人血小板生成素(hTPO)全长 cDNA 克隆及在 CHO 细胞中的表达

以人胎肝ｍＲＮＡ为模板,采用ＲＴ-ＰＣＲ扩增了人ＴＰＯ编码区全长ｃＤＮＡ,全序列测定结果表明与国外报道序列一致;进而构建了ｐｃＤＮＡ３-ＴＰＯ永久表达载体,转染ＣＨＯ细胞后经Ｇ４１８加压筛选、ＴＰＯ表达稳定性等项指标测试,获得了稳定分泌ＴＰＯ的工程细胞株。     

人血小板生成素（hTPO）全长cDNA克隆及其在CHO细胞中的表达

以人胎肝ｍＲＮＡ为模板,采用ＲＴ－ＰＣＲ扩增了人ＴＰＯ编码区全长ｃＤＮＡ,全序列测定结果表明与国外报道序列一致;进而构建了ｐｃＤＮＡ３－ＴＰＯ永久表达载体,转染ＣＨＯ细胞后经Ｇ４１８加压筛选、ＴＰＯ表达稳定性等项指标测试,获得了稳定分泌ＴＰＯ的工程细胞株。     

酵母系统表达TPO与E.coli表达TPO N端结构域生物半衰期比较

为了探讨血小板生成素（ＴＰＯ）糖基化与其生物半衰期的关系,首先纯化了巴氏毕赤酵母系统分泌表达的人ＴＰＯ成熟肽和大肠杆菌表达的人ＴＰＯＮ端结构域蛋白．纯化产物经Ｗｅｓｔｅｒｎｂｌｏｔ鉴定和活性分析后,两种蛋白以相同浓度或相同活性单位给Ｗｉｓｔｅｒ大鼠尾静脉注射,利用酶联免疫分析（ＥＬＩＳＡ）和ＴＦ１细胞测定不同时间点实验动物血浆中ＴＰＯ浓度或生物学活性,求出两种ＴＰＯ的生物半衰期（ｔ１／２）值．结果显示,以相同浓度和相同活性单位给药,ＴＰＯ成熟肽的ｔ１／２均比其Ｎ端结构域的长,前者分别是后者的１．６５倍和１．２７倍．     

重组人血小板生成素在大肠杆菌中表达的研究

采用化学法全合成了编码人血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）成熟肽Ｎ端１５３氨基酸的基因序列,构建基于该合成基因的表达质粒,结果以谷胱甘肽转硫酶－ＴＰＯ１５３（ＧＳＴ－ＴＰＯ１５３）融合蛋白的方式获得了占全菌蛋白４０％的高效表达．进一步采用ＰＣＲ方法分别对ＴＰＯ合成基因及ＴＰＯｃＤＮＡ的翻译起始区（ＴＩＲ）序列进行定点突变,以降低这一区域的Ｇ－Ｃ含量．将突变序列分别插入到ｐＢＶ２２０表达载体中,重组质粒在转化大肠杆菌ＪＭ１０９后,均获得了表达,其中ＴＩＲ区突变后的合成基因表达产物约占全菌蛋白的１５％．为研究基因下游结构对表达的影响,在不改变氨基酸组成的基础上,构建了ＴＰＯ合成基因与ＴＰＯｃＤＮＡ的杂合序列表达质粒．研究结果表明翻译起始效率是影响ｒｈ－ＴＰＯ在大肠杆菌中表达的重要因素之一,同时基因下游序列的组成对表达水平也会产生影响．     

血小板生成素基因在体内的表达及对造血祖细胞增殖活性的影响

探讨了从肌肉组织植入的人血小板生成素（ＴＰＯ）基因在小鼠体内的表达规律,以及对造血祖细胞增殖活性的影响．基因在导入后的２４小时内就开始转录,先于血小板、血液ＴＰＯ浓度、及造血祖细胞的变化．所表达的ＴＰＯ在血液中的聚积可持续４周以上．巨核祖细胞,粒系祖细胞都出现２周左右的增殖增长,长于血小板计数的升高,但红系祖细胞的变化不明显．这些结果显示,基因治疗过程中血小板计数等变化源于ＴＰＯ的表达及刺激,而在此期间一些调控机制被激活,对血小板形成的平衡发挥作用．     

人TPON端结构域在大肠杆菌中的表达、纯化及活性分析

在利用反转录－ＰＣＲ从人胎肝中获得编码人血小板生成素（ｈＴＰＯ）全长ｃＤ－ＮＡ的基础上,综合ＴＰＯ结构与功能的研究信息,在大肠杆菌中表达了成熟肽Ｎ端结构域．目的蛋白在菌体内以包涵体形式存在,表达量约占菌体总蛋白的３０％;包涵体经变性、复性、凝胶过滤、离子交换层析等步骤处理后,所得产物给Ｂａｂｌ／ｃ小鼠腹腔连续注射８ｄ,第９ｄ摘眼球采血,计数血小板的数量．结果表明,ＴＰＯＮ端结构域具有明显促进血小板生成的作用．     

肝细胞导向的促血小板生成素基因转移对化疗后血小板减少症模型治疗的初步研究

为了探讨受体介导的基因转移技术在治疗血小板减少症方面应用的可行性,将促血小板生成素（Ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）基因克隆入质粒型ＥＢ病毒表达载体ｐＤＲ２中,并与半乳糖化组蛋白结合,从而制备了一种为肝细胞表面特异的脱唾液酸糖蛋白受体识别并内吞的核酸－蛋白复合物。在经化疗药物卡铂诱发的血小板减少症的实验动物大鼠中,同时静脉注射该复合物,可将ＴＰＯ基因特异地导入肝细胞并在其中得到表达。从而有效地阻止了血小板减少症的发生,提示了一种以非病毒感染方式对化疗后血小板减少症进行有效基因治疗的可能前景     

人血小板生成素重组载体的构建与体外表达

应用基因克隆技术,以人ＴＰＯｃＤＮＡ为目的基因,构建真核细胞表达重组体ＶＲＴＰＯ,藉脂质体将其转染于ＮＩＨ３Ｔ３细胞,应用ＰＣＲ、ＲＴ－ＰＣＲ及Ｗｅｓｔｅｒｎ印迹等技术对其转染及表达情况进行鉴定．结果表明：ＶＲＴＰＯ构建成功,在ＮＩＨ３Ｔ３细胞可表达人ＴＰＯ,为应用人ＴＰＯｃＤＮＡ进行质粒ＤＮＡ骨骼肌直接注射于动物体内的基因治疗研究奠定了基础     

肝细胞导向的促血小板生成素基因转移对化疗后血小板减少症模型治疗 …

为了探讨受体介导的基因转移技术在治疗血小板减少症方面的可行性,将促血小板生成素（ＴＰＯ）基因克隆入质粒型ＥＢ病毒表达载体ｐＤＲ２中,并与半乳糖化组蛋白结合,从而制备了一种为肝细胞表面特异的脱唾液酸糖蛋白受体识别并内吞的核酸－蛋白复合物。在经化疗药物卡铂诱发的血小板减少症的实验动物大鼠中,同时静脉注射该复合物,可将ＴＰＯ基因特异地导入肝细胞并在其中得到表达。从而有效地阻止了血小板减少症的发生,提示了     

血小板生成素的研究进展

自１９９４年ＤｅＳａｕｖａｇｅ等克隆出了人的血小板生成素（ＴＰＯ）的ＣＤＮＡ后,对ＴＰＯ的结构与功能的研究有了突破性的进展。本综述了近几年对ＴＰＯ研究的最新献,较系统地介绍了ＴＰＯ基本结构、ＣＤＮＡ、ＴＯＰ受体的基因结构特征和ＴＰＯ基因的染色体定位及生物学作用。     

Expression, purification, and characterization of human recombinant thrombopoietin in Chinese hamster ovary cells

Thrombopoietin (TPO) is a primary regulator of megakaryocytopoiesis, a process through which megakaryocytes proliferate and mature into platelets. Recombinant human TPO (rhTPO) was expressed in Chinese hamster ovary (CHO) cells and purified from the culture medium. The cDNA encoding full-length TPO, including the native signal peptide sequence, was amplified by PCR from a human fetal liver cDNA library. The product was cloned into a mammalian expression vector under the control of the SV40 early promoter and enhancer. Secreted rhTPO was purified in three conventional chromatography steps. It migrates on SDS-PAGE as a broad band, characteristic of a heavily glycosylated protein, with an average molecular mass of 85 kDa. rhTPO expressed in CHO cells is biologically active in vitro as demonstrated by its ability to stimulate the proliferation of a megakaryocytic cell line and to trigger the JAK/STAT signal transduction pathway. rhTPO also shows activity in vivo as judged by the elevation of platelet count in treated mice.     

Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis

Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.     

Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells

When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.     

Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

C-terminal region of hTPO is important for secretion and expression in insect cells.

Human thrombopoietin (hTPO) variant cDNAs truncated in the C-terminal regions of wild-type hTPO (332 amino acids) were constructed by PCR and expressed in Trichoplusia ni (Tn5) insect cells using a baculovirus expression system. Each variant, hTPO163 (amino acids 1-163), hTPO198 (1-198) and hTPO245 (1-245), was produced in insect cells with very low efficiency in comparison with wild-type hTPO. Immunoblot analysis showed that the predicted 20, 25 and 34 kDa molecular sizes corresponding to hTPO163, hTPO198 and hTPO245, respectively, were barely detected in culture medium and most of the proteins remained within the cell. These results suggest that C-terminal regions containing potential N-glycosylation sites of hTPO are required for the secretion of hTPO into culture medium as well as expression in insect cells.     

精氨酸脱亚氨酶的表达、纯化和活性研究

目的：建立精氨酸脱亚氨酶的高效表达菌种和纯化工艺路线。方法：人工合成编码支原体精氨酸脱亚氨酶（arginine deiminase, ADI）的基因，构建pBV220-ADI原核表达载体，转染大肠杆菌DH5α中并诱导表达目的蛋白，离子交换层析和分子筛层析法纯化目标蛋白。采用体外精氨酸降解试验测定纯化产物活性。结果：成功构建了原核表达载体pBV220-ADI，基因侧序正确。转化大肠杆菌DH5α后筛选到高水平表达目的蛋白的菌株，目标蛋白以包含体形式存在于胞浆内，表达水平超过全菌体蛋白的35%。采用盐酸胍溶解包含体、低温条件下稀释和透析的方法进行复性。顺次采用阳离子交换和凝胶过滤层析对复性液进行纯化，最终获得纯度达到95%的活性产物。活性测定表明，纯化的ADI比活性为80IU/mg。结论：成功构建了ADI的高效表达菌种，建立了目标物质的分离纯化方法。     

Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli

Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines.     

以融合蛋白形式在大肠杆菌中表达人MCP－1

将编码人单核细胞趋化蛋白－１（ＭＣＰ－１）的基因亚克隆到大肠杆菌表达载体ｐＥＸ３１Ａ中,在大肠杆菌中表达出ＭＳ２／ＭＣＰ－１融合蛋０白,该表达产物约占菌体总蛋白的１５％左右,Ｗｅｓｔｅｒｎｂｌｏｔ检测表明,表达产物可与ＭＣＰ－１抗体特异反应。采用琼脂糖平板法进行活性测定表明,表达产物具有明显的单核细胞趋化活性,说明Ｎ端融合一段细菌蛋白对ＭＣＰ－１有无趋化活性可能没有影响。