摘要
随着社会的发展，由多种原因引起的血小板减症越来越常见。血小板由骨髓中的巨核细胞产生，通过对巨核细胞增殖分化进行调控，可有效促进血小板生成。因此，具有升血小板作用的造血生长因子类基因工程产品倍受推崇。然而，目前临床上用于救治血小板减少症的该类药物种类稀少，且存在着可能产生中和性抗体、副反应突出、价格昂贵等多种缺点，临床上迫切需要能够高效升血小板的新型药物。
血小板生成素(thrombopoietin, TPO)，又称巨核细胞生长发育因子(megakaryocytegrowth and development factor, MGDF)，是调节巨核细胞生成和血小板产生最重要的细胞因子。通过与其受体(c-Mpl)结合，TPO能够激活巨核细胞中一系列的信号通路，发挥其促进巨核细胞增殖分化的功能。据此，人们尝试利用基因工程方法制备出重组人血小板生成素(recombinant human thrombopoietin, rhTPO)和聚乙二醇化重组人巨核细胞生长发育因子(pegylated recombinant human megakaryocyte growth and developmentfactor, PEG-rhMGDF)两种第一代促血小板生长因子类药物。但是，PEG-rhMGDF在临床试验中产生了能够与内源性TPO交叉反应的中和性抗体，美国FDA因此全面禁止了重组人TPO类基因工程产品的临床实验。TPO模拟肽(thrombopoietin mimetic peptide,TMP)是通过噬菌体表面展示技术鉴定出的一种由14个氨基酸组成，与c-Mpl具有高亲和力，但与TPO序列完全不同源的短肽。实验表明，TMP不仅能够在体外有效促进巨核细胞增殖，且在动物体内未发现其能够诱导产生中和性TPO抗体。然而，TMP分子量太小，难以通过基因工程方法制备，且在循环中极易降解，限制了其应用。
多项体内研究发现，人生长激素(human growth hormone, hGH)对造血干/巨核祖细胞增殖分化及促进血小板生成具有重要调控作用。hGH主要通过与其受体(growthhormone receptor, GHR)结合发挥其生物学活性，该受体与c-Mpl同为I类细胞因子受体，能够激活相似的增殖分化信号通路。因此，我们设想将TMP与hGH进行融合，一方面利用与生长激素进行融合，提高分子量，使得TMP能够通过基因工程方法进行制备，同时延长其体内半衰期。另一方面，通过融合发挥TMP与hGH在促进巨核细胞生成及血小板产生方面的协同作用。
基于已有的研究基础，在国家“863”计划和新药创制科技重大专项课题的资助下，我们首先尝试通过巴斯德毕赤酵母表达系统对一个TMP分子与hGH的融合蛋白rTMP-GH进行了发酵表达及纯化制备。之后，为了提高融合蛋白的生物学活性，又设计了一种由两个串联的TMP分子(TMP二联体)与hGH组成的融合蛋白，并利用pMAL-p2x原核表达载体在大肠杆菌中进行可溶性表达与制备。在确认dTMP-GH具有显著促进巨核细胞生成活性的基础上，为了简化制备工艺、提高产量，进一步探索了在大肠杆菌中以温控诱导和包涵体表达方式生产制备dTMP-GH融合蛋白，通过工艺优化最终实现了该重组融合蛋白的高密度发酵表达及放大生产，获得了纯度大于98%，且具有高效促巨核细胞增殖分化活性的dTMP-GH融合蛋白基因工程产品。同时，从信号通路激活的角度对dTMP-GH融合蛋白促巨核细胞增殖分化的作用机制进行了探讨。所取得的主要研究结果与结论如下：
1.通过基因重组成功构建了pPIC9K-rTMP-GH真核重组表达质粒，筛选出高G418抗性的His+/Mut+多克隆表达菌株，实现了rTMP-GH在巴斯德比赤酵母GS115中的高密度发酵表达。经超滤、阴离子柱及分子筛纯化，获得了纯度大于95%的rTMP-GH重组融合蛋白。经体外实验证实rTMP-GH具有一定的促进巨核细胞增殖活性。
2.通过合理化设计酶切位点，成功构建了pMAL-dTMP-GH重组表达质粒。电转化大肠杆菌后，阳性克隆经IPTG诱导，获得可溶性融合蛋白MBP-dTMP-GH。通过亲和层析、Xa酶切、疏水株及分子筛再纯化，最终得到纯度达98.5%，且N端不含任何多余氨基酸的重组融合蛋白dTMP-GH。体外细胞实验证实，dTMP-GH能够以剂量依赖方式促进巨核祖细胞株M07e的增殖，其活性显著优于等摩尔剂量的rTMP-GH融合蛋白。
3.利用温控原核表达载体pBV220，通过包涵体形式实现了dTMP-GH融合蛋白在大肠杆菌中的高密度发酵表达，表达水平约为500mg/L。建立了包括包涵体变性、复性、阳离子柱层析、阴离子柱层析及分子筛层析的纯化方案，获得了纯度大于98%的dTMP-GH融合蛋白，得率为20%。其生物学活性与采用大肠杆菌分泌性表达方式制备的dTMP-GH无显著性差异。
4.体外实验证实，dTMP-GH促进早期巨核细胞增殖的活性较等摩尔剂量dTMP强，GH拮抗剂培维索孟预处理能够显著削弱dTMP-GH的促巨核细胞增殖活性，提示融合蛋白中的hGH具有增强dTMP促巨核细胞增殖分化的作用，而且这种增强作用可能通过巨核细胞表面的GHR介导发挥。
5. Western blot分析结果表明， dTMP作用15min即可显著上调M07e细胞中STAT5和ERK1/2的磷酸化水平，1h左右磷酸化水平恢复正常，而rhGH却无明显促STAT5磷酸化作用，但在其作用1h后可出现ERK1/2的显著磷酸化，并可持续3h以上时间。提示rhGH可能通过缓慢而持续激活ERK1/2信号通路来增强dTMP诱导的巨核细胞增殖与分化。
6.激光共聚焦及Western blot检测分析发现，rhGH处理能够显著上调成熟巨核细胞株Meg-01中肌动蛋白的表达，并能促进肌丝拉伸和细胞伪足伸出，提示rhGH具有促进巨核细胞分化成熟的作用。进一步研究发现，rhGH作用15min即可显著上调Meg-01中Akt的磷酸化水平，而dTMP对Akt无明显激活作用，提示rhGH可能通过激活Akt信号通路促进了巨核细胞的分化成熟。
7. dTMP-GH融合蛋白可同时发挥dTMP与rhGH的生物学活性。在M07e中，dTMP-GH作用可同时上调STAT5和ERK1/2的磷酸化水平，且ERK1/2磷酸化可持续3h以上。给予GH拮抗剂培维索孟能够抑制dTMP-GH对ERK1/2信号通路的持续激活作用。在Meg-01中，dTMP-GH作用可显著上调Akt的磷酸化水平，且该作用能够被培维索孟显著抑制。
8.对人脐血CD34+来源的原代巨核细胞的实验证明，dTMP-GH能够显著上调培养至7d原代巨核细胞STAT5和ERK1/2磷酸化水平，且ERK1/2磷酸化可持续至3h以上。并且dTMP-GH作用可显著激活培养至11d原代巨核细胞中的Akt信号通路。
总之，本研究通过探索不同的表达体系、优化表达条件和纯化工艺，从而成功实现了一种TMP二联体与hGH的重组融合蛋白在大肠杆菌中的高效表达与制备，最终获得纯度大于98%的dTMP-GH融合蛋白。体外实验证实，dTMP-GH具有显著促进巨核细胞增殖分化活性，其中dTMP主要发挥促进巨核细胞早期增殖分化的作用，GH除了可以增强dTMP的促巨核细胞增殖活性外，还具有促进巨核细胞分化成熟的作用。因而，通过融合不仅间接增大了dTMP的分子量，使其可以通过基因工程方法进行制备，而且所形成融合蛋白还具有突出促巨核细胞增殖、分化和成熟的活性，显示出很好的开发应用前景。
With the development of society, thrombocytopenia caused by various reasonsbecomes more and more common. The megakaryocyte (MK) is a bone marrow cellresponsible for the production of platelets. Thrombopoiesis are ligations of the cytoplasmfrom megakaryocytes, can be promoted effectively by regulating the proliferation anddifferentiation of MKs. Therfore, considerable attention has been devoted togene-engineering products of hematopoietic growth factors which could promote plateletsproduction. However, these products for thrombocytopenia treatment in clinic are scarce atthe present stage, and the exisiting products have notable disadvantages, such as theproduction of neutralizing antibodies, prominent side effects and expensive costs. In all,there is an urgent demand for new drugs which can promote thrombopoiesis efficiently.
Thrombopoietin(TPO), which was previously termed as MK growth and developmentfactor(MGDF), is the most important cytokine which regulates megakaryocytopoiesis andthrombopoiesis. Through binding to its receptor(c-Mpl), TPO activates a cascade of signaltransduction pathways which promote MKs proliferation and differentiation. Accordingly,recombinant human thrombopoietin(rhTPO) and pegylated recombinant humanmegakaryocyte growth and development factor(PEG-rhMGDF) as the first generationthrombopoietic growth factors have been developed through gene-engineering approach.However, the emergence of autoantibody against PEG-rhMGDF, which could cross-reactwith and neutralize endogeous TPO, results a total ban on clinical trials of recombinanthuman TPO gene-engineering products by the U.S. FDA. Thrombopoietin mimeticpeptide(TMP), which was identified by phagy display technology, is a14-mer peptide withhigh affinity to c-Mpl and shares no homologous sequences with TPO. Studies showed thatTMP could promote MKs proliferation in vitro, but not induce TPO neutralizing antibody invivo. Nevertheless, TMP is hardly to be prepared by gene-engineering methods due to its small molecular weight, and it is easy to be degraded in the circulation, which together limitits application.
Human growth hormone(hGH) has been reported to play an important role inproliferation and differentiation of hematopoietic and megakaryocytic progenitor cells, andenhance thrombopoiesis. hGH exerts its function mainly by binding to GH receptor(GHR),a kind of type I cytokine receptor as c-Mpl, and in turn activates the proliferation anddifferentiation related signal pathways. Therefore, we envisaged to construct a chimericprotein which fused TMP to hGH. On one hand, fusion with hGH increases molecularweight, which makes preparation of TMP through genetic engineering approach possibleand extends its half life in vivo; on the other hand, by fusion with hGH, there may be asynergetic effect on megakaryocytopoiesis and thrombopoiesis.
Based on our previous works, with funding of national “863” plan and major scienceand technology poject of new drug development, the chimeric protein rTMP-GH was triedto prepared by expression and purification in Pichia pastoris. In order to improve thebioactivity of the fusion protein, a chimeric protein containing a tandem dimmer of TMP(dTMP) fused to hGH was then constructed in Escherichia coli with pMAL-p2x expressionvector. After identifying its significant effect on promotion megakaryocytopoiesis,dTMP-GH was expressed in inclusion body form through temperature control inducing inEscherichia coli, which simplifies the preparation process and improve the yield, Byoptimizing the technology, high densty fermentation and production amplification of thefusion protein were successfully achieved. Finally, a gene-engineering product ofdTMP-GH with high purity over than98%and fuction of promoting the proliferation anddifferentiation efficiently was obtained. At the same time, the mechanism of dTMP-GHpromoting the proliferation and differentiation of MKs was discussed from the perspectiveof signaling pathways. The main results and conclusions are summarized as follows:
1. The recombinant eukaryotic expression plasmid pPIC9K-rTMP-GH was constructedsuccessfully by genetic recombination. The His+/Mut+polyclonal expression strain withhigh G418resistance was screened out, followed by the expression of rTMP-GH throughhigh density fermentation in Pichia pastoris GS115. The recombinant fusion proteinrTMP-GH with purity over than95%was then obtained by purification with ultrafiltration,anion column and molecular sieve. A certain activity on promoting MKs proliferation of rTMP-GH was confirmed in vitro.
2. By rational designed restriction sites, the recombinant expression plasmidpMAL-dTMP-GH was constructed successfully. After electrically transformation toEscherichia coli, the soluble fusion protein MBP-dTMP-GH was obtained through IPTGinduction in positive clone. By application of amylase resin chromatography, Xa factordigestion, hydrophobic chromatography followed by gel filtration, the dTMP-GH fusionprotein with purity of98.5%and without redundant amino acid at N terminal was obtained.The functional assays in vitro showed that dTMP-GH could promote the proliferation ofmegakaryoblast cell line M07e in a dose-dependent manner. Moreover, an enhanced effectof dTMP-GH was found as compared with equimolar concentration of rTMP-GH fusionprotein.
3. By using of prokaryotic expression vector pBV220, the expression of dTMP-GHthrough high densty fermentation in inclusion body form was achieved successfully, withthe yield of500mg/L. A purification strategy including inclusion body denaturation,refolding, cation chromatography, anion chromatography and molecular sievechromatography was established to obtain dTMP-GH with purity over than98%andpencent recovery of20%. There was no significant difference in the bioactivities ofdTMP-GH obtained through the above two approaches.
4. The MKs proliferation activity of dTMP-GH was significantly stronger thanequimolar concentration of dTMP and obviously weaken by GH antagonist Pegvisomant,while there is no notable effect on MKs proliferation of rhGH. It suggested that hGH mayenhanced the effect on promoting MKs proliferation and differentiation of dTMP by GHRmediated.
5. Result of Western blot analyse displayed that the phosphorylation levels of STAT5and ERK1/2in M07e cells were up-regulated in15min and lasted to1h by dTMP. Therewas no detectable promotion of STAT5phosphorylation after rhGH treatment, but itinduced obviously phosphorylation of ERK1/2in1h and continuing for more than3h. Itsuggested that the proliferation and differentiation of MKs induced by dTMP may beenhanced by rhGH through slow and sustained activation of ERK1/2signaling pathway.
6. Confocal laser scanning and Western blot detection showed that rhGH treatmentup-regulated the expression of actin in mature megakaryocytic cell line Meg-01, in turn promoted myoneme stretching and pseudopodia extending. It indicated that rhGH has thefunction of promoting MKs maturation. Further research in Meg-01revealed that rhGHup-regulated the phosphorylation level of Akt significantly, which was not activatedobviously by dTMP. It suggested that rhGH promote MKs differentiation and maturationthrough the activating of Akt signal pathway.
7. The fusion protein dTMP-GH could exert bioactivites of both dTMP and rhGH. Thephosphorylation level of ERK1/2in M07e was up-regulated markedly and lasted more than3h by the treatment of dTMP-GH. And Pegvisomant was able to inhibited the sustainedactivation of ERK1/2by dTMP-GH. In Meg-01, the role of dTMP-GH was to up-regulatethe phosphorylation level of Akt, which could be inhibited by Pegvisomant too.
8. Evidence on human CD34+derived primary megakaryoctes cultured for7dconfirmed that dTMP-GH was able to up-regulated STAT5and ERK1/2phosphorylationlevel, and the phosporylation of ERK1/2sustained for over3h. Additionally, Akt signalpathway in cells cultured for11d could be activated by dTMP-GH.
In this study, by exploring different expression systems, optimizing expressionconditions and purification processes, we successfully fulfilled the efficiently expressionand preparation of a recombinant fusion protein that constructed with a tandem dimmerTMP fused to rhGH in Escherichia coli. A fusion protein of dTMP-GH with high purityover than98%was finally obtained. Experiments in vitro confirmed that dTMP-GH had theactivity of promoting the proliferation and differentiation of MKs. The major effect ofdTMP was to promote the proliferation and differentiation of early MKs, while GHenhanced the activity of dTMP on promoting MKs proliferation, as well as promoteddifferentiation and maturation of MKs. Therefore, the fusion of hGH not only increased themolecular weight of dTMP, which made it possible to be prepared by gene-engineeringapproach, but also had prominent effects on promoting proliferation, differentiation andmaturation of MKs. The novel thrombopoietic fusion protein, dTMP-GH, showed awonderful prospect for the development and application.
引文
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