人乳鐵蛋白、胸腺肽融合基因在畢赤酵母與生菜中表達的研究
發(fā)布時間：2018-11-25 20:55
【摘要】：人乳鐵蛋白(human Lactoferrin, hLf)是具有多種生物學活性的一種鐵結(jié)合性糖蛋白, 廣泛存在于哺乳動物的乳汁、唾液、眼淚等分泌物中。人乳鐵蛋白基因的開放閱讀框為2.14kb,蛋白分子量約為80kD 左右。胸腺肽α1(Thymosin alpha 1,Tα1)是一種廣泛分布于哺乳動物的各個組織器官中的活性肽,含28 個氨基酸殘基,分子量3.108kD,等電點pH=4.2。由于這兩個蛋白在人體免疫調(diào)節(jié)、抗癌和抗病毒等方面具有重要的作用,所以關(guān)于他們的研究越來越受到人們的重視。在進行本論文的工作中首先人工合成人乳鐵蛋白基因的開放閱讀框,并且與本實驗室保存的胸腺肽基因融合,然后對融合基因的酵母和生菜表達系統(tǒng)進行了初步的研究。在融合基因表達系統(tǒng)的研究過程中為了提高融合基因的表達量,又引入了透明顫菌血紅蛋白基因(Vitreoscilla hemoglobin gene,vgb)進行了研究。 在融合基因的研制中:依據(jù)已經(jīng)公布的人乳鐵蛋白基因序列并同時考慮到可實現(xiàn)分別克隆拼接的酶切位點,設(shè)計并合成51 個相互重疊的寡核苷酸單鏈,分9 組退火形成7個基因片段和兩個Linker。然后通過克隆、拼接將7 個基因片段合成完整的人乳鐵蛋白基因并與胸腺肽基因(100bp 左右)融合,成功的合成了人乳鐵蛋白、胸腺肽融合基因(Tα1hLf)。通過測序證明了合成的約2.3kb 的融合基因序列正確,閱讀框未發(fā)生改變。 在畢赤酵母表達融合基因?qū)嶒炛?利用畢赤酵母分泌型表達載體pPIC9K 構(gòu)建可以在胞內(nèi)表達vgb 和胞外分泌表達融合基因的載體pPIC9K-vgbTα1hLf。轉(zhuǎn)化酵母后,通過PCR、SDS-PAGE 檢測證實融合基因已經(jīng)整合于酵母基因組并且表達出約83kD 左右的融合蛋白。Western blotting 和抑菌實驗表明分泌表達的融合蛋白中人乳鐵蛋白具有天然蛋白的活性,從而驗證了合成的乳鐵蛋白基因和融合基因的正確性。差示光譜分析和貧氧實驗表明VHb 具有活性并在貧氧環(huán)境中可以促進畢赤酵母的生長和融合蛋白的表達。搖瓶表達實驗表明融合蛋白的表達量可以達到30mg/L。 在生菜表達融合基因?qū)嶒炛?用pGΩ4A 構(gòu)建了兩種分別由RuBP 啟動子和35S 啟動子啟動融合基因的中間載體,然后將融合基因表達盒與VHb 基因表達盒反向串聯(lián)并克隆于pBI121 構(gòu)建出植物表達載體pGBIVHbTα1hL(fvgb 和融合基因均由35S 啟動子啟動)和pGBIRVHbTα1hLf(vgb 由35S 啟動子啟動,融合基因由RuBP 啟動子啟動),利用農(nóng)桿菌介導(dǎo)法將兩種植物表達載體轉(zhuǎn)化生菜并獲得46株再生苗。對再生苗進行PCR 檢測和用乳鐵蛋白抗體ELISA 檢測,結(jié)果表明融合蛋白已經(jīng)獲得表達。差示光譜分析表明vgb 基因也已經(jīng)表達且具有活性。
[Abstract]:Human lactoferrin (human Lactoferrin, hLf) is an iron-binding glycoprotein with many biological activities, which is widely found in mammalian milk, saliva, tears and other secretions. The open reading frame of human lactoferrin gene is 2.14 kb and the molecular weight of protein is about 80kD. Thymosin 偽 1 (Thymosin alpha 1 T 偽 1) is an active peptide widely distributed in various tissues and organs of mammals. It contains 28 amino acid residues with molecular weight of 3.108 KD and isoelectric point pH=4.2.. Because these two proteins play an important role in immunomodulation, anticancer and antivirus, so more and more attention has been paid to their research. In this paper, the open reading frame of human lactoferrin gene was synthesized and fused with the thymosin gene stored in our laboratory. Then the yeast and lettuce expression systems of the fusion gene were studied. In order to improve the expression of fusion gene, (Vitreoscilla hemoglobin gene,vgb was introduced into the fusion gene expression system. In the development of fusion genes, 51 overlapping oligonucleotide single strands were designed and synthesized based on the published sequence of human lactoferrin gene and taking into account the restriction sites that could be cloned and spliced separately. Seven gene fragments and two Linker. after annealing in 9 groups By cloning and splicing 7 fragments of human lactoferrin gene and fused with thymosin gene (100bp), human lactoferrin and thymosin fusion gene (T 偽 1hLf) were successfully synthesized. The sequence of fusion gene of synthetic 2.3kb was confirmed by sequencing, and the reading frame was not changed. In the experiment of Pichia pastoris fusion gene expression: Pichia pastoris secretory expression vector pPIC9K was used to construct the vector pPIC9K-vgbT 偽 1hLf. which can express vgb in cells and express fusion gene in extracellular secretion. After transforming yeast through PCR, SDS-PAGE analysis confirmed that the fusion gene had been integrated into yeast genome and expressed about 83kD fusion protein. Western blotting and bacteriostasis experiments showed that human lactoferrin in secreted and expressed fusion protein had the activity of natural protein. Therefore, the correctness of the synthesized lactoferrin gene and the fusion gene was verified. Differential spectroscopic analysis and oxygen deficiency experiments showed that VHb was active and could promote the growth of Pichia pastoris and the expression of fusion protein in oxygen-deficient environment. Shake flask expression experiment showed that the expression of fusion protein could reach 30 mg / L. In the experiment of expressing fusion gene in lettuce, two intermediate vectors of RuBP promoter and 35s promoter were constructed by using pG 惟 4A. Then the fusion gene expression box and VHb gene expression box were connected in reverse and cloned into pBI121 to construct the plant expression vector pGBIVHbT 偽 1hL (fvgb and fusion genes were both initiated by 35s promoter) and pGBIRVHbT 偽 1hLf (vgb was initiated by 35s promoter and fusion gene was initiated by RuBP promoter). Two plant expression vectors were transformed into lettuce by Agrobacterium tumefaciens and 46 regenerated seedlings were obtained. The recombinant vaccine was detected by PCR and lactoferrin antibody ELISA. The results showed that the fusion protein had been expressed. Differential spectroscopic analysis showed that vgb gene was also expressed and active.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：Q78
本文編號：2357347
[Abstract]:Human lactoferrin (human Lactoferrin, hLf) is an iron-binding glycoprotein with many biological activities, which is widely found in mammalian milk, saliva, tears and other secretions. The open reading frame of human lactoferrin gene is 2.14 kb and the molecular weight of protein is about 80kD. Thymosin 偽 1 (Thymosin alpha 1 T 偽 1) is an active peptide widely distributed in various tissues and organs of mammals. It contains 28 amino acid residues with molecular weight of 3.108 KD and isoelectric point pH=4.2.. Because these two proteins play an important role in immunomodulation, anticancer and antivirus, so more and more attention has been paid to their research. In this paper, the open reading frame of human lactoferrin gene was synthesized and fused with the thymosin gene stored in our laboratory. Then the yeast and lettuce expression systems of the fusion gene were studied. In order to improve the expression of fusion gene, (Vitreoscilla hemoglobin gene,vgb was introduced into the fusion gene expression system. In the development of fusion genes, 51 overlapping oligonucleotide single strands were designed and synthesized based on the published sequence of human lactoferrin gene and taking into account the restriction sites that could be cloned and spliced separately. Seven gene fragments and two Linker. after annealing in 9 groups By cloning and splicing 7 fragments of human lactoferrin gene and fused with thymosin gene (100bp), human lactoferrin and thymosin fusion gene (T 偽 1hLf) were successfully synthesized. The sequence of fusion gene of synthetic 2.3kb was confirmed by sequencing, and the reading frame was not changed. In the experiment of Pichia pastoris fusion gene expression: Pichia pastoris secretory expression vector pPIC9K was used to construct the vector pPIC9K-vgbT 偽 1hLf. which can express vgb in cells and express fusion gene in extracellular secretion. After transforming yeast through PCR, SDS-PAGE analysis confirmed that the fusion gene had been integrated into yeast genome and expressed about 83kD fusion protein. Western blotting and bacteriostasis experiments showed that human lactoferrin in secreted and expressed fusion protein had the activity of natural protein. Therefore, the correctness of the synthesized lactoferrin gene and the fusion gene was verified. Differential spectroscopic analysis and oxygen deficiency experiments showed that VHb was active and could promote the growth of Pichia pastoris and the expression of fusion protein in oxygen-deficient environment. Shake flask expression experiment showed that the expression of fusion protein could reach 30 mg / L. In the experiment of expressing fusion gene in lettuce, two intermediate vectors of RuBP promoter and 35s promoter were constructed by using pG 惟 4A. Then the fusion gene expression box and VHb gene expression box were connected in reverse and cloned into pBI121 to construct the plant expression vector pGBIVHbT 偽 1hL (fvgb and fusion genes were both initiated by 35s promoter) and pGBIRVHbT 偽 1hLf (vgb was initiated by 35s promoter and fusion gene was initiated by RuBP promoter). Two plant expression vectors were transformed into lettuce by Agrobacterium tumefaciens and 46 regenerated seedlings were obtained. The recombinant vaccine was detected by PCR and lactoferrin antibody ELISA. The results showed that the fusion protein had been expressed. Differential spectroscopic analysis showed that vgb gene was also expressed and active.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：Q78
【引證文獻】
相關(guān)碩士學位論文 前1條
1 武樹華;高賴氨酸基因Lys、CFLR和人乳鐵蛋白基因hLF轉(zhuǎn)化水稻的研究[D];福建農(nóng)林大學;2012年
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