docتولید آنتی بادی تک زنجیرهای انسانی شده ضد مارکر CD20 در E.coli
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تولید آنتی بادی تک زنجیرهای انسانی شده ضد مارکر CD20 در E.coli
ƱŚººŬƳŻƾĪºƃżěƭƺºƬƗƵŚĮºƄƳřŵƾºƄƷƹĦěƾºưƬƗƽƶºƬŬƯ ÏÎŚţÎÏšŚŰƠƇÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 E.coliŹŵCDÏÍźƧŹŚƯŶƋƵŶƃƾƳŚƀƳřƽřƵźǀŬƳŻƦţƽŵŚŝƾŤƳōŶǀƫƺţ ÑŵřĦƳƽŹƹŚųƾƬƘƳŚƌƯŹźŤƧŵÐƾƌǀƟŹƺĜƴǀƀůƾƬƗŶưŰƯźŤƧŵÏŚǀƳũźƟ źƠƇźŤƧŵÎƵŵřŻŶưůřƵŶǀůƹ vahideh_ ahmadzadeh @ yahoo .com ƾſŚƴƃ ŢƀƿŻ ƵƹźĭƱřźƸţšŚƤǀƤŰţ ƹ ƭƺƬƗ Ŷůřƹ ƾƯLjſř ŵřŻō ƵŚĮƄƳřŵƱřźƸţ ƩƹƺƀƯ ƽƵ ŶƴƀƿƺƳ ÖÏÒÏÎƁźƿŸěÖÏÏ ÏÖŢƟŚƿŹŵ ƵŶǀĪģ ƱŚºƯŹŵƽřźºŝƵŵźŤƀºĭŹƺƏƶŝƵŵƺŝ CDÏÍ ƱĥƾŤƳōŶƋźŝĨƿźưƿŚĩ ƩŚƳƺƬĩƺƳƺƯƽŵŚŝƾŤƳō Rituximab ŜƯƾƀƧƺţŹ ƝŶƷƹƶƴ ǀ ƯŻ ŹŵƪƨƄºƯƱřƺºƴƗƶºŝƵŹřƺºưƷƾºƃƺƯƪƯŚƧƽŚƷƽŵŚŝƾŤƳōƾƿřŻƾƴưƿřƹīŹżŝżƿŚſŵƺūƹƲƿřŚŝ ŵŵźĭƾƯƵŵŚƠŤſř BƽŚƷƩƺƬſ ƽŚƯƺƠƴƫ ƢºǀƤŰţƲƿřŻřƝŶƷŵƺưƳƵŵŚƠŤſřƵŶƃƾƳŚƀƳřƽŵŚŝƾŤƳōĨģƺĩšŚƘƐƣŻřƱřƺţƾƯƪĪƄƯƲƿřƪůŢƸūŢſřƵŵƺŝ ŭźƐƯƾěřźţƺƳƺưƿř ŢſřBƩƺƬſŚŝƎŞţźƯƽŚƷƽŹŚŬƴƷŚƳƱŚƯŹŵŹŵƱōŵźŝŹŚĩŹƺƔƴƯƶŝƾƳŚƀƳřƽřƵźǀŬƳŻĨţƽŵŚŝƾŤƳōŶǀƫƺţ ƶºŝƾºƃƺƯCDRƾůřƺºƳCDR-graftingƁƹŹŻřƵŵŚƠŤºſřŚºŝƵŶƃƾƳŚƀƳřRituximab ƽŵŚŝƾŤƳōƾůřźƏ ŹƺƔƴƯƶŝ ƾ ſŹźŝ ƁƹŹ ƾƫřƺţŶƳŶƃƪƤŤƴƯƾƃƺƯƽŚŤưƷŚŝŢƷŚŞƃƲƿźŤƄǀŝƽřŹřŵŜŴŤƴƯƾƳŚƀƳř Germline ƽŚƷƲǀƫƺŝƺƬĭƺƳƺưƿřFrameworkƽŚƷŢưƀƣ ƂƴºĩřƹƹSDS-PAGEƁƹŹŚºŝŜǀĩźţƺƳƽŵŚŝƾŤƳōƱŚǀŝƱřżǀƯŶƿŵźĭƆǀƬŴţžĜſƵŶƃƱŚǀŝE.coli ŹŵƵŶƃƾůřźƏƽŶǀţƺŘƬĩƺƳ ŢƟźĭŹřźƣƾŝŚƿŻŹřŵŹƺƯDot blotĨǀƴĪţŻřƵŵŚƠŤſřŚŝRajiƾƫƺƬſƽƵŵŹŚŝƱō ƹ IGHVÎ-ÑÓ*ÍÐƽŚºƷƲǀƫƺºŝƺƬĭƺƳƺưƿřCDR GraftingƭŚºŬƳřŹƺºƔƴƯƶºŝƾƳŚƀºƳřFrameworkƾůřƺºƳśŚºŴŤƳřŹŵ ŚºƷ ƶ ºŤƟŚƿ ƽŵŚºŝ ƾºŤƳō ŵźºĩƆŴƄºƯSDS-PAGEƁƹŹŚºŝƱŚǀŝ ƾſŹźŝŶƳŵřŵƱŚƄƳƶǀƫƹřƽŵŚŝƾŤƳōŚŝřŹŢƷŚŞƃƲƿźŤƄǀŝ IGKVÎ-ÐÖ*ÍÎ ƂƴºĩřƹRajiƽŚºƷƩƺƬºſŮƐſŹŵƵŶƃƱŚǀŝCDÏÍƱĥƾŤƳōƶŝŢŞƀƳŜſŚƴƯƾŤǀƴǀƟřŚŝƵŶƿŵźĭƱŚǀŝE.coliŹŵ LJŚŝ ŢƔƬƛ Śŝ ƽŶǀƫƺţ ŵřŵƱŚƄƳ ŹřźºƣƝŶƷƽřźŝƾƳŚƯŹŵƁƹŹƱřƺƴƗƶŝƵŶƿŵźĭƪƈŤƯCDÏÍźĩŹŚƯƶŝŶƳřƺţƾƯƵŶƃŶǀƫƺţƾƳŚƀƳřƽřƵźǀŬƳŻĨţƽŵŚŝƾŤƳō ƽź ǀ ĭ ƶŬ ǀ ŤƳ ŵƹŹŹŚĩƶŝƎŞţźƯƽŚƷƱŚƏźſƱŚƯŹŵŹŵƱĥƾŤƳōƲƿřƱŵřŵ CDÏÍ ƽř Ƶź ǀ ŬƳŻĨţ ƽŵŚŝƾ ŤƳōƱŵźĩ ƾ ƳŚƀƳř ƽŶ ǀ ƬĩƱŚĭĥřƹ ƱŚººƯŹŵŹŵƮºƸƯƽŚºƷźƧŹŚƯŻřƾººƨƿŶºƴƿŚưƳƾºƯƩŚºưƗřřŹ ƲºƿřÏƹÐŢºſř CDÏÍNHLƲǀĪģƺººƷźºǀƛƭƺºƠƴƫ ƵŶºƃŵŚºŬƿřƚƫŚºŝƹŻŚſƂǀěB ƽŚƷƩƺƬſŮƐſŹŵźĩŹŚƯ ƶƯŶƤƯ ºƸū ƾ޺ſŚƴƯ ƹ Ƣǀƣŵ Źřżŝř ƩŚƳƺƬĩƺƳƺƯ ƽŚƷƽŵŚŝƾŤƳō Ţ ƶºŝƩŚƈºţřŚºŝƹÎƵŵƺºŝƾƳŚºƯŹŵƹ ƾĪƿĥƺƫƺǀŝ šŚƤǀƤŰţ ŵƺųšřźŧřƽŹƺƯƺţƽŚƷƩƺƬſŮƐſŹŵƞƬŤŴƯƽŚƷźƧŹŚƯ ƾſŚƴƃ ŢƀƿŻ ƵƹźĭƱřźƸţšŚƤǀƤŰţ ƹ ƭƺƬƗ ŶůřƹƾƯLjſř ŵřŻō ƵŚĮƄƳřŵƾƫƺĪƫƺƯƹƾƫƺƬſƾſŚƴƃŢƀƿŻƾƈƈŴţƽřźŤĩŵƽƺŬƄƳřŵÎ żƿźŞţ ƾƨƃżě ƭƺƬƗ ƵŚĮƄƳřŵ ƾƿƹŹřŵ ƽŵźŝŹŚƧ šŚƤǀƤŰţ żƧźƯŹŚǀƄƳřŵƽĥƺƫƺƴĪţƺǀŝƾƈƈŴţƽřźŤĩŵÏ ƭƺƬƗ ƵŶƨƄƳřŵ ƾſŚƴƃ ŢƀƿŻ Ƶƹźĭ ŵŚŤſřƽĥƺƫƺǀŝƺƿŵřŹƾƈƈŴţƽřźŤĩŵÐ żƿźŞţ ƵŚĮƄƳřŵ ƾƘǀŞƏ ŵřŻō ƵŚĮƄƳřŵ ƾſŚƴƃ ŢƀƿŻ Ƶƹźĭ ŵŚŤſřƾƷŚǀĭ ƽĥƺƫƺƿżǀƟƾƈƈŴţƽřźŤĩŵÑ ƱřźƸţšŚƤǀƤŰţ ƹ ƭƺƬƗ Ŷůřƹ ƾƯLjſř ƵŶƃƾƳŚƀƳřƽř ƵźǀŬƳŻƦţƽŵŚŝ ƾŤƳōŶǀƫƺţ źƿŸºěƝŚºƐƘƳřƽŶǀŤĜěƶƘƐƣĨƿƎſƺţƶĩ Ƶŵƺŝ(VH)ƲǀĮƴſ ƾƿŚưºſLjěƽŚºƷƩƺƬºſƶŝ BƽŚƷƩƺƬſƪƯŚƨţƭŚĮƴƷŹŵƹ ŶºƳřƵŶºƃƪƈºŤƯƮºƷƶŝŢſř ƽźƯ ÎÒ ƩƺƏƽřŹřŵLJƺưƘƯ ŚƄºƛŻř CaÏ ŹƺºŞƗƵŶºƴƴƧƮǀƔƴţƾƫƺƬſŮƐſƦƿźưǀŤƫƺƯ ƹ ƶƴǀƯōŶǀºſř ÒÍ Śºţ ÏÒƾºŞƿźƤţƩƺºƏƶºŝźºĪƴǀƫƱřƺºƴƗƶŝ ƭźºƟƲƿřŢſř(GlyÑ Ser)ÐƾƿŚţÎÒŶǀŤĜěźĪƴǀƫƲƿźţƩƺưƘƯ Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 ˺˼ ƾƳŚǀŝƮŤƀǀſŹŵƾŤůřŹƶŝƵŵƺŝFCƽƶƤƐƴƯŶƣŚƟ ƽŵŚŝƾ ŤƳōŻř ŢºƸūŹŵƾĪǀŤƳĥšřźǀǀƜţƱŚĪƯřƹŢſřŶǀƫƺţƪŝŚƣE. coli ƹƩŚƈºţřƪƿŚưţƂƿřżƟřƱƺĤưƷƽŵŚŝƾ ŤƳōšŚǀƇƺƈųƽŚƤţŹř žƨƬĜººưƧŻřƾºƿżū CDÏÍÑŵƹŹƾºƯƲǀººŝŻřƱōƱŚºǀŝ źŧƺºƯ B ƽŚƷƩƺƬſźǀŨĪţƹŢǀƫŚƘƟƮǀƔƴţŹŵLJŚưŤůř Ƶŵƺŝ ƽŚºƷƩƺƬºſŻřƪƤŤƀºƯƽŵŚºŝƾºŤƳōŲºſŚěŵŚŬƿřŹŵƹŢſř ƩŚºƳƺƬĩƺƳƺƯƽŚºƷƽŵŚºŝƾŤƳōÒŵŹřŵƾưƸƯƂƤƳ(TI) 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ƪºƇŚůŢƳŚţŚƳźěƺſ žĜſŶƳŶƿŵźĭ ƾſŹźŝ SDS-PAGE ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ ƵŶƃƾƳŚƀƳřƽř ƵźǀŬƳŻƦţƽŵŚŝ ƾŤƳōŶǀƫƺţ ÎƩƹŶūŶƳŶƿŵźĭśŚŴŤƳřRituximabĨƿźưƿŚĩƽŵŚŝƾŤƳō ŚºŝƶŝŚƄºƯChothia ˺˾ CanonicalStructuresƽřŹřŵƹƲǀĮƴſ ƾƳŚƀƳřGermline ƽŚƷ ƲǀƫƺŝƺƬĭƺƳƺưƿřƲǀĮƴſƹƦŞſƽŚƷƵźǀŬƳŻCDRCanonicalClassƾŝŚƿŻŹřÎƩƹŶū Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 Cothia et al. (ÎÖÖÏ)ƽƶƬǀſƹƶŝƵŶƃƲǀǀƘţIGKVÎ-ÐÖ*ÍÎ ƹIGHVÎ-ÑÓ*ÍÐ Canonical Class ? Similar to class 1/10A but:H102 (Chothia Numbering) = L (allows: YHVISDG) ? Similar to class 2/10A, but:H50 (Chothia Numbering) = I (allows: REWYGQVLNKA) H71 (Chothia Numbering) = R (allows: VAL) CDR Canonical class CDR H1 2/11A L1 H2 1/7A L2 1/9A L3 ? 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pETÏÏbscFvŶǀưſLjěŻřƶƘƐƣ ƵŶƃƮƌƷŜǀĩźţƺƳŶǀưſLjěÏƵŶƄƳƮƌƷŜǀĩźţƺƳ źĩŹŚƯżƿŚſDNAM ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ ƵŶƃƾƳŚƀƳřƽř ƵźǀŬƳŻƦţƽŵŚŝ ƾŤƳōŶǀƫƺţ IGKVÎ-ÐÖ*ÍÎƾƳŚƀººƳřGermlineƽŚººƷƲ ǀƫƺººŞƬĭƺƳƺưƿř ƾůřƺººƳƽƵŶººƳźƿŸěƱřƺººƴƗƶººŝŜººŴŤƴƯIGHVÎ-ÑÓ*ÍÐ ƹ ƱŵƹżºƟř Żř ŶºƘŝźºƔƳ ŵŹƺƯ ƲǀŘţƹźěŶƿŵźĭƆŴƄƯƹŶƃ ƺºƬǀĩ ÏÓ ƽƵŵƹŶºŰƯ Źŵ ƾŰºƋřƹ ŶºƳŚŝ šŹƺºƇƶŝIPTG ƽƶººǀůŚƳƹŵźººƷŹŵLJŚººŝƽĥƺººƫƺưƷƽřŹřŵƾººƃƺƯCDR ŻřƪºƇŚůŢƳŚţŚƳźěƺºſŹƺºŞƗŢſřƵŶƿŵźĭŶǀƫƺţ ƾƳƺŤƫřŵ ƾƳŚƀºƳřŵŹřƺºƯŻřƽŹŚǀƀºŝŹŵŶºƳŵƺŝƶºǀƫƹřƽŵŚŝƾ ŤƳōŚŝƶŝŚƄƯ ƵŶºƃƽźºǀĭƵŻřŶƳřÓÓÓ g/ml ŢƔƬƛ Śŝ ƹ LJŚŝ ƅƺƬųŹŵ ŚºŝƾƃƺƯƮƸƯƽŚƷƶƴ ǀƯřŶǀſřƒƠůŚŝƲƿřźŝŚƴŝŢſřƝŶƷƱĥ ƆŴƄƯ ƺƬŝƾſŚƯƺƧ Śŝ ƵŶƃ ƽżǀƯō ĬƳŹ SDS-PAGE Ʃĥ ƽŵŚºŝƾºŤƳōƱŵźĩƾƳŚƀƳřŹŵƂƳřŹŚĪưƷƹRemy Robert ƾºŤǀƴǀƟřƽřŹřŵƽŶºǀƫƺţŜºǀĩźţƺƳƽŵŚºŝƾºŤƳō ŵźĩƆŴƄƯ ƾƳƺƳŚºĩŹŚŤųŚºſƹƾƃƺƯƽŚŤưƷŚŝ CDRs ƹFrameworks Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 ˺̀ ƾºŤƳōƶºŝŢŞƀƳŚƷƱō ƾŤǀƴǀƟřƂƷŚĩŚŝƵřźưƷŚƷƽ ŵŚŝƾ ŤƳōƱŵźĩ ŵƺºưƳƝźƏźŝřŹƪĪƄƯƲƿřƱřƺţƾ ƯƾŤƄĭźŝƱƺǀſŚţƺƯƩŚưƗř ƢƏŚººƴƯŹŵƖººƣřƹƾº ƃƺƯƮººƸƯƶººƴǀƯřƽŚººƷŶǀº ſřWO-Ï ŶƳŵźĩƒƠůƵŶƃ ƾůřźƏ ƾƿŚƸƳ ƽŵŚŝƾŤƳōŹŵřŹ Vernier ƽŚººƷƽŵŚººŝƾººŤƳōĨººģƺĩšŚººƘƐƣƲǀººŝŹŵÎÒÏÓƹÏÔ źºƔƳ ŵŹƺƯ ƲǀŘţƹźě ƆǀƬŴţƶŝ źŬƴƯƱƺŤſŻřƱƺǀſŚĪǀƳƺſ Źŵ ƾƳƺºŤƫřŵƺƬǀĩ ÏÓ ŶºƳŚŝ šŹƺƇƶŝ ƶƧ ŶƿŵźĭęřŹŵƺƳŚƳŚŝ Dot blotĨºǀƴĪţƵŵŚƠŤººſřŚººŝżǀƫŚººƳōÐƪĪººƃŶººƿŵźĭ ŮƐºſŹŵ ƵŶºƃƱŚºǀŝ CDÏÍƱĥƾºŤƳōƶºŝŢŞƀºƳŜºſŚƴƯ ÑƪĪƃŶƳŵƺŝRajiƽŚƷƩƺƬſ ƖƿźſŢƧźůƾƿŚƷƩƺƨƫƺƯƲǀƴģŵŹřŵŚƷƽŹŚưǀŝƆǀŴƄţ ŦŰŝ ƶº ǀƬƗƽŵŚº ƿŻƾº ƃƺƯƽŚººƷƽ ŵŚººŝƾ º ŤƳōƱƺººƴĩŚººţƶººģźĭř ŹŵƽŹřŶºƷŚĮƳƂƷŚƧƮƧƾŤǀƀǀƳĥƺƳƺưƿřƝŶƷƽŚƷŹƺƯƺţ īŹżººŝżƿŚººſƹŚººƷƽ ŵŚººŝƾ º ŤƳōƲº ƿřŶººƋźººŝƾº ƴưƿřŲººſŚě źƋŚů ƽƶƘƫŚƐƯŹŵÎ ŶƴŤƀƷŵŚƿŻŽŚǀƤƯŚŝŢųŚſźŤưƧ ƲººƿřźººŝƶººŞƬƛƽřźººŝŢººſřƵŵźººĩŵƹŶººŰƯřŹƾƈººǀŴƄţ šŹƺƇƶŝŚƷƱĥƽźǀĭŢƸūšŚƘƫŚƐƯƢŞƏŶƃƶŤųŚſƱō ƾƳŚƀºƳřƽřźºŝ ƽŵŶºƘŤƯ ƽŚºƷƁƹŹ ŢƟŚƿ ũřƹŹźŤưĩ ƾƫƺĪƬƯ ƹƱŚºƯŹŵ šŚƘƫŚƐƯŹŵřŹŵźŝŹŚƧƲƿźŤƄǀŝ scFv ŜǀƧźţƺƳ ŹŵśƺººųŷƺººƠƳšŹŶººƣƽřŹřŵƶŤººƃřŵƱƺººųƱŚººƿźūŹŵ ŵŚºŬƿř ŶºƴƳŚƯƽŵŹřƺºƯŚºƯřŶƳřƵŶƃŶǀƫƺţ ƽŵŶƘŤƯ ƽŚƷƱĥ ƾ ŤƳō ƶºƴƿżƷƹƱŵƺºŝƱŚºſō ƝŶºƷźºǀƛƽŚƷƱŚĭŹřźĮƿŵƹƶǀƬƧ ƹƾƳŚºƯŹŵŶºƇŚƤƯŢºƸūŚºƷƱōŵźŝŹŚºƧƪºƯŚƧƽŚƷƽŵŚŝ ƾŤƳō ŽŚºſřźºŝscFv ƹśŚºŴŤƳřVH-linker-VLƽźǀĭŢƸū ƽŚºƷƱŻƹ ŚºŝƵŶºƃ ƾƳŚƀºƳř ƽŚºƷƽ ŵŚŝƾ ŤƳō Ŷǀƫƺţ ŚƷŢ ƿŵƹŶŰƯ ƽřŹřŵ scFvƶŤºƃřŵ ƽźºŤƸŝƱŚºǀŝƱřżǀƯVH-linker-VL ŚººƷƱōƲ ǀººŝŻřƶººĩŵƹŹƾ ººƯŹŚººĩƶººŝŚººƷƽ ŵŚººŝƾ ººŤƳōƱŵźººĩ ŹƺƔƴƯƶŝ ŶƷŵƾƯƱŚƄƳE.coliŹŵřŹƽźŤƄǀŝƾŤǀſƺŤǀƀĩƺţ ƾůřƺºƳƶºŝ ƾºƃƺƯ CDRƾůřƺºƳƁƹŹƲºƿřŹŵŵŵźºĭƾ Ư ƲǀƀºĩƺţƲŤºƃřŵšŹƺºƇŹŵƹźºŤƸŝƩŚƈţřŹƺĩŸƯźĪƴǀƫ pETÏÏbƾƳŚºǀŝ ƪºƯŚů E.coliƾƳŚºǀŝ ƮŤƀºǀſ ŹŵƱĥ ƱŚºǀŝ ŹŚǀƀŝ ŹƺţƺƯƹźěpET ƮŤƀǀſŹŵŢƟźĭ Źřźƣ ƵŵŚƠŤſř ŵŹƺƯ ƶŤųŚƴºƃƩƹřŶºŤƯƽŚƷƁƹŹŻřƾĪƿƱřƺƴƗƶŝ CDR-Grafting ŶƳƺººƃƾººƯƵŵŻŶººƳƺǀěƾƳŚƀººƳřƽŵŚººŝƾººŤƳōFramework ƁƹŹŻřƂƳřŹŚºººººººĪưƷƹWei-Gang HuÏÑƹÏÒ E.coliŻřźºưǀƬěRNAƎºſƺţ T Ô ĥŚºƟ ƾƇŚƈŤųř ƹƽƺƣ ƩŚºƳƺƬĩƺƳƺƯƽŵŚºŝƾ ºŤƳōƱŵźºĩƾƳŚƀƳřƽřźŝCDR-grafting Ĩºţ ƽŚºƷƽŵŚºŝƾºŤƳō ƽƶºƴǀƯŻŹŵŦºŰŝƪºŝŚƣ šŚƗƺƋƺƯ ƽƵŶºƳźƿŸěƱřƺƴƗƶŝřŹ ƾƳŚƀƳř GermlineƽŚƷƲ ǀƫƺŝƺƬĭƺƳƺưƿř ƽŻŚºǀƳ ƹƵŵƺŝƽŶǀƠƫƺſ ƽŵ ƽŚƷŶƳŚŝ ŶƣŚƟ ƹ ƶƬƿŻƺĪǀƬĭźǀƛ ƽřŹřŵƲƿřźŝŚºƴŝƵŵƺºŝĨǀţŚƯƺºſ ƽŚºƷƱƺ ǀºſŚţƺƯŶºƣŚƟ ƾƳŚƀƳř ƲƿźººţƮººƸƯŻřscFvƱŚº ǀŝŢº ǀƠǀĩŵƺººƃƾººưƳƾƀººƿƺƳƹŹ ŚºººƷƱōƶºººƘƫŚƐƯƲ ºººƿřŹŵŶºººƳŵźĩƵŵŚƠŤºººſřRicinŶºººƋ ƾºƴǀŘţƹźěƕřƺºƳřŻřscFvƶĪƴƿř ƶŝ ƶūƺţ ŚŝŶƃŚŝƾƯƵźǀŬƳŻ Germline ƽŚºƷƲ ǀƫƺŞƬĭƺƳƺưƿřŶƴŤƟźĭźƔƳŹŵCDRƾůřƺƳ ƱƺǀºſLjǀſō ƱƺǀſLjǀŤſřźǀƔƳ ƶưūźţ Żř ŶƘŝ šŚůLjƇř ƶŝ Ƣº ǀƤŰţƲº ƿřŹŵŶƴŤƀººƷƱŚƀººƳřŹŵƮººĩƾººƿřŻƾ º ƴưƿřŢº ǀƬŝŚƣ ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ ƱřŹŚƨưƷƹƵŵřŻŶưůřƵŶǀůƹ ˺́ ŜºſŚƴƯƾºŤǀƴǀƟř ƽřŹřŵƵŶƃ ƾƳŚƀƳřŜǀĩźţƺƳ ƽŵŚŝƾŤƳōŵřŵ E.coli źºǀƔƳƾƿŚƿźŤĩŚŝƮŤƀǀſŹŵŵŹřŶƳƱƺǀſLjǀƀƧƺŝźƧ ŚºŝƶƀºƿŚƤƯŹŵřŹŵƺºų ƾºŞǀĩźţƪºǀƯƲƿřźŝŚºƴŝƵŵƺŝRaji ƎºſƺţCTLAÑ (CDÎÒÏ)ŶºƋźºŝƵŶƃƾƳŚƀƳřƽřƵźǀŬƳŻ Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 ƽŚºƷƩƺƬºſŮƐºſŹŵ ƵŶºƃƱŚǀŝ CDÏÍ Ʊĥ ƾŤƳōƶŝŢŞƀƳ ĨººţƽŵŚººŝƾº ŤƳō.ÏÕÐÍŢººſřƱŚº ǀŝƪººŝŚƣƾŝƺººųƶººŝ ŢſřƵŵƺưƳƒƠůĨƿźưƿŚĩƶǀƫƹřƽŵŚŝƾŤƳō ŶºǀƫƺţƾƿŚºƿźŤĩŚŝƾƳŚǀŝƮŤƀǀſƽƶƬǀſƹƶŝ ƂƳřŹŚĪưƷƹƲģ ƽźǀĭƶŬǀŤƳ ƽŵŚºŝƾ ºŤƳō ƶºƧ ŵřŵƱŚƄºƳźƋŚůƶƘƫŚƐƯŪƿŚŤƳ ƾƬĩŹƺƏƶŝ ƾƳŚºǀŝƩƺƬºſpETÏÏbƾƳŚºǀŝŹƺŤĩƹ ƽŚƷƾĭĦƿƹƲǀƴĤưƷ E.coliƽźŤĩŚºŝƾƳŚºǀŝƮŤƀºǀſŹŵƵŶƃ ƾƳŚƀƳř ƽřƵź ǀŬƳŻĨţ ƾºŤƳōƾƿŚºſŚƴƃƶºŝŹŵŚºƣƽŻŚºſƆƫŚºųŻřžºě ƵŶƃŶǀƫƺţ ƲƿřźŝŚºƴŝŢſř Raji ƽŚƷƩƺƬſŮƐſŹŵƵŶƃƱŚ ǀŝCDÏÍ Ʊĥ ƾºŞǀĩźţƪºǀƯƱŵƺºŝřŹřŵƪºǀƫŵ ƶºŝ ŜºǀĩźţƺƳ ƽŵŚŝƾŤƳōƲƿř ŹŵƮĩ ƾƿřŻƾƴưƿřšŹŶƣƹƝŶƷƱĥ ƾŤƳōƶŝŢŞƀƳŜſŚƴƯ ŹřźºƣƝŶºƷ ƽřźºŝ ƾƳŚºƯŹŵƁƹŹƱřƺƴƗ ƶŝŶƳřƺţƾƯƱŚƀƳř ŵƹŹŹŚĩƶŝŚƯƺƠƴƫƱŚƏźſŹŵźĩŹŚƯƲƿřƱŵřŵ ƹźºƔƳŵŹƺƯƱĥšŚǀƇƺƈųƶŝƶūƺţŚŝƲƿřźŝŚƴŝÐÎ Ŷƃ ƱŚºǀŝŵƺºŝ ŜſŚƴƯƲǀŘţƹźěƱŚǀŝ ƽřźŝ BLÏÎƶƿƺſ E.coli ŹřŶºƤƯźŨĩřŶºůƶºŝIPTG ƎſƺţŚƤƫřŻřŶƘŝŢƗŚſƶſŹŵ ƶƐºſřƹ ŚŝŜǀĩźţƺƳƽŵŚŝƾŤƳōƱĥ ƱŚǀŝ ƽƶŬǀŤƳŶǀſŹŵƺų ƲǀŘţƹźººěŶººǀƫƺţpelBƽƶƳŚƄººƳŶºǀŤĜěƹ pETÏÏbƪººƯŚů Źŵ ƽŶºƳŚŝ šŹƺƇƶŝ řŹ ŵƺų ƶƧ ŵƺŝƮſLjěƽźěŹŵƩƺƬŰƯ ŹŵÓHis-tagźººĭƱŚƄººƳŵƺººūƹŵřŵ ƱŚƄººƳSDS-PAGE ƾƟřźĭƺţŚƯƹźĩŻřƵŵŚƠŤſřŚŝƱōƆǀƬŴţƝŶƷ ƽŵŚŝƾ ŤƳō ƽŚƸŤƳř ƱŚƄºƳ Dot BlotũŚºŤƳŵƺưƳƪǀƸƀţřŹNi-NTAƾƬƿŚưţ References cycle progression of B lymphocytes. Immunol 1-Pucca MB, Bertolini TB, Barbosa JE, Ribeiro Today. 1994; 15: 450-454 SV, monoclonal 5- Kuijpers TW, Bende RJ, Baars PA, et al. CD20 antibodies: scFv patents as a marker of a new deficiency in humans results in impaired T cell- class of potential biopharmaceuticals . Braz J Pha independent antibody responses. J Clin Invest. Sci. 2011; 47: 31-38 2010; 120: 214-222 2- Liang Y, Buckley TR, Tu L, Langdon SD, 6- Schirrmann T, Meyer T, Schutte M, Frenzel A, Tedder TF. Structural organization of the human Hust M. Phage display for the generation of MS4A gene cluster on chromosome 11q12. antibodies for proteome research, diagnostics and Immunogenetics. 2001; 53: 357-368 therapy. Molecules. 2011; 16: 412-426 3- Jazirehi 7- Lonberg N. Fully human antibodies from Porto molecular GS. Therapeutic AR, Bonavida B. Cellular and signal transduction pathways transgenic mouse and phage display platforms. modulated by rituximab (rituxan, anti-CD20 Curr mAb) innon-Hodgkin's lymphoma: implications 8- Shibasaki S, Ueda M. Therapeutic antibodies in therapeutic and other proteins obtained by molecular display intervention. Oncogene. 2005; 24: 2121-2143 technologies. Recent Pat Biotechnol. 2009; 3: 4- Tedder TF, Engel P. CD20: a regulator of cell- 19-27 chemosensitization and Opin Immunol. 2008; 20: 450-459 ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 ƵŶƃƾƳŚƀƳřƽř ƵźǀŬƳŻƦţƽŵŚŝ ƾŤƳōŶǀƫƺţ ˺̂ 9- Tiller T, Meffre E, Yurasov S, Tsuiji M, single-chain Fv antibodies from Drosophila S2 Nussenzweig MC, Wardemann H. Efficient cells. Protein Eng Des Sel. 2012; 25: 59-66 generation of monoclonal antibodies from single 17- Weisser NE, Hall JC. Applications ofsingle- human B cells by single cell RT-PCR and chain variable fragment antibodies in therapeutics expression vector cloning. J Immunol Methods. and diagnostics. Biotechnol Adv. 2009; 27: 2008; 329: 112-124 502-20 10-Peterson NC. Advances in monoclonal 18- Abhinandan KR, Martin AC. Analysis and antibody technology: genetic engineering of mice, prediction of VH/VL packing in antibodies. cells, and immunoglobulins. ILAR J.2005; 46: Protein Eng Des Sel. 2010; 23: 689-697. 314-319. 19-Asano R, Nakayama M, Kawaguchi H, 11- Meerten T, Hagenbeek A. CD20-targeted Kubota T, Nakanishi T, Umetsu M, et al. therapy: a breakthrough in the treatment of non- Construction and humanization of a functional Hodgkin's lymphoma. Neth J Med. 2009; 67: 251- bispecific EGFR x CD16 diabody using a 259 refolding system. FEBS J. 2012; 279: 223-233 12- Chiu WC, Lai YP, Chou MY. Humanization 20- Foote J, Winter G. Antibody framework and characterization of ananti-human TNF-alpha residues affecting the conformation of the murine monoclonal antibody. Plos One. 2011; 6: hypervariable loops. J Mol Biol. 1992; 224: 487- e16373 499 13- Jayaram N, Bhowmick P, Martin AC. 21- MacCallum RM, Martin AC, Thornton JM. Germline VH/VL pairing in antibodies. Protein Antibody-antigen interactions: contact analysis Eng Des Sel. 2012; 25: 523-529 and binding site topography. J Mol Biol. 1996; 14- Parker AS, Zheng W, Griswold KE, Bailey- 262: 732-745 Kellogg C. Optimization algorithms for functional 22- Martin ACR. Protein sequence and structure deimmunization of therapeutic proteins. BMC analysis of antibody variable domains. antibody Bioinformatics. 2010; 11:180 engineering. Antibody Engineering. 2010; 8: 35- 15-Robert R, Streltsov VA, Newman J, Pearce 51. LA, Wark KL, Dolezal O. Germline humanization 23- de BB, Madera M, Chothia C. VH gene of a murine Aâ antibody and crystal structure of segments in the mouse and human genomes. J the humanized recombinant Fab fragment. Protein Mol Biol. 2004; 342: 131-143. Sci. 2010; 19: 299-308. 24- Hwang WY, Almagro JC, Buss TN, Tan P, 16-Gilmartin AA, Lamp B, Rumenapf T, Persson Foote J. Use of human germline genes in a CDR MA, Rey FA, Krey T. High-level secretion of homology-based recombinant monomeric murine and human humanization. Methods. 2005; 36: 35-42 approach to antibody ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 ˻˹ ƱřŹŚƨưƷƹƵŵřŻŶưůřƵŶǀůƹ 25- Kim HY, Tsai S, Lo SC, Wear DJ, Izadjoo structural variations on biological activity of anti- MJ. Production and characterization of chimeric PSMA scFv and immunotoxins targeting prostate monoclonal cancer. Anticancer Res. 2010; 30: 3373-3379 antibodies against Burkholderia pseudomallei and B. mallei using the DHFR 29- Zhou N, Paemen L, Opdenakker G, Froyen G. expression system. Plos One. 2011; 6: 19867 Cloning and expression in Escherichia coli of a 26-Makabe K, Nakanishi T, Tsumoto K, Tanaka human Y, Kondo H, Umetsu M et al. Thermodynamic immunoglobulin variable fragment (scFv). FEBS consequences of mutations in vernier zone Lett. 1997; 414: 562-566 residues of a humanized anti-human epidermal 30- Su YC, Lim KP, Nathan S. Bacterial growth factor receptor murine antibody. J Biol expression of the scFv fragment of a recombinant Chem. 2008; 283: 1156-1166 antibody specific for Burkholderia pseudomallei 27- Wei-Gang Hu, Junfei Yin, Damon Chau, exotoxin. J Biochem Mol Biol. 2003; 36: 493-498. Laurel M, Negrych, John W. Humanization and 31- Chen LH, Huang Q, Wan L, et al. Expression, characterization of an anti-ricin neutralization purification, and in vitro refolding of a humanized monoclonal antibody. Plos One. 2012; 7 single-chain Fv antibody against human CTLA4 28- Buhler P, Wetterauer D, Gierschner D, (CD152). Protein Expr Purif. 2006; 46: 95-502. gelatinase B-inhibitory single-chain Wetterauer U, Beile UE, Wolf P. Influence of ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ ƵŶƃƾƳŚƀƳřƽř ƵźǀŬƳŻƦţƽŵŚŝ ƾŤƳōŶǀƫƺţ ˻˺ Generation of Humanized Single Chain anti-CD20Antibody Marker in E.coli Ahmadzadeh V1, Farajnia S2, Hosseinpour Feizi MA3, Khavarinejad RA1 1 Dept. of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. Downloaded from zums.ac.ir at 17:47 IRST on Wednesday March 8th 2017 2 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3 Dept. of Biology, University of Tabriz, Tabriz, Iran Corresponding Author: Ahmadzadeh V, Dept. of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. E-mail: [email protected] Received: 19 May 2013 Accepted: 12 Aug 2013 Background and Objectives: Rituximab is an anti-CD20 chimeric monoclonal antibody widely used for the treatment of malignant B cells lymphoma. However, the immunogenicity of murine-derived monoclonal antibodies and the large size of full length antibodies restrict cancer immunotherapy. Humanized single chain antibodies can be a solution and a promising alternative for application in immunotherapy. The aim of this study was to produce a humanized scFv antibody for a potential use in the diagnosis and treatment of B cell lymphoma. Materials and Methods: We used a CDR graftingbased approach to design a humanized scFv gene fragment. The CDRs were grafted onto the closest human frameworks. The designed sequence was expressed in E.coli then purified. The level of expression was analyzed by SDS-PAGE and the reactivity to CD20 expressing cell line was explored by immunoblotting. Results: Similarity analyses revealed that human germline gene IGHV1-46*03 and IGKV1-39*01 have the highest homology with their murine counterparts. Analysis by SDS-PAGE exhibited a high expression level in E. coli. Reactivity to CD20 expressing Raji cells showed that the produced antibody maintained the binding capacity to human CD20 marker. Conclusion: In our study, humanized anti- CD20 scFv indicated an original antigen-binding affinity. The findings serve as a basis for the development of novel therapeutic strategies in the treatment of CD20- expressing cancers. Keywords: Humanization, Single-chain antibody variable fragment, CD20 ÎÐÖÏƽŵƹŹŷōÕÕƽƵŹŚưƃ ÏÎƽƵŹ ƹŵ ƱŚºŬƳŻƾƳŚƯŹŵƾŤƃřŶƸŝšŚƯŶųƹƾĪƃżěƭƺƬƗƵŚĮƄƳřŵƾƄƷƹĦěƾưƬƗƽƶƬŬƯ
