陽春砂單萜合酶基因AvTPS1載體構(gòu)建和原核表達(dá)
發(fā)布時間：2018-11-19 19:11
【摘要】：目的構(gòu)建AvTPS1的原核表達(dá)載體并進(jìn)行蛋白表達(dá),為該基因在原核表達(dá)系統(tǒng)中的功能鑒定奠定基礎(chǔ)。方法用In-Fusion方法構(gòu)建AvTPS1截掉轉(zhuǎn)運(yùn)肽的表達(dá)載體;用Gateway方法構(gòu)建AvTPS1包含完整閱讀框的表達(dá)載體;兩種表達(dá)載體分別轉(zhuǎn)化至大腸桿菌BL21(DE3)和BL21DE3plys中進(jìn)行誘導(dǎo)表達(dá),用變性聚丙烯酰氨凝膠電泳(SDS-PAGE)以及Western雜交檢測蛋白的表達(dá)情況。結(jié)果構(gòu)建了原核表達(dá)載體pET-32a(+)-(-tp)AvTPS1和pDEST17-AvTPS1;這兩個表達(dá)載體在大腸桿菌BL21(DE3)和BL21DE3plys中均沒有表達(dá)出明顯的相應(yīng)蛋白,但是這兩個載體在大腸桿菌BL21(DE3)中大量表達(dá)后,用Western雜交檢測有蛋白存在。結(jié)論成功構(gòu)建了陽春砂單萜合酶基因AvTPS1的表達(dá)載體和工程菌。
[Abstract]:Objective to construct the prokaryotic expression vector of AvTPS1 and express the protein in order to lay a foundation for the functional identification of the gene in the prokaryotic expression system. Methods the expression vector of AvTPS1 truncated peptide was constructed by In-Fusion method, and the expression vector containing complete reading frame of AvTPS1 was constructed by Gateway method. The two expression vectors were transformed into Escherichia coli BL21 (DE3) and BL21DE3plys to induce expression, and the protein expression was detected by denatured polyacrylamide gel electrophoresis (SDS-PAGE) and Western hybridization. Results the prokaryotic expression vectors pET-32a ()-(- tp) AvTPS1 and pDEST17-AvTPS1; were constructed. The two expression vectors did not express the corresponding proteins in E. coli BL21 (DE3) and BL21DE3plys, but after the two vectors were expressed in E. coli BL21 (DE3), the proteins were detected by Western hybridization. Conclusion the expression vector and engineering strain of AvTPS1 were successfully constructed.
【作者單位】： 廣州中醫(yī)藥大學(xué)中藥資源科學(xué)與工程研究中心嶺南中藥資源教育部重點(diǎn)實(shí)驗室(廣州中醫(yī)藥大學(xué))國家中成藥工程技術(shù)研究中心南藥研發(fā)實(shí)驗室;廣州中醫(yī)藥大學(xué)中藥學(xué)院;
【基金】：國家自然科學(xué)基金青年科學(xué)基金(81303163) 廣東省高等學(xué)校優(yōu)秀青年教師培養(yǎng)計劃項目(Yq2013042) 廣州中醫(yī)藥大學(xué)青年英才培養(yǎng)項目(AAC414124A08)
【分類號】：S567.239
本文編號：2343181
[Abstract]:Objective to construct the prokaryotic expression vector of AvTPS1 and express the protein in order to lay a foundation for the functional identification of the gene in the prokaryotic expression system. Methods the expression vector of AvTPS1 truncated peptide was constructed by In-Fusion method, and the expression vector containing complete reading frame of AvTPS1 was constructed by Gateway method. The two expression vectors were transformed into Escherichia coli BL21 (DE3) and BL21DE3plys to induce expression, and the protein expression was detected by denatured polyacrylamide gel electrophoresis (SDS-PAGE) and Western hybridization. Results the prokaryotic expression vectors pET-32a ()-(- tp) AvTPS1 and pDEST17-AvTPS1; were constructed. The two expression vectors did not express the corresponding proteins in E. coli BL21 (DE3) and BL21DE3plys, but after the two vectors were expressed in E. coli BL21 (DE3), the proteins were detected by Western hybridization. Conclusion the expression vector and engineering strain of AvTPS1 were successfully constructed.
【作者單位】： 廣州中醫(yī)藥大學(xué)中藥資源科學(xué)與工程研究中心嶺南中藥資源教育部重點(diǎn)實(shí)驗室(廣州中醫(yī)藥大學(xué))國家中成藥工程技術(shù)研究中心南藥研發(fā)實(shí)驗室;廣州中醫(yī)藥大學(xué)中藥學(xué)院;
【基金】：國家自然科學(xué)基金青年科學(xué)基金(81303163) 廣東省高等學(xué)校優(yōu)秀青年教師培養(yǎng)計劃項目(Yq2013042) 廣州中醫(yī)藥大學(xué)青年英才培養(yǎng)項目(AAC414124A08)
【分類號】：S567.239
【相似文獻(xiàn)】
相關(guān)期刊論文 前2條
1 梁燕;嚴(yán)建萍;譚湘陵;;通育粳1號水稻乙醇脫氫酶基因克隆與原核表達(dá)[J];生物技術(shù)通報;2011年03期
2 俞樂;劉擁海;彭新湘;;水稻L-半乳糖內(nèi)酯脫氫酶基因的克隆和原核表達(dá)及抗體的制備[J];湖南農(nóng)業(yè)大學(xué)學(xué)報(自然科學(xué)版);2010年04期
相關(guān)碩士學(xué)位論文 前1條
1 馬泉芳;單萜合酶AtTPS03基因在湘油15中的過表達(dá)研究[D];湖南農(nóng)業(yè)大學(xué);2011年
，本文編號：2343181
本文鏈接：http://sikaile.net/kejilunwen/jiyingongcheng/2343181.html
最近更新
教材專著
