【摘要】： 目的:構(gòu)建OY-TES-1氨基端截短蛋白(OY-TES-1-N)重組質(zhì)粒、體外表達(dá)OY-TES-1-N蛋白,對(duì)表達(dá)產(chǎn)物進(jìn)行純化和鑒定,為OY-TES-1后續(xù)研究奠定基礎(chǔ)。 方法:(1)提取人睪丸組織總RNA,逆轉(zhuǎn)合成cDNA;(2)擴(kuò)增OY-TES-1氨基末端268(A6-R273)個(gè)氨基酸(AA)的cDNA序列;(3)將PCR擴(kuò)增產(chǎn)物插入原核表達(dá)載體pMAL-C2,構(gòu)建pMAL-C2-OY-TES-1-N重組質(zhì)粒并轉(zhuǎn)化DH5a菌;(4)通過藍(lán)白斑篩選、DNA測(cè)序篩出正確的pMAL-C2-OY-TES-1-N重組質(zhì)粒,再將其轉(zhuǎn)化TB_1菌進(jìn)行表達(dá);(5)用IPTG對(duì)重組菌進(jìn)行誘導(dǎo)表達(dá),并對(duì)IPTG濃度和加入時(shí)機(jī)以及誘導(dǎo)溫度和誘導(dǎo)時(shí)間進(jìn)行優(yōu)化;(6)通過SDS-PAGE電泳鑒定表達(dá)的MBP-OY-TES-1-N融合蛋白;(7)融合蛋白通過Amylose-resin親和層析柱純化,將純化的MBP-OY-TES-1-N蛋白進(jìn)行Western Blot初步鑒定及蛋白質(zhì)串聯(lián)質(zhì)譜分析鑒定。 結(jié)果:重組質(zhì)粒pMAL-C2-OY-TES-1-N測(cè)序與已知序列相同;成功誘導(dǎo)表達(dá)出融合蛋白MBP-OY-TES-1-N。確定了pMAL-C2.OY-TES-1-N體外表達(dá)的優(yōu)化條件:37℃振蕩培養(yǎng)3小時(shí),加入IPTG(終濃度為0.3mmol/L),然后在32℃振蕩培養(yǎng)4小時(shí);蛋白質(zhì)飛行質(zhì)譜儀分析所得的肽段與目的蛋白相符。 結(jié)論:成功構(gòu)建了pMAL-C2-OY-TES-1-N重組質(zhì)粒,并高效表達(dá)了MBP-OY-TES-1-N融合蛋白。為后續(xù)OY-TES-1基因的研究打下基礎(chǔ)。
[Abstract]:Aim: to construct the recombinant plasmid of OY-TES-1 amino terminal truncated protein (OY-TES-1-N) and express OY-TES-1-N protein in vitro. Methods: (1) Total RNAs of human testis were extracted to reverse the synthesis of cDNAs; (2) the cDNA sequence of amino terminal 268 (A6-R273) amino acid (AA) of OY-TES-1 was amplified; (3) the PCR amplification product was inserted into the prokaryotic expression vector pMAL-C2, and the recombinant pMAL-C2-OY-TES-1-N plasmid was constructed and transformed into DH5a bacteria. The correct recombinant plasmid of pMAL-C2-OY-TES-1-N was screened by sequencing. (5) the recombinant strain was induced to express by IPTG. The concentration and timing of IPTG, induction temperature and induction time were optimized. (6) the expressed MBP-OY-TES-1-N fusion protein was identified by SDS-PAGE electrophoresis; (7) the fusion protein was purified by Amylose-resin affinity chromatography. The purified MBP-OY-TES-1-N protein was identified by Western Blot and tandem mass spectrometry. Results: the sequence of the recombinant plasmid pMAL-C2-OY-TES-1-N was the same as the known sequence, and the fusion protein MBP-OY-TES-1-Nwas successfully induced. The optimal conditions for the expression of pMAL-C2.OY-TES-1-N in vitro were determined as follows: shaking culture at 37 鈩，

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