本文選題：降鈣素原 + 原核表達(dá)�。� 參考：《臨床檢驗(yàn)雜志》2017年03期

【摘要】：目的構(gòu)建人降鈣素原(procalcitonin,PCT)原核表達(dá)載體,獲得高純度PCT重組蛋白。方法根據(jù)NCBI上PCT基因序列設(shè)計(jì)引物,利用PCR技術(shù)擴(kuò)增PCT基因,構(gòu)建PCT/p ET-22b(+)重組表達(dá)載體,轉(zhuǎn)化至大腸埃希菌(E.coli)BL21中誘導(dǎo)表達(dá);采用鎳柱親和層析法純化重組蛋白,用western blot和膠體金法對(duì)其進(jìn)行鑒定。結(jié)果瓊脂糖凝膠電泳結(jié)果顯示PCR擴(kuò)增產(chǎn)物約為350 bp。同源性比對(duì)分析結(jié)果表明,PCT基因片段(348 bp)成功插入p TG19-T載體,未出現(xiàn)堿基突變。用Bam HⅠ和HindⅢ對(duì)重組表達(dá)載體雙酶切,得到約為350 bp和5 500 bp片段。SDS-PAGE電泳顯示PCT重組蛋白以可溶性形式存在,Mr約14 000,經(jīng)鎳柱親和層析法純化后即可獲得。western blot和PCT膠體金法結(jié)果呈陽(yáng)性,顯示融合蛋白含組氨酸標(biāo)簽(His-tag),成功表達(dá)出PCT重組蛋白。結(jié)論應(yīng)用重組DNA技術(shù)成功構(gòu)建PCT基因融合重組表達(dá)載體,通過(guò)蛋白質(zhì)表達(dá)純化技術(shù)獲得PCT融合蛋白。
[Abstract]:Objective to construct a prokaryotic expression vector of procalcitonin PCT (PCT) and obtain a high purity PCT recombinant protein. Methods according to the sequence of PCT gene on NCBI, the PCT gene was amplified by PCR. The recombinant expression vector PCT / p ET-22b () was constructed and transformed into E. coli BL21 to induce expression, and the recombinant protein was purified by nickel column affinity chromatography. It was identified by western blot and colloidal gold method. Results agarose gel electrophoresis showed that the PCR amplification product was about 350 BP. The results of homology analysis showed that pTG19-T vector was successfully inserted into pTG19-T vector without base mutation. The recombinant expression vector was digested with Bam H 鈪，

本文編號(hào)：2028546