人乳铁蛋白、胸腺肽融合基因在毕赤酵母与生菜中表达的研究
发布时间：2018-11-25 20:55
【摘要】：人乳铁蛋白(human Lactoferrin, hLf)是具有多种生物学活性的一种铁结合性糖蛋白, 广泛存在于哺乳动物的乳汁、唾液、眼泪等分泌物中。人乳铁蛋白基因的开放阅读框为2.14kb,蛋白分子量约为80kD 左右。胸腺肽α1(Thymosin alpha 1,Tα1)是一种广泛分布于哺乳动物的各个组织器官中的活性肽,含28 个氨基酸残基,分子量3.108kD,等电点pH=4.2。由于这两个蛋白在人体免疫调节、抗癌和抗病毒等方面具有重要的作用,所以关于他们的研究越来越受到人们的重视。在进行本论文的工作中首先人工合成人乳铁蛋白基因的开放阅读框,并且与本实验室保存的胸腺肽基因融合,然后对融合基因的酵母和生菜表达系统进行了初步的研究。在融合基因表达系统的研究过程中为了提高融合基因的表达量,又引入了透明颤菌血红蛋白基因(Vitreoscilla hemoglobin gene,vgb)进行了研究。 在融合基因的研制中:依据已经公布的人乳铁蛋白基因序列并同时考虑到可实现分别克隆拼接的酶切位点,设计并合成51 个相互重叠的寡核苷酸单链,分9 组退火形成7个基因片段和两个Linker。然后通过克隆、拼接将7 个基因片段合成完整的人乳铁蛋白基因并与胸腺肽基因(100bp 左右)融合,成功的合成了人乳铁蛋白、胸腺肽融合基因(Tα1hLf)。通过测序证明了合成的约2.3kb 的融合基因序列正确,阅读框未发生改变。 在毕赤酵母表达融合基因实验中:利用毕赤酵母分泌型表达载体pPIC9K 构建可以在胞内表达vgb 和胞外分泌表达融合基因的载体pPIC9K-vgbTα1hLf。转化酵母后,通过PCR、SDS-PAGE 检测证实融合基因已经整合于酵母基因组并且表达出约83kD 左右的融合蛋白。Western blotting 和抑菌实验表明分泌表达的融合蛋白中人乳铁蛋白具有天然蛋白的活性,从而验证了合成的乳铁蛋白基因和融合基因的正确性。差示光谱分析和贫氧实验表明VHb 具有活性并在贫氧环境中可以促进毕赤酵母的生长和融合蛋白的表达。摇瓶表达实验表明融合蛋白的表达量可以达到30mg/L。 在生菜表达融合基因实验中:用pGΩ4A 构建了两种分别由RuBP 启动子和35S 启动子启动融合基因的中间载体,然后将融合基因表达盒与VHb 基因表达盒反向串联并克隆于pBI121 构建出植物表达载体pGBIVHbTα1hL(fvgb 和融合基因均由35S 启动子启动)和pGBIRVHbTα1hLf(vgb 由35S 启动子启动,融合基因由RuBP 启动子启动),利用农杆菌介导法将两种植物表达载体转化生菜并获得46株再生苗。对再生苗进行PCR 检测和用乳铁蛋白抗体ELISA 检测,结果表明融合蛋白已经获得表达。差示光谱分析表明vgb 基因也已经表达且具有活性。
[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 伪 1 (Thymosin alpha 1 T 伪 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 伪 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 伪 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 伪 1hL (fvgb and fusion genes were both initiated by 35s promoter) and pGBIRVHbT 伪 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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78
本文编号：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 伪 1 (Thymosin alpha 1 T 伪 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 伪 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 伪 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 伪 1hL (fvgb and fusion genes were both initiated by 35s promoter) and pGBIRVHbT 伪 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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78
【引证文献】
相关硕士学位论文 前1条
1 武树华;高赖氨酸基因Lys、CFLR和人乳铁蛋白基因hLF转化水稻的研究[D];福建农林大学;2012年
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