【摘要】：目的 構(gòu)建非糖基化、抗凝血酶激活的高分子量尿激酶原突變體。 方法 使用PCR擴(kuò)增的方法,將302位天冬酰胺(Asn302)突變?yōu)楸彼?Ala302),去掉糖基化位點(diǎn);將156位精氨酸(Arg156)突變?yōu)橘嚢彼?Lys156),去掉凝血酶作用位點(diǎn)。 結(jié)果 PCR產(chǎn)物經(jīng)瓊脂糖電泳鑒定分子量為1296Da(道爾頓,dalton)。突變體與pUC19克隆載體用HindⅢ、XbaⅠ雙酶切。二者連接后,轉(zhuǎn)化入DH5α感受態(tài)細(xì)胞(大腸桿菌)中。用藍(lán)白斑篩選法識(shí)別PCR產(chǎn)物是否與pUC19載體連接。包含PCR產(chǎn)物的克隆呈現(xiàn)白色。將白色克隆提質(zhì)粒送測(cè)序。測(cè)序正確的基因與pIPES雙表達(dá)載體連接后備用。 結(jié)論 非糖基化抗凝血酶的高分子量尿激酶原突變體構(gòu)建成功。
[Abstract]:Objective to construct a non-glycosylated, anti-thrombin-activated high molecular weight urokinase mutant. Methods using PCR amplification, 302-site asparagine (Asn302) was mutated to alanine (Ala302) to remove glycosylation sites, and 156-arginine (Arg156) was mutated to lysine (Lys156) to remove thrombin action sites. Results the molecular weight of PCR product was identified as 1296Da (Dalton, dalton).) by agarose electrophoresis. The mutant and pUC19 clone vector were digested with Hind 鈪，

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