµŸ¨š¸Éŗo¤µÁž} œ o°¤¼¨ÁºÊ °Š˜oœÄœ„µ¦Á¨º°„čo¦³ host-vector system °¥nµŠ¤¸
ž¦³·š›·£µ¡˜n°Åž
°»ž„¦–r¨³ª·›¸ „µ¦
1. ª·›¸„µ¦Á¨¸Ê¥ŠÁºÊ°
Á¨¸Ê¥ŠÁŽ¨¨r Escherichia coli DH5v ®¦º° INVvFc š¸É¤¸¡¨µ¤·— (pBluescript II KS ®¦º° pHY300PLK
®¦º° pCHIB1 ®¦º° pHYB43) Ĝ°µ®µ¦Á¨¸Ê¥ŠÁºÊ ° Luria-Bertani (LB) medium ž¦·¤µ˜¦ 5 ¤·¨¨·¨·˜¦ (¤¨.) š¸É¤¸
ž’·¸ªœ³Â°¤¡·Ž·¨¨·œ‡ªµ¤Á o¤ oœ 100 ŤÇ¦„¦´¤˜n°¤·¨¨·¨·˜¦ (¦µ¥¨³Á°¸¥— °ŠÁºÊ°Â¨³¡¨µ¤· ——ŠÄœ˜µ¦µŠš¸É
1) ‹µ„œ´ÊœœÎµÅžn ¤Á¡µ³š¸É°»–®£¼¤· 37qC oµ¤‡ºœ
2. „µ¦„´—¡¨µ¤·—‹µ„ÁºÊ ° Escherichia coli strains
„µ¦„´—¡¨µ¤·—‹µ„ÁŽ¨¨rčoª·›¸ alkaline lysis (Sambrook et al., 1989) Ž¹ÉŠ¤¸ ¦µ¥¨³Á°¸¥—×¥¥n °—´Š˜n°Åž
œ¸Ê: ™n µ¥ÁºÊ°š¸ÉÁ¨¸Ê¥ŠÅ—o‹µ„ ´Êœ˜°œš¸É 1 ž¦·¤µ˜¦ 1.5 ¤·¨¨· ¨·˜¦ ¨ŠÄœ microcentrifuge tube œÎµÅžž{~ œš¸É‡ªµ¤Á¦Èª¦°
5,000 rpm (Microcentrifuge EBA 12, Hettich) Áž}œÁª¨µ 2 œµš¸ šÎµ„µ¦„¦³‹µ¥˜³„°œÁºÊ°—oª¥ Solution I (50 mM
glucose, 25 mM Tris-HCl ¨³ 10 mM EDTA pH 8.0) ž¦·¤µ˜¦ 100 ŤÇ¦¨·˜¦ ¨o ªÂnŪoĜœÊε ȊÁž}œÁª¨µ
5 œµš¸ ¨oªÁ˜·¤ Solution II (0.2 N NaOH ¨³ 1% SDS) ž¦·¤µ˜¦ 200 ŤÇ¦¨·˜¦ Ÿ¤Ä®oÁ oµ„´œÃ—¥„µ¦„¨´®¨°—