【摘要】：旨在構(gòu)建雞熱休克蛋白70(heat shock protein 70,HSP70)表達質(zhì)粒p ColdⅡ-HSP70,誘導表達、純化后檢測HSP70蛋白的質(zhì)量。將目的基因擴增后克隆至表達載體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表達并純化。經(jīng)SEC-FPLC和HPLC進行純度鑒定,采用鱟試劑檢測蛋白內(nèi)毒素含量(將HSP70蛋白按1 200、600、120倍稀釋);Western blot及質(zhì)譜方法檢測蛋白分子量。結(jié)果顯示:酶切和測序結(jié)果均證實HSP70表達載體構(gòu)建正確,可誘導表達。經(jīng)IPTG誘導表達后確定BL21(DE3)在37℃條件下表達蛋白效果最佳,經(jīng)檢測HSP70蛋白的相對分子質(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基因的表達載體,獲得了純化的HSP70蛋白,為進一步研究HSP70在熱應(yīng)激中的作用機制提供了基礎(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)科大學生命科學研究院;南京農(nóng)業(yè)大學動物醫(yī)學院;錦州醫(yī)科大學畜牧獸醫(yī)學院;遼寧省畜產(chǎn)品質(zhì)量與安全工程重點實驗室;
【基金】：遼寧省自然科學基金項目(2015020765) 遼寧省科學技術(shù)計劃項目(2015211003) 遼寧醫(yī)學院第二批領(lǐng)軍人物團隊項目
【分類號】：S831

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本文編號：2125964