癌睾丸抗原OY-TES-1氨基端截短蛋白的基因克
发布时间：2018-08-12 11:49
【摘要】： 目的:构建OY-TES-1氨基端截短蛋白(OY-TES-1-N)重组质粒、体外表达OY-TES-1-N蛋白,对表达产物进行纯化和鉴定,为OY-TES-1后续研究奠定基础。 方法:(1)提取人睾丸组织总RNA,逆转合成cDNA;(2)扩增OY-TES-1氨基末端268(A6-R273)个氨基酸(AA)的cDNA序列;(3)将PCR扩增产物插入原核表达载体pMAL-C2,构建pMAL-C2-OY-TES-1-N重组质粒并转化DH5a菌;(4)通过蓝白斑筛选、DNA测序筛出正确的pMAL-C2-OY-TES-1-N重组质粒,再将其转化TB_1菌进行表达;(5)用IPTG对重组菌进行诱导表达,并对IPTG浓度和加入时机以及诱导温度和诱导时间进行优化;(6)通过SDS-PAGE电泳鉴定表达的MBP-OY-TES-1-N融合蛋白;(7)融合蛋白通过Amylose-resin亲和层析柱纯化,将纯化的MBP-OY-TES-1-N蛋白进行Western Blot初步鉴定及蛋白质串联质谱分析鉴定。 结果:重组质粒pMAL-C2-OY-TES-1-N测序与已知序列相同;成功诱导表达出融合蛋白MBP-OY-TES-1-N。确定了pMAL-C2.OY-TES-1-N体外表达的优化条件:37℃振荡培养3小时,加入IPTG(终浓度为0.3mmol/L),然后在32℃振荡培养4小时;蛋白质飞行质谱仪分析所得的肽段与目的蛋白相符。 结论:成功构建了pMAL-C2-OY-TES-1-N重组质粒,并高效表达了MBP-OY-TES-1-N融合蛋白。为后续OY-TES-1基因的研究打下基础。
[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，
本文编号：2178966
[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，
本文编号：2178966
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