利用煙草表達重組牛凝乳酶及人表皮生長因子的研究
發(fā)布時間：2018-11-26 06:42
【摘要】：植物生物反應(yīng)器以植物細胞為載體,利用植物細胞的各種組分來合成人類需要的產(chǎn)品。相較于其他生物反應(yīng)器,植物生物反應(yīng)器具有綜合成本低、產(chǎn)品安全性高、生產(chǎn)易于規(guī)�；葍�(yōu)點,現(xiàn)已廣泛應(yīng)用于工業(yè)用蛋白(酶)和醫(yī)藥蛋白的生產(chǎn)。本研究以轉(zhuǎn)基因煙草植株作為植物生物反應(yīng)器,對在煙草葉片中表達的重組牛凝乳酶和重組人表皮細胞生長因子進行了探索和嘗試,獲得以下研究成果：構(gòu)建了牛凝乳酶原和前牛凝乳酶原基因的植物細胞核轉(zhuǎn)化載體四個,分別為p33cym11、p33cyml2、p33cym21和p33cym22。這四個載體中,(前)牛凝乳原基因由于不同的信號肽而有所區(qū)別,重組凝乳酶的羧基端(C端)融合有6×組氨酸標簽,目的是為了表達后通過親和層析分離重組凝乳酶提供方便。通過農(nóng)桿菌介導法,把上述基因?qū)霟煵?經(jīng)過草甘膦篩選,獲得了抗性再生植株；經(jīng)過PCR和Southern雜交檢測,證實抗性再生植株中(前)牛凝乳酶原基因已經(jīng)整合到基因組中,經(jīng)RT-PCR檢測,證明了外源基因在煙草細胞中得到轉(zhuǎn)錄。利用金標免疫試紙條檢測,證明了轉(zhuǎn)基因煙草中標記基因得到了表達；Western檢測表明,重組牛凝乳酶已經(jīng)得到表達；經(jīng)ELISA實驗表明其表達量占總可溶性蛋白的比例在0.12%～0.52%之間,也就是35.2～83.2 ng/g鮮重；凝乳實驗表明,重組凝乳酶具有良好的凝乳活性,與市場上的產(chǎn)品活性相當。構(gòu)建了人表皮生長因子基因的煙草葉綠體轉(zhuǎn)化表達載體pWX-Nt02。該載體中含有報告基因smGFP、選擇標記基因aadA以及目的基因hEGF;載體中目的基因下游添加了6×組氨酸標簽序列,便于目的蛋白的分離純化。通過基因槍法將載體pWX-Nt02導入煙草葉綠體基因組中,經(jīng)過篩選,獲得了轉(zhuǎn)基因植株。結(jié)合分子檢測和報告基因表達分析,經(jīng)過4輪篩選,獲得了同質(zhì)化的葉綠體轉(zhuǎn)基因煙草植株。經(jīng)過PCR和Southern雜交分析,證實外源基因已插入煙草葉綠體基因組中,并且達到同質(zhì)化。同質(zhì)化的轉(zhuǎn)基因煙草有很好的遺傳穩(wěn)定性,嚴格地遵循母性遺傳特性。熒光分析和SDS-PAGE分析證明,報告基因綠色熒光蛋白(GFP)成功表達；ELISA檢測表明,成熟葉片中GFP的表達量占總可溶性蛋白的22.38%,即GFP的積累量達到4.22 mg/g鮮重；通過硫酸銨分級沉淀和凝膠過濾層析的方法,分離純化了綠色熒光蛋白。Western檢測證實目的蛋白重組人表皮生長因子已經(jīng)得到表達。ELISA實驗表明,其表達量為可溶性蛋白的0.124%～0.165%,即23.16～25.77 ng/g鮮重。細胞增值實驗和延伸實驗表明,煙草表達的重組人表皮生長因子具有很好的生物活性。
[Abstract]:Plant bioreactor uses plant cells as carrier and uses various components of plant cells to synthesize products needed by human beings. Compared with other bioreactors, plant bioreactor has many advantages, such as low cost, high product safety, easy to scale production, and has been widely used in the production of industrial protein (enzyme) and pharmaceutical protein. In this study, transgenic tobacco plants were used as plant bioreactor to explore the expression of recombinant bovine condensate enzyme and recombinant human epidermal growth factor in tobacco leaves. The following results were obtained: four plant nuclear transformation vectors of bovine prothrombin gene and procoagulinogen gene were constructed, which were p33cym11p33cyml2p33cym21 and p33cym22, respectively. Among the four vectors, the bovine procoagulant gene is different because of different signal peptides, and the carboxyl terminal (C-terminal) of the recombinant coagulase has a 6 脳 histidine label. The aim of this study was to provide convenience for the separation of recombinant curd by affinity chromatography. The above genes were introduced into tobacco by Agrobacterium tumefaciens, and the resistant regenerated plants were obtained by glyphosate screening. The results of PCR and Southern hybridization showed that bovine procoagulase genes in resistant regenerated plants had been integrated into the genome, and that exogenous genes were transcribed in tobacco cells by RT-PCR detection. The expression of marker gene in transgenic tobacco was proved by using gold labeled immunosorbent strip test, and the expression of recombinant bovine curd enzyme was confirmed by Western assay. ELISA test showed that the proportion of the total soluble protein was 0.12%, that is, the fresh weight of 35.2% (83.2 ng/g). The result of curd experiment showed that the recombinant coagulase had good curd activity, which was equivalent to the product activity in the market. The expression vector pWX-Nt02. of tobacco chloroplast transformation of human epidermal growth factor gene was constructed. The reporter gene smGFP, selective marker gene aadA and the downstream target gene 6 脳 histidine label sequence were added to the vector of the target gene hEGF;, which was convenient for the separation and purification of the target protein. The vector pWX-Nt02 was introduced into the chloroplast genome of tobacco by gene gun method, and the transgenic plants were obtained by screening. After four rounds of screening, homogenized chloroplast transgenic tobacco plants were obtained by molecular detection and reporter gene expression analysis. By PCR and Southern hybridization analysis, it was confirmed that the exogenous gene had been inserted into the chloroplast genome of tobacco and reached homogenization. Homogenized transgenic tobacco has good genetic stability and strictly follows maternal genetic characteristics. Fluorescence analysis and SDS-PAGE analysis showed that the reporter gene green fluorescent protein (GFP) was successfully expressed, and ELISA analysis showed that the expression of GFP in mature leaves accounted for 22.38% of the total soluble protein, that is, the accumulation of GFP reached 4.22 mg/g fresh weight. Green fluorescent protein was isolated and purified by ammonium sulfate fractionation and gel filtration chromatography. Western assay confirmed that the recombinant human epidermal growth factor (EGF) of the target protein had been expressed. ELISA assay showed that the recombinant human epidermal growth factor (EGF) was expressed. Its expression was 0.124% of the soluble protein 0.165, that is 23.16525.77 ng/g fresh weight. Cell proliferation and extension experiments showed that recombinant human epidermal growth factor (HEGF) expressed by tobacco had good biological activity.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q943.2
本文編號：2357608
[Abstract]:Plant bioreactor uses plant cells as carrier and uses various components of plant cells to synthesize products needed by human beings. Compared with other bioreactors, plant bioreactor has many advantages, such as low cost, high product safety, easy to scale production, and has been widely used in the production of industrial protein (enzyme) and pharmaceutical protein. In this study, transgenic tobacco plants were used as plant bioreactor to explore the expression of recombinant bovine condensate enzyme and recombinant human epidermal growth factor in tobacco leaves. The following results were obtained: four plant nuclear transformation vectors of bovine prothrombin gene and procoagulinogen gene were constructed, which were p33cym11p33cyml2p33cym21 and p33cym22, respectively. Among the four vectors, the bovine procoagulant gene is different because of different signal peptides, and the carboxyl terminal (C-terminal) of the recombinant coagulase has a 6 脳 histidine label. The aim of this study was to provide convenience for the separation of recombinant curd by affinity chromatography. The above genes were introduced into tobacco by Agrobacterium tumefaciens, and the resistant regenerated plants were obtained by glyphosate screening. The results of PCR and Southern hybridization showed that bovine procoagulase genes in resistant regenerated plants had been integrated into the genome, and that exogenous genes were transcribed in tobacco cells by RT-PCR detection. The expression of marker gene in transgenic tobacco was proved by using gold labeled immunosorbent strip test, and the expression of recombinant bovine curd enzyme was confirmed by Western assay. ELISA test showed that the proportion of the total soluble protein was 0.12%, that is, the fresh weight of 35.2% (83.2 ng/g). The result of curd experiment showed that the recombinant coagulase had good curd activity, which was equivalent to the product activity in the market. The expression vector pWX-Nt02. of tobacco chloroplast transformation of human epidermal growth factor gene was constructed. The reporter gene smGFP, selective marker gene aadA and the downstream target gene 6 脳 histidine label sequence were added to the vector of the target gene hEGF;, which was convenient for the separation and purification of the target protein. The vector pWX-Nt02 was introduced into the chloroplast genome of tobacco by gene gun method, and the transgenic plants were obtained by screening. After four rounds of screening, homogenized chloroplast transgenic tobacco plants were obtained by molecular detection and reporter gene expression analysis. By PCR and Southern hybridization analysis, it was confirmed that the exogenous gene had been inserted into the chloroplast genome of tobacco and reached homogenization. Homogenized transgenic tobacco has good genetic stability and strictly follows maternal genetic characteristics. Fluorescence analysis and SDS-PAGE analysis showed that the reporter gene green fluorescent protein (GFP) was successfully expressed, and ELISA analysis showed that the expression of GFP in mature leaves accounted for 22.38% of the total soluble protein, that is, the accumulation of GFP reached 4.22 mg/g fresh weight. Green fluorescent protein was isolated and purified by ammonium sulfate fractionation and gel filtration chromatography. Western assay confirmed that the recombinant human epidermal growth factor (EGF) of the target protein had been expressed. ELISA assay showed that the recombinant human epidermal growth factor (EGF) was expressed. Its expression was 0.124% of the soluble protein 0.165, that is 23.16525.77 ng/g fresh weight. Cell proliferation and extension experiments showed that recombinant human epidermal growth factor (HEGF) expressed by tobacco had good biological activity.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q943.2
【參考文獻】
相關(guān)期刊論文 前10條
1 朱玉英;王存芳;;凝乳酶的發(fā)展及其在奶酪生產(chǎn)中的應(yīng)用[J];乳業(yè)科學與技術(shù);2015年04期
2 廖小金;肖清江;莊露;周衛(wèi)東;陳清西;;重組人表皮生長因子在畢赤酵母中的表達純化及鑒定[J];廈門大學學報(自然科學版);2014年06期
3 楊藝;普燕;張富春;;牛凝乳酶原基因在畢赤酵母中的表達及其酶學特性的研究[J];食品工業(yè)科技;2014年15期
4 周婷婷;王瀝浩;王文慧;馮雪;楊晶;;轉(zhuǎn)人表皮生長因子基因紅花研究[J];西北農(nóng)林科技大學學報(自然科學版);2013年12期
5 ;Optimization of Medium Composition for Production of Recombinant Calf Chymosin from Kluyveromyces lactis in Submerged Fermentation[J];Journal of Northeast Agricultural University(English Edition);2011年01期
6 張衛(wèi)兵;宋曦;賀曉玲;甘伯中;;Bacillus licheniformis產(chǎn)凝乳酶培養(yǎng)基的優(yōu)化[J];中國釀造;2011年02期
7 劉洋;徐成武;趙德剛;;重組人表皮生長因子基因遺傳轉(zhuǎn)化煙草研究[J];分子植物育種;2008年03期
8 白杰英;龐曉斌;曾林;尚世臣;時彥勝;;人表皮生長因子在煙草中的表達及其重組蛋白的初步鑒定[J];河南大學學報(醫(yī)學版);2007年03期
9 智慶文;張鳳英;柴敏;于新惠;孫曼霽;;人表皮生長因子在轉(zhuǎn)基因番茄中成功表達[J];中國藥理學通報;2007年05期
10 王關(guān)林,易慶,方宏筠;人egf基因在突變枯草芽孢桿菌WYBS2001中的轉(zhuǎn)化、分泌表達及其產(chǎn)物功能研究(英文)[J];遺傳學報;2003年02期
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