【作者】 高德民；

【导师】 王凤山；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2009， 博士

【摘要】 胸腺五肽（thymopentin,TP5）是位于胸腺生成素Ⅱ的第32～36位氨基酸残基片段,具有与TPⅡ相似的生物学活性,能双向调节失衡的免疫系统。TP5能诱导T细胞的分化,促进T淋巴细胞亚群发育并活化,是一种重要的免疫调节剂,临床上主要用于免疫缺陷症和自身免疫性疾病的治疗。因TP5半衰期较短（30 s）,人们制备了许多TP5的类似物或模拟肽来延长TP5的半衰期并提高其活性,如在TP5之间插入稳定性成分、用D-型氨基酸取代L-型氨基酸,或者制成环肽等,尽管这些工作取得了一定的成绩,但结果却难以令人满意。Tα1是一种天然存在于多种生物组织的多肽物质,由28个氨基酸残基组成,等电点4.2,分子量3108 Da。Tα1具有与TP5类似的免疫活性,主要是增强细胞免疫,用于免疫缺陷或免疫功能低下相关的疾病。Tα1的血浆半衰期约1.5 h。目前,Tα1主要从动物组织中提取或化学合成,但从组织中提取存在产量低、产物不稳定等缺点,而化学合成则价格昂贵。为了提高TP5的活性,延长其半衰期,本研究依据肽链延长可提高多肽类药物体内半衰期的事实,将具有相似生物活性与临床用途的TP5和Tα1借助基因工程方法连接在了一起,表达制备了Tα1-TP5融合肽。1 Tα1-TP5融合肽的基因克隆及在毕赤酵母中的表达本研究根据Tα1-TP5的氨基酸序列,选用毕赤酵母偏爱的密码子,人工设计合成了Tα1-TP5全基因序列;借助Primer软件,设计了上下游引物,通过PCR反应、酶切、连接等步骤,构建了融合基因Tα1-TP5表达载体pGAPZαA-Tα1-TP5。由于在毕赤酵母中表达融合肽可能出现N-端和C-端不均一的现象,在构建表达载体时时采取了两种方法:第一,将目的基因直接插在α-信号肽后面的Glu-Ala-Glu-Ala处;第二,在目的基因C-端增加了凝血酶酶切位点的编码基因,保留了6×His纯化标签。这样,既能简便快捷的用金属鳌合亲和填料纯化表达产物,又能通过凝血酶高效完全地切除N-端多余氨基酸残基和6×His标签,得到Tα1-TP5融合肽,从而提高Tα1-TP5融合肽的纯化效率。采用电激法用BlnⅠ线性化载体pGAPZαA-Tα1-TP5转化P.pastoris GS115,筛选Zeocine高抗性的阳性克隆,用煮-冻-煮法提取酵母基因组DNA,用PCR方法对P.pastoris GS115的基因表型进行了鉴定。重组转化子经摇瓶培养,Tricine-SDS-PAGE证明上清液中含有Tα1-TP5融合肽,其中两个转化子表达量较高。考查了发酵时间、培养温度对Tα1-TP5融合肽表达量的影响。结果显示,在30℃、72 h后发酵上清中的Tα1-TP5表达量最高,达32.2 mg/L。2 Tα1-TP5融合肽的分离纯化研究了Tα1-TP5融合肽的分离纯化工艺。发酵混合物经高速离心取上清,用超滤法去除大分子的蛋白酶、金属螯和层析纯化融合肽、冷冻干燥浓缩、SephadexG-25凝胶过滤脱盐、凝血酶酶切后用SephadexG-70凝胶过滤分离,再干燥浓缩,目的产物Tα1-TP5融合肽在Tricine-SDS-PAGE上呈现单一条带。ESI-MS也证实了Tα1-TP5融合肽在毕赤酵母中得到正确的分泌表达。3 Tα1-TP5融合肽的二级结构研究利用圆二色谱（Circular Dichroism,CD）与傅立叶变换红外光谱（fouriertransform infrared spectroscopy,FTIR）对Tα1-TP5融合肽的二级结构进行了分析。CD结果显示Tα1-TP5融合肽中二级结构的各构象分别是α-helix为32.01%,β-antiparallel为8.50%,β-parallel为8.86%,β-turn为17.02%,random coil为33.61%,说明Tα1-TP5融合肽分子结构中,α-helix和random coil是二级结构的主要部分。该结果FTIR的分析结果基本一致,二种分析方法相互补充,相互印证。4 Tα1-TP5融合肽体外半衰期的研究从家兔心脏取血后进行肝素抗凝,加入一定浓度的多肽药物,37℃水浴,于不同的时间点取样,用盐酸丙酮（盐酸:丙酮:水=1:40:5,v/v）对血浆样品进行处理,高速离心得到沉淀,用醋酸溶解。用RP-HPLC测定不同保温时间血浆中Tα1-TP5、Tα1及TP5的浓度,计算Tα1-TP5、Tα1及TP5体外血浆半衰期。结果表明,Tα1-TP5、Tα1及TP5的血浆半衰期分别为140±14 min、127±11 min和5.6±0.7 min。Tα1-TP5融合肽较TP5的体外血浆半衰期有明显差异（p＜0.01）。5 Tα1-TP5融合肽的活性研究小鼠脾细胞增殖实验表明,在系列浓度为10^-11～10^-6mol/L时,Tα1-TP5融合肽对昆明小鼠脾淋巴细胞的的最大增殖率为50.97±5.19%,Tα1的最大增殖率为45.38±8.26%,TP5融合肽的最大增殖率为40.61±5.17%,Tα1-TP5融合肽与TP5之间有显著性差异（p＜0.01）。在巨噬细胞的吞噬实验中,Tα1-TP5融合肽组、Tα1组、TP5组和对照组的廓清指数K分别为0.020±0.0010、0.016±0.0007、0.013±0.0008和0.005±0.0006,而对应的吞噬指数依次为3.95±0.19、3.73±0.56、3.43±0.29和2.48±0.40。结果表明,各药物组均能影响正常小鼠单核巨噬细胞吞噬功能,且融合肽的廓清指数K和吞噬指数α也显著高于其它组（P＜0.01）。通过研究Tα1-TP5、Tα1和TP5对小鼠血清中IL-2的表达的影响表明,Tα1-TP5融合肽组的平均水平为848.81±46.70 pg/mL,Tα1组的平均水平为761.50±31.33 pg╱mL,而TP5组的平均水平为749.62±29.08 pg/mL,对照组为718.35±32.16 pg/mL,融合肽较其它实验组有显著性差异（P＜0.01）。这表明Tα1-TP5融合肽较Tα1和TP5能更有效地促进淋巴细胞分泌IL-2。考虑到Tα1-TP5融合肽的结构与功能的关系,认为Tα1-TP5融合肽免疫活性的提高是由于Tα1-TP5构象的微小变化,尤其是α-helix含量的升高引起的。因为α-helix被认为是Tα1的主要活性中心,α-helix含量的升高,不仅显著延长了Tα1-TP5融合肽的半衰期,而且Tα1-TP5融合肽与靶细胞的持久作用更有利于免疫活性的增强。本实验结果表明,Tα1-TP5融合肽具有潜在的临床应用价值。本研究取得的主要成果有:（1）构建了pGAPZαA-Tα1-TP5表达载体,并采用毕赤酵母表达系统对Tα1-TP5融合肽进行了分泌表达,表达量可达32.2 mg/L,用ESI-MS证实了其结构的正确性。（2）建立了实验室水平的Tα1-TP5融合肽分离纯化工艺,经过超滤、亲和层析和凝胶过滤三步骤,所获得产品的纯度可达到99%以上。（3）用CD和FTIR研究了Tα1-TP5融合肽的二级结构,表明α-helix和random coil是其二级结构的主要组成部分。推测Tα1-TP5融合肽二级结构中的α-helix构象是影响其活性的重要因素。（4）与TP5和Tα1相比较进行了Tα1-TP5融合肽体外血浆t_1╱2研究,显示Tα1-TP5融合肽的半衰期约为140±14 min,高于同比试验TP5（5.6±0.7 min）和Tα1（127±11min）。（5）进行了胸腺Tα1-TP5融合肽体内外免疫活性研究,表明融合肽Tα1-TP5在促进昆明小鼠脾细胞的增殖、增加巨噬细胞的吞噬功能,和促进IL-2表达的实验中均显示了比TP5和Tα1更高的活性,是一个活性较好的潜在的免疫调节药物。本研究为一种新型免疫调节剂的开发奠定了基础。更多还原

【Abstract】 Thymopentin（TP5）,corresponding to the position 32-36 of thymopoietin,which exhibits similarly biological activities as thymopoietin,can have a function of two-ways regulation on imbalanced immune system.As an important immunomodulator,TP5 can induce T cell differentiation and promote the development and activation of T-lymphocyte subsets,resulting in curing immune deficiency and autoimmune disorders in clinical application.Because its half-life is very short（30 s）,many TP5 analogs and peptidomimetics such as the insertion of stable components,D-amino acid substitution or cyclic peptide,have been prepared to enhance their half-lives and activities.However,all the results were still not satisfactory.Thymosonα1（Tα1）,a natural polypeptide existing in a variety of biological tissues,is composed of 28 amino acid residues with a molecular weight of 3100 Da, pI 4.2,and has similar immunocompetence as TP5.As an immunomodulator,Tα1 has been used clinically for the treatment of immune defects.Plasm half-life of Tα1 was about 1.5 h.Currently,Tα1 is prepared from the animal tissues or chemical synthesis, so it has many shortcomings,e.g.low yields,and high price.Based on the facts that the proper extension of peptide chain can improve half-lives of peptide drugs,to elongate the half-life and activity of TP5,Tα1 was fused with TP5 and Tα1-TP5 fusion peptide was prepared by genetic engineering.1 Cloning and expression of Tα1-TP5 gene in P.pastorisIn this dissertation,a synthetic gene encoding a Tα1-TP5 was synthesized on the basis of its amino acid sequence and P.pastoris preferred codons.Upstream and downstream primers were designed by primer software,and then Tα1-TP5 gene was manipulated by PCR amplification,restriction enzyme digestion and ligation,and cloned into the P.pastoris expression vector pGAPZαA.The recombinant plasmid pGAPZαA-Tα1-TP5 was transformed into E.coli JM109,and then colony screening, restriction enzyme digestion,PCR reaction and sequence analysis showed that the synthesized gene and cloned gene were consistent completely with designed gene.There are some cases where unnecessary amino acids are left on the N- and C-terminus of the expressed fusion peptide.In our study,we inserted target gene rightly behind the Glu-Ala-Glu-Ala ofα-factor signal sequence,added a thrombin site gene between the C-terminus and stop condon,remaining 6×his tag.In this way, fusion peptide could be easily purified by metal chelating affinity chromatography, and the purified products could be further cleaved by thrombin to obtain Tα1-TP5 fusion peptide.BlnⅠ-lineared pGAPZαA-Tα1-TP5 was transformed into P.pastoris GS115 strain and positive colonies were screened with high concentration of Zeocin.Yeast genomic DNA was extracted by boiling-freezing-boiling and identified by PCR amplification.The recombinant transformants were cultured in the shake flask. Tricine-SDS-PAGE showed the production of Tα1-TP5 and found two transformants had high yields.The influence of culture time and temperature were studied.When cultured at 30℃to optical density at 72 h,the recombinant Tα1-TP5 fusion peptide reached 32.2 mg/L.2 Separation and purification of Tα1-TP5 fusion peptideThe technology of separation and purification of the fusion peptide was studied. After gaining the supematant by centrifugation,the macromolecules including proteases were removed by ultrafiltration,the fusion peptide was separated by metal chelate chromatography,and desalted and purified by G-25 gel filtration.Fusion peptide was digested by thrombin and was concentrated again.The purified Tα1-TP5 fusion peptide sample showed a single band by Tricine-SDS-PAGE.The correct molecular weight of Tα1-TP5 was further confirmed with ESI-MS.3 The secondary structure study of Tα1-TP5 fusion peptideThe secondary structure of Tα1-TP5 fusion peptide was determined by circular dichroism（CD） spectroscopy and Fourier transform infrared（FTIR） spectroscopy. The CD results showed that the secondary structure of Tα1-TP5 fusion peptide constituted of 32.01%α-helix,8.50%β-antiparallel,8.86%β-parallel,17.02%β-tum, and 33.61%random coil.Theα-helix and random coil covered the most part of the Tα1-TP5 fusion peptide,which was consistent with tendency of the Fourier transform infrared（FTIR） spectroscopy.4 In vitro plasma half life of Tα1-TP5 fusion peptideThe in vitro plasma half-life of each target peptide was determined by reversed-phase high-performance liquid chromatography（RP-HPLC） following incubation at 37℃in heparinized rabbit plasma at different time points.The results showed that the in vitro half-life of Tα1-TP5 fusion peptide,Tα1 and TP5 were （140±14） min,（127±11） min and（5.6±0.7） min respectively.The half-life of Tα1-TP5 fusion peptide is obviously longer than that of TP5 and Tα1 alone.5 Activities of Tα1-TP5 fusion peptideMouse spleen cell proliferation experiment showed that when the concentration was in the range of 10^-11 to 10^-6mol/L,the largest proliferation ratio of Tα1-TP5,Tα1 and TP5 were（50.97±5.19）%,（45.38±8.257）%and（40.61±5.17）%respectively. Tα1-TP5 has significantly higher activity（P＜0.01） than TP5 in promoting the proliferation of mouse splenocytes in vitro,but the activity difference between Tα1-TP5 and Tα1 was not significant.The in vivo macrophage phagocytosis experiments showed that the clearance index K of Tα1-TP5 fusion peptide,Tα1,TP5 and control were 0.020±0.0010, 0.016±0.0007,0.013±0.0008 and 0.005±0.0006 respectively.And their phagocytic index corresponded 3.95±0.19,3.73±0.56,3.43±0.29 and 2.48±0.40.All the peptide drugs could enhance the normal mouse macrophage phagocytosis,and Tα1-TP5 had better activity in promoting the phagocytosis of macrophages than TP5.The quantitative analysis of IL-2 in the serum of the peptide drug treated mice showed that the average IL-2 level of Tα1-TP5,Tα1,TP5 and control group were 848.81±46.70 pg/mL,761.50±31.33 pg/mL,749.62±29.08 pg/mL and 718.35±32.16 pg/mL respectively.These demonstrated that Tα1-TP5 fusion peptide has significantly higher activity in promoting T lymphocytes to secrete IL-2 than Tα1 or TP5 alone does.In view of the relations betweem structure and function of Tα1-TP5,enhanced activity may attribute to the small conformational changes of Tα1-TP5,compared with Tα1,especially the small increase ofα-helix structure,because in previous studies,it was presumed thatα-helix structure played a main role in the activity of Tα1.In summary,this study showed Tα1-TP5 might be used as a potential therapeutic agent for the human aliments.In this study,the main research results are as follows:（1） The vector pGAPZαA-Tα1-TP5 was constructed.Tα1-TP5 fusion peptide was expressed in P.pastoris and its expression level was detected to be 32.2 mg/L in the culture.ESI-MS confirmed its correctness of its structure.（2） A method of separation and purification of Tα1-TP5 fusion peptide in laboratory level was establishe.Tα1-TP5 fusion peptide was well purified by a serial of ultrafiltration,affinity adsorbtion and gel filtration and the purity of the product was over 99%.（3） The secondary structure of Tα1-TP5 was studied by CD spectroscopy and FTIR pectroscopy.Theα-helix and random coil covered the most part of its secondary structure.Further speculated theα-helix conformation was an important factor for its immunocompetence.（4） The in vitro plasma half-life of Tα1-TP5 was studied,and the results conformed that its half-life（140±14 min） is significantly longer than that of TP5 （5.6±0.7 min） or Tα1（127±11min）.（5） The in vitro activity assay presented that Tα1-TP5 fusion peptide has greater activity in promoting proliferation of Kunming mouse splenocytes,and in vivo experiment showed that Tα1-TP5 fusion peptide had better activity in promoting the phagocytosis of macrophages and secretion of IL-2 than both Tα1 and TP5.Our findings suggested that Tα1-TP5 fusion peptide might be a potential therapeutic agent.In a word,this study has provided a basis for the further medicinal development of Tα1-TP5 fusion peptide.更多还原