SARS小包膜蛋白表達、純化及單克隆抗體的制備
發(fā)布時間：2018-10-19 15:17
【摘要】：SARS-CoV是一種新型冠狀病毒,小包膜蛋白(E蛋白)是重要的結(jié)構(gòu)蛋白,在病毒侵染宿主過程中起重要作用。 本研究中目的基因SARS-E基因由大連寶生物公司按照SARS冠狀病毒CUHK-W1株編碼E蛋白的基因全序列合成。 選用pET28a原核表達載體進行表達。將E基因克隆于重組表達載體pET28a上,轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)IPTG誘導(dǎo)獲得了高效表達的分子量約為27KD的可溶性融合蛋白。Western blot證實表達的目的蛋白是SARS-E蛋白。 本研究大規(guī)模發(fā)酵培養(yǎng)了重組SARS-E蛋白,探討了合適的發(fā)酵條件,并嘗試用乳糖代替IPTG誘導(dǎo)表達。通過DEAE Sepharose FF、SPSepharose FF和BLUE Sepharose CL 6B三步純化,純度達到95%以上。ELISA結(jié)果顯示,純化SARS-E蛋白能與SARS病人恢復(fù)期血清發(fā)生特異性反應(yīng),進而又制備了鼠源性抗SARS-E蛋白單克隆抗體。
[Abstract]:SARS-CoV is a new type of coronavirus. Small envelope protein (E protein) is an important structural protein and plays an important role in the infection of the virus. In this study, the target gene SARS-E gene was synthesized by Dalian Bao Biological Company according to the gene sequence encoding E protein of SARS coronavirus CUHK-W1 strain. PET28a prokaryotic expression vector was selected for expression. The E gene was cloned into recombinant expression vector pET28a and transformed into Escherichia coli BL21 (DE3). The highly expressed soluble fusion protein. Western blot with a molecular weight of about 27KD was confirmed to be SARS-E protein by IPTG induction. In this study, recombinant SARS-E protein was cultured on a large scale, and suitable fermentation conditions were discussed, and lactose instead of IPTG was used to induce expression. The purity of purified SARS-E protein was over 95% through three steps purification of DEAE Sepharose FF,SPSepharose FF and BLUE Sepharose CL 6B. The results of ELISA showed that the purified SARS-E protein could react specifically with the recovered serum of SARS patients, and then the monoclonal antibody against SARS-E protein was prepared.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392
本文編號：2281523
[Abstract]:SARS-CoV is a new type of coronavirus. Small envelope protein (E protein) is an important structural protein and plays an important role in the infection of the virus. In this study, the target gene SARS-E gene was synthesized by Dalian Bao Biological Company according to the gene sequence encoding E protein of SARS coronavirus CUHK-W1 strain. PET28a prokaryotic expression vector was selected for expression. The E gene was cloned into recombinant expression vector pET28a and transformed into Escherichia coli BL21 (DE3). The highly expressed soluble fusion protein. Western blot with a molecular weight of about 27KD was confirmed to be SARS-E protein by IPTG induction. In this study, recombinant SARS-E protein was cultured on a large scale, and suitable fermentation conditions were discussed, and lactose instead of IPTG was used to induce expression. The purity of purified SARS-E protein was over 95% through three steps purification of DEAE Sepharose FF,SPSepharose FF and BLUE Sepharose CL 6B. The results of ELISA showed that the purified SARS-E protein could react specifically with the recovered serum of SARS patients, and then the monoclonal antibody against SARS-E protein was prepared.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前7條
1 陳志南,徐靜;SARS冠狀病毒的研究現(xiàn)狀[J];第四軍醫(yī)大學(xué)學(xué)報;2003年11期
2 車小燕,丘立文,潘玉先,徐華,郝衛(wèi),廖志勇,梅亞波,張麗婭,萬卓越,袁國勇,黃震;SARS冠狀病毒N蛋白單克隆抗體的快速、高效制備的方法研究[J];第一軍醫(yī)大學(xué)學(xué)報;2003年07期
3 陳彥濤,羅鐘琳,丁建東;SARS病毒E蛋白的計算機模擬的初步研究[J];高等學(xué)�；瘜W(xué)學(xué)報;2003年08期
4 陶澤新,王志玉,溫紅玲,任桂杰,宋艷艷,王桂亭,姚蘋,許洪芝;SARS病毒E蛋白基因與EGFP基因在BHK-21細胞中的融合表達[J];山東大學(xué)學(xué)報(醫(yī)學(xué)版);2004年05期
5 祝慶余,秦鄂德,王翠娥,于曼,司炳銀,范寶昌,常國輝,彭文明,楊保安,姜濤,李豫川,鄧永強,劉洪,甘永華;非典型肺炎病例標(biāo)本中新型冠狀病毒的分離與鑒定[J];中國生物工程雜志;2003年04期
6 張建芳,蘇明權(quán),馮群嶺,岳喬紅,郝曉柯;SARS冠狀病毒E蛋白基因的克隆與表達[J];生物技術(shù)通訊;2003年05期
7 陳蘊佳,高歌,鮑一明,Rodrigo LOPEZ,吳健民,蔡濤,葉志強,顧孝誠,羅靜初;SARS冠狀病毒全基因組序列初步分析[J];遺傳學(xué)報;2003年06期
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