【摘要】：旨在構(gòu)建雞熱休克蛋白70(heat shock protein 70,HSP70)表達(dá)質(zhì)粒p ColdⅡ-HSP70,誘導(dǎo)表達(dá)、純化后檢測(cè)HSP70蛋白的質(zhì)量。將目的基因擴(kuò)增后克隆至表達(dá)載體p Cold II,構(gòu)建重組載體p ColdⅡ-HSP70,在2種大腸桿菌菌株(BL21(DE3)和Rosetta(DE3)p Lys S中經(jīng)IPTG在不同溫度與不同濃度條件下誘導(dǎo)表達(dá)并純化。經(jīng)SEC-FPLC和HPLC進(jìn)行純度鑒定,采用鱟試劑檢測(cè)蛋白內(nèi)毒素含量(將HSP70蛋白按1 200、600、120倍稀釋);Western blot及質(zhì)譜方法檢測(cè)蛋白分子量。結(jié)果顯示:酶切和測(cè)序結(jié)果均證實(shí)HSP70表達(dá)載體構(gòu)建正確,可誘導(dǎo)表達(dá)。經(jīng)IPTG誘導(dǎo)表達(dá)后確定BL21(DE3)在37℃條件下表達(dá)蛋白效果最佳,經(jīng)檢測(cè)HSP70蛋白的相對(duì)分子質(zhì)量為71 988.04,蛋白純度大于90%,內(nèi)毒素小于500 Eu/mg。結(jié)果表明:成功構(gòu)建了大骨雞HSP70基因的表達(dá)載體,獲得了純化的HSP70蛋白,為進(jìn)一步研究HSP70在熱應(yīng)激中的作用機(jī)制提供了基礎(chǔ)。
[Abstract]:The aim of this study was to construct the expression plasmid P Cold II -HSP70 of the chicken heat shock protein 70 (heat shock protein 70, HSP70) expression plasmid, and to detect the quality of HSP70 protein after purification. The expression was induced and purified under different temperatures and different concentrations. The purity of the protein was identified by SEC-FPLC and HPLC. The content of protein endotoxin was detected by limulus reagent (diluted HSP70 protein at 1200600120 times). The molecular weight of the protein was detected by Western blot and mass spectrometry. The results showed that the results of enzyme digestion and sequencing confirmed that the HSP70 expression vector was constructed positive. The expression of BL21 (DE3) at 37 centigrade was the best, the relative molecular weight of the HSP70 protein was 71988.04, the protein purity was more than 90% and the endotoxin was less than 500 Eu/mg.. The results showed that the expression vector of the HSP70 gene was successfully constructed and the purified HSP70 protein was obtained, and the purified HSP70 protein was obtained. Further studies on the mechanism of HSP70 in heat stress provide the basis.
【作者單位】： 錦州醫(yī)科大學(xué)生命科學(xué)研究院;南京農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院;錦州醫(yī)科大學(xué)畜牧獸醫(yī)學(xué)院;遼寧省畜產(chǎn)品質(zhì)量與安全工程重點(diǎn)實(shí)驗(yàn)室;
【基金】：遼寧省自然科學(xué)基金項(xiàng)目(2015020765) 遼寧省科學(xué)技術(shù)計(jì)劃項(xiàng)目(2015211003) 遼寧醫(yī)學(xué)院第二批領(lǐng)軍人物團(tuán)隊(duì)項(xiàng)目
【分類號(hào)】：S831

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 王天友;;文昌雞熱休克蛋白70的克隆和原核表達(dá)[J];中國(guó)家禽;2015年03期

2 彭昊;唐林生;李軍;陶立;陳澤祥;潘艷;謝永平;許力干;楊威;黃維義;;隱孢子蟲(chóng)截短熱休克蛋白70(HSP70)基因的原核表達(dá)及其抗原性分析[J];畜牧與獸醫(yī);2014年12期

3 孔麗娜;甘建伉;張德祥;張細(xì)權(quán);張文武;羅慶斌;;HSP70基因多態(tài)性對(duì)矮小型黃羽肉雞熱耐受性的影響[J];南京農(nóng)業(yè)大學(xué)學(xué)報(bào);2013年06期

4 張啟耀;田文儒;高善頌;張志宏;侯春霞;李輝輝;;預(yù)熱誘導(dǎo)HSP70表達(dá)對(duì)高溫引致胚胎損傷的保護(hù)作用[J];畜牧與獸醫(yī);2009年09期

5 程維杰;李秋玲;孫延鳴;王洪梅;李建斌;仲躋峰;;熱休克蛋白70(HSP70)研究進(jìn)展[J];畜牧獸醫(yī)雜志;2008年06期

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 趙頌;楊卉新;朱弘焱;陶曉莉;蘇玉虹;田玉民;;大骨雞HSP70蛋白的表達(dá)、純化及質(zhì)量檢測(cè)[J];畜牧與獸醫(yī);2017年01期

2 胡苗;何茵如;楊霞;朱家勇;梅寒芳;;HSPA1L在H_2O_2誘導(dǎo)SH-SY5Y細(xì)胞損傷中的作用[J];廣東藥學(xué)院學(xué)報(bào);2016年06期

3 尹音;李冬霞;馮巖;陳新;王U，

本文編號(hào)：2125964