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Áž°¦rÁŽÈœ˜r°Š ampicillin resistant colony ž¦µ„’ªnµÅ¤n¤¸‡ªµ¤Â˜„˜nµŠ„´œ °Š E. coli š´ÊŠ 2 µ¥¡´œ›»r˜n°„µ¦Á„·—
segregrational instability Ž¹ÉŠ°µ‹ÁœºÉ°Š¤µ‹µ„Áž}œÁºÊ °Äœ„»¨Á—¸¥ª„´œ‹¹ Š¤¸‡»–¤´˜·Ä„¨oÁ‡¸¥Š„´œ (˜µ¦µŠš¸É 1) ¨³Äo
Á¡ºÉ°Áž}œ cloning host Á®¤º°œ„´œ (Gibco BRL, Invitrogen) ¨³¡ªnµ¡¨µ¤·—š¸ÉŤn¤¸·Êœ¥¸ œÅ‡˜·Áœ (pBluescript II
KS ¨³ pHY300PLK) ¤¸ segregrational stability Ą¨oÁ‡¸¥Š„´œ Ž¹ÉŠ¼Š„ªnµ¡¨µ¤·—š¸É¤¸·Êœ¥¸œÅ‡˜· Áœ (pCHIB1 ®¦º°
pHYB43) ˜n™oµ¥´Š‡ŠÁ¨¸Ê¥ŠÁºÊ°š¸É¤¸¡¨µ¤·——´Š„¨nµªš´ÊŠ®¤—Äœ°µ®µ¦Á¨¸Ê¥ŠÁºÊ° LB š¸É¤¸¥µž’·¸ªœ³Â°¤¡·Ž· ¨¨·œ„È
¡ªnµ¡¨µ¤·—š»„œ·—¥´Š‡Š¤¸ segregrational stability š¸É—¸˜¨°—„µ¦Á¨¸Ê¥ŠÁºÊ°Áž} œÁª¨µ 30 ª´œ —´Šœ´Êœ„µ¦š¸É¡¨µ¤·—
˜n¨³œ·—¤¸ segregrational stability ˜„˜nµŠ„´œÁ¤ºÉ°Á¨¸Ê¥ŠÄœ£µª³ non selective pressure °µ‹ ¹Êœ°¥¼n„´ œµ—
°Š¡¨µ¤·—×¥¡ªn µ¡¨µ¤·—š¸É¤¸œµ—Á¨È „‹³¤¸ segregrational stability ¼Š„ªnµ¡¨µ¤·— œµ—Ä®n (Gamel
and Piot, 1992; Lertcanawanichakul and Wiwat, 2000) ¨³¥´Š ¹Êœ°¥¼n„´ copy number °Š¡¨µ¤·— (Old and
Prim rose, 1989) „¨nµª‡º °¡¨µ¤·—š¸É¤¸ copy number ¼Š‹³¤¸ segregrational stability ¼Š˜µ¤Åž—o ª¥Ž¹ÉŠ°µ‹
ÁœºÉ°Š¤µ‹µ„¡¨µ¤· —¤¸‹Îµœªœ¤µ„ÄœÁŽ¨¨r šÎµÄ®o¤¸Ã°„µ¼ŠÄœ„µ¦™nµ¥š°—Åž¥´ŠÁŽ¨¨r¦»nœ¨¼„Å—o¤µ„„ªnµ¡¨µ¤·—š¸É¤¸
copy number ˜Éε (¤¸ž¦·¤µ–¡¨µ¤·—œo°¥) ˜n Ĝ„µ¦š—¨°Š¡ªnµ pHY300PLK Ž¹ÉŠ¤¸œµ—Ä®n„ªnµÂ¨³¤¸ copy
number ˜É優n µ pBluescript II KS ˜n¤¸ segregrational stability š¸É ¼Š„ªnµ Ž¹ÉŠ°µ‹Áž}œÁ¡¦µ³‡»–¤´˜·°Š¡¨µ¤·—
¡µ®³„¨nµª‡º° pBluescript II KS µ¤µ¦™ÄoÁž}œ expression vector ŗoÁœºÉ°Š‹µ„¤¸ Promoter °Š¥¸œ lacZ (Plac) ¨³
¤¸ copy number ¼ŠšÎµÄ®o°µ‹¤¸„µ¦Äo ¡¨´ŠŠµœ¤µ„Äœ‹Î µ¨°Š˜´ªÂ¨³Â—Š°°„ °Š¥¸œš¸É°µ‹Áž}œ¡·¬˜n°ÁŽ¨¨r šÎµÄ®o
¡¨µ¤·—™¼„„ε‹´—°°„Åž (Mari et al., 1999; Vassileva-Atanassova et al., 1999 ) ®¦º°°µ‹ÁœºÉ°Š¤µ‹µ„„µ¦ÄoÁºÊ°
Á¦·É¤˜oœÄœ„µ¦n¤Á¨¸Ê¥ŠÅ¤nÁšnµ„´œ (Brigidi et al., 1996) œ°„‹µ„œ¸Ê segregrational stability ¥´ŠÁ„¸É¥ª o°Š„´ „µ¦š¸É
¡¨µ¤·—¡µ®³¤¸·Êœ¥¸œ°—ÄnÁ oµÅž (inserted gene) (Gruss and Ehrlich, 1988) —´ŠÁn œÄœ„¦–¸°Š pHYB43 ®¦º°
pCHIB1 š¸É¤¸¥¸œÅ‡˜·Áœ°—ÄnÁ oµÅžÄœ¡¨µ¤·—¡µ®³ pHY300PLK ®¦º° pBluescript II KS ˜µ¤¨Îµ—´ šÎµÄ®o¤¸
segregrational stability ¨—˜É娊 (˜µ¦µŠš¸É 2) Ž¹ÉŠ°µ‹ÁœºÉ°Š¤µ‹µ„Þ¦˜¸œÅ‡˜·Áœš¸É ™¼„´Š‡´Ÿ¨·˜°°„¤µÃ—¥¥¸œ
Ҙ·Áœ°µ‹¤¸‡ªµ¤Áž}œ¡·¬˜n°ÁŽ¨¨rÁ‹oµoµœŽ¹ÉŠ°—‡¨o°Š„´¦µ¥Šµœ °Š Sirirattanathon (1997) ¨³
Lertcanawanichakul (2000) Ž¹ÉŠ¡ªnµ™oµÁŽ¨¨rÁ‹o µoµœœ·— E. coli ®¦º° Bacillus thuringiensis š¸É¦´Á°µ¡¨µ¤·—¨¼„
Ÿ¤Å‡˜·ÁœÁ oµ¼nÁŽ¨¨r‹³šÎµÄ®o¤¸„µ¦Ÿ¨·˜Á°œÅŽ¤rҘ·Áœ °°„¤µÅ—o˜n‹³šÎµÄ®o segregrational stability ¨—˜É娊
Á¡¦µ³Ã—¥ž„˜·Â¨oª„µ¦Ÿ¨·˜ heterologous protein Ĝ‡š¸Á¦¸ ¥¤´„‹³nŠŸ¨Ä®oÁ„·—‡ªµ¤Áž} œ¡·¬˜n°ÁŽ¨¨r (Mari et al.,
1999) ¨³šÎµÄ®o¤¸„µ¦œÎµ¡¨µ¤·—°°„œ°„ÁŽ¨¨r¦ª¤š´ÊŠšÎµÄ®o°´˜¦µ„µ¦°¥¼n¦°— °ŠÁŽ¨¨r¨—˜É娊—oª¥ œ°„‹µ„œ¸Ê¥´Š
¤¸¦µ¥Šµœªnµ segregrational stability °Š¡¨µ¤·—¥´Š ¹Êœ°¥¼n„´°»–®£¼¤·Â¨³„µ¦Á˜·¤°µ„µ«Äœ„µ¦n¤Á¨¸Ê¥ŠÁºÊ° pH ¨³
°Š‡rž¦³„° °Š°µ®µ¦Á¨¸Ê¥ŠÁºÊ° Áž} œ˜oœ (Brigidi et al., 1996)
¦»žŸ¨„µ¦ª·‹´¥
„µ¦Á¨¸Ê¥ŠÁºÊ°š¸É¤¸¡¨µ¤·—œ·—˜nµŠÇ Ĝ£µª³ non selective pressure šÎµÄ®oÁ„·— structural stability š¸É˜„˜nµŠ
„´œ ¹Êœ°¥¼n„´ œµ—¨³ copy number °Š¡¨µ¤·— ¦ª¤š´ÊŠ heterologous protein š¸É™¼„Ÿ¨·˜°°„¤µ‹µ„¡¨µ¤·—
¨¼„Ÿ¤¥¸œÅ‡˜· Áœ„È nŠŸ¨˜n° segregrational stability —oª¥Ánœ„´œŽ¹ÉŠ°µ‹ÁœºÉ°Š¤µ‹µ„‡ªµ¤Áž}œ¡·¬˜n°ÁŽ¨¨r°Š
heterologous protein (Mari et al., 1999) —´ Šœ´ÊœÁ¤ºÉ°Áž¦¸¥Áš¸¥¦³®ªnµŠ¡¨µ¤· —¡µ®³ pHY300PLK ®¦º°
pBluescript II KS „´¡¨µ¤· —¨¼„Ÿ¤¥¸œÅ‡˜·Áœ pHYB43 (pHY300PLK+¥¸œÅ‡˜·Áœ) ®¦º ° pCHIB1
(pBluescript II KS+¥¸œÅ‡˜·Áœ) ‹¹Šµ¤µ¦™Á¦¸¥Š¨Îµ—´ segregrational stability ŗo—´Šœ¸Ê pHYB43 < pHY300PLK