胸腺素α1-胸腺五肽融合肽的制备、体内外免疫调节与辅助抗肿瘤作用及与TLR2结合性质研究
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摘要
恶性肿瘤致死率高,是当前严重影响人类健康、威胁人类生命的主要疾病之一。目前,临床上主要采用的非手术治疗手段有化学治疗、放射治疗等,但这些手段在治疗肿瘤的同时也使患者的免疫功能受到严重破坏。因此,寻找能够在肿瘤治疗中提高机体免疫功能的免疫调节剂具有重大的社会和经济意义。胸腺五肽(Thymopentin, TP5)和胸腺素α1(Thymosin, Tα1)是从胸腺中获得的两种多肽,二者具有相似的免疫调节活性,是重要的免疫调节剂,已成功用于临床肿瘤辅助治疗,能提高化疗或放疗病人的免疫应答及耐受性,抑制或降低病情的进展或复发,延长病人的生存期,提高病人的生活质量。但是,TP5的体内半衰期(t1/2)很短,人体内药代学研究发现TP5的t1/2只有30s。需要频繁的给药才能维持疗效。Tα1l的体内t1/2较长(约为100min),但目前主要以化学合成的方法生产,价格昂贵,难以被广大患者所接受。
为了提高TP5的活性,延长其体内t1/2,本课题组已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,在毕赤酵母Pichia pastoris中表达制备了融合肽Tα1-TP5。研究证实Tα1l-TP5具有比TP5更强的体外刺激小鼠脾淋巴细胞增殖及体内促进巨噬细胞吞噬的能力,Tα1-TP5也具有比TP5及Tα1更长的体外血浆t1/2。但是,Tα1-TP5经Pichia pastoris分泌表达的量很少,无法满足进一步体内试验研究的需求,并且需要凝血酶切割去掉融合标签,纯化成本高,纯化步骤繁琐。切割后,Tα1-TP5的N端带有2个多余的氨基酸残基,C端带有多余的4个氨基酸残基,可能存在免疫原性。
故为了提高产量并降低纯化成本,本课题换用大肠杆菌表达体系,采用内含肽介导的纯化系统,加入二硫苏糖醇(DTT)诱导内含肽自切割,分别从细菌裂解上清及包涵体中纯化Tα1-TP5。考察Tα1-TP5是否具有比TP5及Tαl更强的体内免疫调节活性及辅助抗肿瘤作用,并进一步采用表面等离子共振技术(SPR)研究Tal-TP5与Toll样受体2(TLR2)的结合情况,为将Tα1-TP5开发成一种新型的免疫调节剂提供重要的基础与依据。
本课题的研究内容及取得的主要成果有以下几个方面。
1Ta1-TP5在大肠杆菌中的表达及纯化
采用大肠杆菌表达系统对Tα1-TP5进行了融合表达。选用pTYB2作为表达载体,将Tα1-TP5基因插入至内含肽(Sce intein)基因的N端,Sce intein基因的C端与几丁质结合域(Chitin binding domain, CBD)相连,构建重组质粒pTYB2-Tal-TP5-Sce intein-CBD,转入E.coli ER2566中诱导表达重组蛋白。超声裂解菌体后SDS-PAGE考察重组蛋白的可溶表达情况。考察不同诱导温度及诱导剂异丙基硫代-β-D-半乳糖苷(IPTG)浓度对重组蛋白可溶表达量的影响。采用亲和层析对重组蛋白进行纯化,加入DTT诱导intein柱上自切割,从而释放Tα1-TP5。为了提高Tα1-TP5的产量,也从包涵体中纯化了Tα1-TP5。摸索了包涵体的洗涤条件,采用直接稀释法对包涵体蛋白Tα1-TP5-Sce intein-CBD进行复性,并考察还原剂和氧化剂的比例、L-精氨酸(L-Arg)及最终尿素的浓度对复性效率的影响。复性后的蛋白过亲和层析柱,加入DTT诱导intein柱上自切割,从而获得Tα1-TP5。Tα1-TP5进一步经Superdex30(?)钝化并脱盐后,ESI-MS鉴定其分子量的正确性。Tricine-SDS-PAGE鉴定Tα1-TP5的纯度。
结果显示,Tα1-TP5成功在E.coli ER2566中表达,融合蛋白部分以包涵体的形式存在,诱导温度由37℃降低至16℃时可使可溶蛋白的表达量由原来的15.9%增加至18.9%,而IPTG浓度由1mmol/L降至0.1mmol/L对可溶蛋白表达量影响不大。复性体系中还原剂和氧化剂的比例为1：22.5、尿素终浓度为0.8mol/L时复性效率最高。L-Arg的加入对复性效率影响不大。Superdex30进一步纯化和脱盐后,从菌体裂解上清中获得了约4.6mg Tα1-TP5,从包涵体中获得了约3.0mg Tα1-TP5。 ESI-MS分别鉴定分子量,发现与理论值相当(3785.02Da), Tricine-SDS-PAGE检测Tal-TP5的纯度大于95%。最终我们运用此方法从每L发酵液中得到了约7.6mgTal-TP5。
2Tal-TP5体内外免疫调节活性研究
采用CCK-8法测定了Tal-TP5对小鼠脾淋巴细胞增殖的促进作用,采用流式细胞术考察了Tα1-TP5对小鼠脾淋巴细胞表面CD69分子表达的促进作用,采用RT-PCR技术检测Tα1-TP5对小鼠脾淋巴细胞IFN-y表达的的影响。结果表明：从上清及包涵体中纯化的Tα1-TP5均能促进小鼠脾淋巴细胞的增殖,且作用呈浓度依赖性,浓度为40μg/mL时,增殖率分别为26.21%、21.84%,与阴性组比较差异具有显著性意义(p<0.01,p<0.01)。Tα1-TP5能促进CD69的表达,与阴性对照组比较差异具有显著性意义(21.51%vs.9.5%,p<0.05),Tα1-TP5促进淋巴细胞表面CD69表达的能力也较TP5(16.06%)和Tα1(16.65%)高。RT-PCR结果显示Tα1-TP5能在基因水平上促进IFN-y的表达,且促进IFN-y表达的能力高于TP5及Tα1。说明我们纯化的Tα1-TP5具有比TP5及Tα1更强的免疫调节活性。
连续2天腹腔注射剂量为250mg/kg氢化可的松(HC),建立小鼠体内免疫抑制模型,皮下注射Tα1-TP5连续治疗2周,考察Tα1-TP5对小鼠免疫抑制的抵抗作用。结果发现,Tα1-TP5能明显增强免疫抑制小鼠的胸腺指数,与模型组比较差异具有显著性意义(2.253±0.427vs.0.444±0.407,p<0.05),与TP5及Tα1单独给药组比较差异也具有显著性意义(p<0.05)。但Tα1-TP5X(?)脾脏指数的影响不大。HE染色分析Tα1-TP5能改善HC导致的胸腺萎缩,增加胸腺组织中胸腺细胞的数量。另外Tα1-TP5能使CD4+CD8+胸腺细胞的比例恢复至正常水平。与模型组比较,Tα1-TP5显著增加外周血中IFN-γ的含量,抵抗HC造成的IFN-γ水平降低。总之,Tα1-TP5具有改善HC诱导的免疫抑制状态的能力,且活性比TP5及Tα1l更强。
3Tal-TP5辅助抗肿瘤作用研究
采用CCK-8法测定了Tal-TP5(?)(?)乳腺癌细胞MDA-MB-231、肝癌细胞HepG2、肺癌细胞A549及黑色素瘤细胞B16四种肿瘤细胞增殖的抑制作用,结果表明：Tα1-TP5对四种肿瘤细胞生长的抑制作用呈时间依赖性,对MDA-MB-231细胞的抑制作用最强,72h时细胞活力只占阴性组的64.6%,显著高于TP5组(64.6%vs.90.8%,p<0.05),但是与Tαl组比较没有显著性差异(64.6%vs.70.2%,p>0.05)。Tα1-TP5对HepG2细胞和A549细胞的抑制作用稍弱于对MDA-MB-231细胞,72h时细胞活力分别占阴性组的73.9%及69.9%,抑制作用强于TP5但与Tα1差别不大。Tα1-TP5对B16细胞也有一定的抑制作用,72h时细胞活力只占阴性组的82.5%,抑制作用与TP5及Tα1相比没有显著性差异。
建立C57BL/6小鼠荷黑色素瘤模型,考察Tα1-TP5联合环磷酰胺(CY)对肿瘤生长的抑制作用。结果表明：与CY单独给药相比,Tα1-TP5联合CY明显降低肿瘤体积及肿瘤重量,药物治疗2周后肿瘤重量减少了将近88.1%。Tα1-TP5也能逆转CY导致的外周血中白细胞数量减少,治疗2周后外周血中白细胞数量增加至正常水平。流式细胞术检测肿瘤组织中组织相容性复合体I(MHC I)的表达,发现CY组MHC I的表达率只有11.65%,而Tα1-TP5能显著增加肿瘤组织中MHC I的表达,与CY组比较差异具有显著性(28.71%vs.11.65%,p<0.05)。免疫组化结果显示Tα1-TP5较TP5及Tα1更能促进肿瘤组织中CD8+T细胞的浸润及CD86的表达。可见Tα1-TP5具有弱的直接杀伤B16细胞的功能,主要通过激活免疫系统而发挥辅助抗肿瘤作用。
4Tα1-TP5与TLR2结合活性研究
采用激光共聚焦显微镜观察考察Tα1-TP5与骨髓来源的巨噬细胞(BMDMs)的结合情况。将Tα1-TP5、TP5及Tα1分别用异硫氰酸荧光素(FITC)标记,并分别与分离的BMDMs孵育2h,再用Hoechst33342及DiI分别标记细胞核及细胞膜,激光共聚焦显微镜观察药物结合情况。结果发现,Tα1-TP5、P5及Tα1均能结合在BMDMs的细胞膜上。
采用SPR技术检测Tα1-TP5与存在于BMDMs上的TLR2(?)的结合。将TLR2作为配体以氨基偶联的方式偶联到CM5芯片上,Tα1-TP5、TP5及Tα1作为待分析物流过芯片表面,考察三种多肽与TLR2的结合能力及亲和力大小。结果显示：SPR技术能够准确反映三种多肽与TLR2(?)的结合水平。Tα1-TP5、TP5及Tα1与TLR2的解离平衡常数(KD)分别为6.84×10-6mol/L、1.57×10-6mol/L及35.4×10-6mol/L。 Tα1-TP5与TLR2的结合能力显著强于Tα1,但与TP5相比没有显著性差异。到目前为止,三种多肽可能的受体首次被确定。
本研究取得的主要成果和结论如下：
(1)首次构建了表达Tα1-TP5的大肠杆菌表达系统,对重组蛋白质成功进行了表达,并利用内含肽介导的纯化系统分别从细菌裂解上清及包涵体中分离纯化了Tα1-TP5, Superdex30进一步纯化得到了高纯度的目的多肽。获得的Tal-TP5C(?)(?)只含有一个多余的甘氨酸(Gly)残基。此研究提供了一种高效率、低成本的纯化策略,适合Tα1-TP5的大规模生产。
(2)纯化的Tα1-TP5能明显促进小鼠脾淋巴细胞的增殖、CD69及IFN-γ的表达,展示了较好的体外免疫调节活性。
(3)首次进行了Tα1-TP5的体内免疫调节活性研究。Tα1-TP5能抵抗HC诱导的免疫抑制,且效果较TP5及Tα1强。
(4)首次进行了Tα1-TP5的辅助抗肿瘤作用研究。Tα1-TP5联合CY展现了很好的抗肿瘤活性,同时Tα1-TP5激活了被CY抑制的免疫系统,增强对肿瘤的杀伤。Tα1-TP5(?)的辅助抗肿瘤活性较TP5及Tα1强。
(5)首次研究了Tα1-TP5的受体,Tal-TP5能与TLR2结合,提示Tα1-TP5介导免疫应答可能是通过TLR2信号通路。
Malignant tumors, which have high mortality, are one kind of the major diseases that seriously affect human health and threaten human life. Currently, the commenly used methods in the treatment of tumors in clinic are chemotherapy and radiotherapy, but, these remedies result in the severe damage on the immune function of patients. Therefore, searching for immunomodulators which can improve the immune function in cancer therapy has significant social and economic significance. Thymopentin (TP5) and thymosin alpha1(Tα1) are two thymic peptides with similar immunoregulatory activities and are being used in clinic for the treatment of some malignancies. They can improve the immune response and tolerance of patients to chemotherapy or radiotherapy, inhibit or reduce the progression of the diseases and improve the patient's quality of life. However, the in vivo half-life (t1/2) of TP5is very short, which is only30s. So. frequent administration is required to maintain its efficacy, which brings great inconvenience to patients. Tα1is mainly synthesized and purified by the method of solid phase synthesis, which is very expensive. The high price of Tal is a burdern for the majority of patients.
In order to prolong the t1/2and enhance the activity of TP5. a Tα1-TP5fusion peptide was designed and expressed in Pichia pastor is by our research team. Previous study showed that Tα1-TP5had higher activity than TP5in inducing T-lymphocyte proliferation in vitro. In in vivo carbon clearance test, the abilities of Tα1-TP5in promoting phagocytic capability of macrophages as well as the secretion of IL-2in peripheral blood of mice were also higher than those of TP5and Tα1. Besides. Tal-TP5had longer in vitro plasma t1/2than TP5and Tα1. However, the amount of Tα1-TP5secreted from Pichia pastor is was small and could not meet the demand of further in vivo study. Besides, the thrombin was needed to remove the fusion tag to obtain Tα1-TP5, which resulted in the high purification costs and complex purification steps. After thrombin cutting, two extra amino acids existed in the N-terminal of Tal-TP5and four extra amino acids existed in the C-terminal of Tα1-TP5, which might increase immunogenicity.
To obtain large quantity of active Tα1-TP5with low cost, a new intein-fused expression and purification strategy was used for the production of Tα1-TP5fusion peptide from the soluble fraction and inclusion bodies. The in vivo immune regulating activity and synergistic anti-tumor activity of Tα1-TP5were also investigated. Besides, the potential receptor of Tα1-TP5was investigated by surface plasmon resonance (SPR) binding studies. In sum, the research may provide important basis for developing Tα1-TP5as a novel immunomodulatory agent.
The main contents and results obtained were summarized as follows:
1Expression and purification of Tα1-TP5in Escherichia coli
Tα1-TP5was expressed in Escherichia coli using pTYB2as the expression vector. The gene of Tα1-TP5was inserted into the N-terminal of the gene of Sce intein-Chitin binding domain (CBD). Then the recombinant plasmid pTYB2-Tα1-TP5-Sce intein-CBD was transformed into Escherichia coli ER2566and recombinant protein Tα1-TP5-Sce intein-CBD was expressed in ER2566. After sonication, SDS-PAGE was used to confirm whether the recombinant protein was soluble. The influences of different induction temperature and the concentration of isopropyl-β-D-galactoside (IPTG) on the amount of soluble recombinant protein were examined. Affinity chromatography was used to purify the recombinant protein. The target peptide was released from the chitin beads via self-cleaving of intein induced by dithiothreitol (DTT). In order to increase production, Tal-TP5was also obtained from inclusion bodies. The condition of washing inclusion body was investated. Subsequently, the inclusion body was renatured by direct dilution method. Besides, the effect of redox ratios, L-Arg and final urea concentration on refolding efficiency was evaluated. The refolded protein was applied to affinity chromatography columns, and Tα1-TP5was released from the chitin beads via self-cleaving of intein induced by DTT. Further, Tα1-TP5was purified and desalted by Superdex30. The correctness of its molecular weight was identified by ESI-MS and the purity of Tα1-TP5was evaluated by Tricine-SDS-PAGE.
Tα1-TP5was successfully expressed in E.coli ER2566. After sonication, the fusion protein was partially in the form of inclusion bodies. As the induction temperature decreased from37℃to16℃, the amount of soluble protein increased from15.9%to18.9%of total bacterial protein. Change of IPTG concentration from1mmol/L to0.lmmol/L showed no effect on soluble expression level. When redox ratio (reduced:oxidized) was1:22.5and the final urea concentration was0.8mol/L, the fusion protein has the highest refolding efficiency. L-Arg showed no effect on the refolding efficiency. After further purified and desalted by Superdex30,4.6mg Tal-TP5was obtained from the supernatant and3.0mg Tal-TP5was obtained from inclusion bodies per liter medium. The purity was up to95%determined by16.5%Tricine-SDS-PAGE. Finally, this approach reported here allowed the production of approximately7.6mg of Tα1-TP5from1L shaking flask culture of E. coli.
2Study on the in vitro and in vivo immunomodulatory activity of Tal-TP5
The ability of Tal-TP5on promoting mouse spleen lymphocytes proliferation was detected by CCK-8assay. The effect of Tα1-TP5on promoting the expression of CD69on mouse spleen lymphocytes was investigated by flow cytometry. Then the effect of Tal-TP5on promoting the expression of IFN-γ was detected by RT-PCR. The result showed that Tal-TP5purified from the supernatant and inclusion bodies could promote the proliferation of mouse spleen lymphocytes in a concentration-dependent manner. When the concentration of Tα1-TP5was40μg/mL. the proliferation rate was26.21%and21.84%, respectively. Compared to negetive control, the difference was significant (p<0.01, p<0.01). Tα1-TP5could promote the expression of CD69and compared to negative control, the difference was significant (21.51%vs.9.5%, p<0.05). The ability of Tα1-TP5in promoting CD69expression was also higher than that of TP5(16.06%) and Tα1(16.65%). RT-PCR results showed that Tα1-TP5could promote IFN-γ expression at the gene level, and the ability was higher than that of TP5and Tα1. These all indicated that purified Tal-TP5possessed stronger immune regulating activity than TP5and Tal.
The immunosuppressive mice model was established by intraperitoneal injection of hydrocortisone (HC) at a dose of250mg/kg for two consecutive days. Tα1-TP5was subcutaneously injected for two consecutive weeks to evaluate the resistance of Tal-TP5to immunosuppression induced by HC. The results showed that, Tal-TP5could significantly enhance the thymus index of immunesuppressed mice, and compared with the model group, the difference was significant (2.253±0.427vs0.444±0.407,p<0.05). Compared with the TP5and Tal alone group, the difference was also significant (p<0.05). However, the effect of Tα1-TP5on spleen index was little. HE staining indicated that Tal-TP5could antagonize thymic atrophy induced by HC and increase the number of thymocytes in the thymus tissue. In addition, Tal-TP5made CD4+CD8+thymocytes returned to normal levels. Compared with model group, Tal-TP5significantly increased the concentration of IFN-γ in the peripheral blood. In sum, Tal-TP5has the ability to alleviate the immunosuppression induced by HC, and the activity was stronger than that of TP5and Tα1.
3Study on the synergistic anti-tumor activity of Tal-TP5
The inhibitory effect of Tal-TP5on breast cancer cell MDA-MB-231, lung cancer cell A549, hepatoma carcinoma cell HepG2and melanoma cell B16was evaluated by CCK-8assay. Tal-TP5could inhibit the proliferation of these four tumor cells in a time-dependent manner and Tal-TP5showed the highest inhibitory effect on MDA-MB-231. After72h incubation, cell viability was only64.6%of that of the negetive control, and compared to TP5group, the difference was significant (64.6%vs.90.8%, p<0.05), but compared to Tal group, there was no significant difference (64.6%vs.70.2%, p>0.05). The inhibitory effect of Tal-TP5on HepG2cell and A549cell was weaker than on MDA-MB-231cell, and cell viabilities were73.9%and69.9%of that of the negative group after72h incubation, which was stronger than that of TP5but had no significant difference from that of Tα1. Tα1-TP5also could inhibit the proliferation of B16cells, and after72h incubation, the cell viability was82.5%of that of the negative group. Compared to TP5and Tal group. there were no significant differences.
C57BL/6mice were inoculated subcutaneously with2×105of B16melanoma cells/mouse in the forelimb armpit. The effect of Tα1-TP5in combination with cyclophosphamide (CY) on inhibition of tumor growth was investigated. Compared to CY alone, Tα1-TP5combined with CY significantly reduced tumor volume and tumor weight, and the tumor weight was reduced by nearly88.1%after two weeks treatment. Tal-TP5also increased leukocyte number lowered by CY treatment, and leukocyte number in peripheral blood increased to normal levels after two weeks treatment of Tα1-TP5. In CY group, MHC I expression rate was only11.65%detected by flow cytometry, however, Tal-TP5significantly increased MHC I expression in the tumor tissue, and compared to CY group, the difference was statistically significant (28.71%vs.11.65%, p<0.05). Immunohistochemistry results showed that Tal-TP5promoted more CD8+T cell infiltration and CD86expression in tumor tissue than TP5and Tα1. From all the results above it was concluded that Tal-TP5alone had weak killing effect on tumor cells and mainly activated the immune system in combination therapy.
4Study on the binding character of Tal-TP5to TLR2
Confocal microscope was used to observe the binding character of Tα1-TP5to bone marrow-derived macrophages (BMDMs). Tα1-TP5. TP5and Tα1were labeled with fluorescein isothiocyanate (FITC) and incubated with cultured BMDMs for2h. Then, the cell nucleus and cell membrane were stained by Hoechst33342and Dil. respectively. The results showed that all the three peptides were located on the cell membrane of BMDMs.
SPR was used to detect the binding of Tα1-TP5to TLR2. which exist on the cell membrane of BMDMs. TLR2was immobilized by standard amine coupling using an amine coupling kit. The peptides were dissolved in running buffer, and a flow rate of30μL/min was employed for association and dissociation at a constant. SPR technology could accurately reflect the level of the binding of the three polypeptides with TLR2. The KD of Tal-TP5, TP5and Tal were6.84×10-6mol/L,1.57×10-6mol/L and35.4×10-6mol/L, respectively. The KD of Tal-TP5was much smaller than that of Tα1, indicating that Tα1-TP5had higher affinity to TLR2than Tα1. Compared with TP5, the affinity of Tal-TP5to TLR2was slightly low but there was no statistical significance. Thus, the potential receptor of the three peptides was first confirmed as TLR2.
The main results and conclusions are as follows:
(1) The expression strain of Tal-TP5was firstly constructed. The fusion protein was successfully expressed in E.coli and Tal-TP5was purified from the supernatant and inclusion bodes with intein-mediated purification systems. High purity of Tal-TP5was obtained by further purification with Superdex30. There was only a Gly residue at the end of Tal-TP5, which might greatly reduce the immunogenicity. The approach described here is a low-cost, convenient and potential way for obtaining a large amount of Tα1-TP5.
(2) Purified Tal-TP5can significantly promote the proliferation of mouse spleen lymphocytes, as well as the expression of CD69and IFN-y. Tal-TP5showed good in vitro immunoregulatory activity.
(3) The in vivo immunomodulatory activity of Tal-TP5was evaluated for the first time. Tα1-TP5can antagonize immune suppression induced by HC, and the effect was stronger than TP5and Tα1.
(4) The synergistic anti-tumor activity of Tal-TP5was evaluated for the first time. Tα1-TP5combined with CY exhibited good anti-tumor activity. Moreover, Tα1-TP5activated the immune system, which was responsible for killing tumor cells. The synergistic anti-tumor activity of Tα1-TP5was stronger than that of TP5and Tal.
(5) The receptor of Tα1-TP5was first studied. Tα1-TP5could bind to TLR2, suggesting that Ta-TP5may mediate immune response through TLR2signaling pathway.
为了提高TP5的活性,延长其体内t1/2,本课题组已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,在毕赤酵母Pichia pastoris中表达制备了融合肽Tα1-TP5。研究证实Tα1l-TP5具有比TP5更强的体外刺激小鼠脾淋巴细胞增殖及体内促进巨噬细胞吞噬的能力,Tα1-TP5也具有比TP5及Tα1更长的体外血浆t1/2。但是,Tα1-TP5经Pichia pastoris分泌表达的量很少,无法满足进一步体内试验研究的需求,并且需要凝血酶切割去掉融合标签,纯化成本高,纯化步骤繁琐。切割后,Tα1-TP5的N端带有2个多余的氨基酸残基,C端带有多余的4个氨基酸残基,可能存在免疫原性。
故为了提高产量并降低纯化成本,本课题换用大肠杆菌表达体系,采用内含肽介导的纯化系统,加入二硫苏糖醇(DTT)诱导内含肽自切割,分别从细菌裂解上清及包涵体中纯化Tα1-TP5。考察Tα1-TP5是否具有比TP5及Tαl更强的体内免疫调节活性及辅助抗肿瘤作用,并进一步采用表面等离子共振技术(SPR)研究Tal-TP5与Toll样受体2(TLR2)的结合情况,为将Tα1-TP5开发成一种新型的免疫调节剂提供重要的基础与依据。
本课题的研究内容及取得的主要成果有以下几个方面。
1Ta1-TP5在大肠杆菌中的表达及纯化
采用大肠杆菌表达系统对Tα1-TP5进行了融合表达。选用pTYB2作为表达载体,将Tα1-TP5基因插入至内含肽(Sce intein)基因的N端,Sce intein基因的C端与几丁质结合域(Chitin binding domain, CBD)相连,构建重组质粒pTYB2-Tal-TP5-Sce intein-CBD,转入E.coli ER2566中诱导表达重组蛋白。超声裂解菌体后SDS-PAGE考察重组蛋白的可溶表达情况。考察不同诱导温度及诱导剂异丙基硫代-β-D-半乳糖苷(IPTG)浓度对重组蛋白可溶表达量的影响。采用亲和层析对重组蛋白进行纯化,加入DTT诱导intein柱上自切割,从而释放Tα1-TP5。为了提高Tα1-TP5的产量,也从包涵体中纯化了Tα1-TP5。摸索了包涵体的洗涤条件,采用直接稀释法对包涵体蛋白Tα1-TP5-Sce intein-CBD进行复性,并考察还原剂和氧化剂的比例、L-精氨酸(L-Arg)及最终尿素的浓度对复性效率的影响。复性后的蛋白过亲和层析柱,加入DTT诱导intein柱上自切割,从而获得Tα1-TP5。Tα1-TP5进一步经Superdex30(?)钝化并脱盐后,ESI-MS鉴定其分子量的正确性。Tricine-SDS-PAGE鉴定Tα1-TP5的纯度。
结果显示,Tα1-TP5成功在E.coli ER2566中表达,融合蛋白部分以包涵体的形式存在,诱导温度由37℃降低至16℃时可使可溶蛋白的表达量由原来的15.9%增加至18.9%,而IPTG浓度由1mmol/L降至0.1mmol/L对可溶蛋白表达量影响不大。复性体系中还原剂和氧化剂的比例为1：22.5、尿素终浓度为0.8mol/L时复性效率最高。L-Arg的加入对复性效率影响不大。Superdex30进一步纯化和脱盐后,从菌体裂解上清中获得了约4.6mg Tα1-TP5,从包涵体中获得了约3.0mg Tα1-TP5。 ESI-MS分别鉴定分子量,发现与理论值相当(3785.02Da), Tricine-SDS-PAGE检测Tal-TP5的纯度大于95%。最终我们运用此方法从每L发酵液中得到了约7.6mgTal-TP5。
2Tal-TP5体内外免疫调节活性研究
采用CCK-8法测定了Tal-TP5对小鼠脾淋巴细胞增殖的促进作用,采用流式细胞术考察了Tα1-TP5对小鼠脾淋巴细胞表面CD69分子表达的促进作用,采用RT-PCR技术检测Tα1-TP5对小鼠脾淋巴细胞IFN-y表达的的影响。结果表明：从上清及包涵体中纯化的Tα1-TP5均能促进小鼠脾淋巴细胞的增殖,且作用呈浓度依赖性,浓度为40μg/mL时,增殖率分别为26.21%、21.84%,与阴性组比较差异具有显著性意义(p<0.01,p<0.01)。Tα1-TP5能促进CD69的表达,与阴性对照组比较差异具有显著性意义(21.51%vs.9.5%,p<0.05),Tα1-TP5促进淋巴细胞表面CD69表达的能力也较TP5(16.06%)和Tα1(16.65%)高。RT-PCR结果显示Tα1-TP5能在基因水平上促进IFN-y的表达,且促进IFN-y表达的能力高于TP5及Tα1。说明我们纯化的Tα1-TP5具有比TP5及Tα1更强的免疫调节活性。
连续2天腹腔注射剂量为250mg/kg氢化可的松(HC),建立小鼠体内免疫抑制模型,皮下注射Tα1-TP5连续治疗2周,考察Tα1-TP5对小鼠免疫抑制的抵抗作用。结果发现,Tα1-TP5能明显增强免疫抑制小鼠的胸腺指数,与模型组比较差异具有显著性意义(2.253±0.427vs.0.444±0.407,p<0.05),与TP5及Tα1单独给药组比较差异也具有显著性意义(p<0.05)。但Tα1-TP5X(?)脾脏指数的影响不大。HE染色分析Tα1-TP5能改善HC导致的胸腺萎缩,增加胸腺组织中胸腺细胞的数量。另外Tα1-TP5能使CD4+CD8+胸腺细胞的比例恢复至正常水平。与模型组比较,Tα1-TP5显著增加外周血中IFN-γ的含量,抵抗HC造成的IFN-γ水平降低。总之,Tα1-TP5具有改善HC诱导的免疫抑制状态的能力,且活性比TP5及Tα1l更强。
3Tal-TP5辅助抗肿瘤作用研究
采用CCK-8法测定了Tal-TP5(?)(?)乳腺癌细胞MDA-MB-231、肝癌细胞HepG2、肺癌细胞A549及黑色素瘤细胞B16四种肿瘤细胞增殖的抑制作用,结果表明：Tα1-TP5对四种肿瘤细胞生长的抑制作用呈时间依赖性,对MDA-MB-231细胞的抑制作用最强,72h时细胞活力只占阴性组的64.6%,显著高于TP5组(64.6%vs.90.8%,p<0.05),但是与Tαl组比较没有显著性差异(64.6%vs.70.2%,p>0.05)。Tα1-TP5对HepG2细胞和A549细胞的抑制作用稍弱于对MDA-MB-231细胞,72h时细胞活力分别占阴性组的73.9%及69.9%,抑制作用强于TP5但与Tα1差别不大。Tα1-TP5对B16细胞也有一定的抑制作用,72h时细胞活力只占阴性组的82.5%,抑制作用与TP5及Tα1相比没有显著性差异。
建立C57BL/6小鼠荷黑色素瘤模型,考察Tα1-TP5联合环磷酰胺(CY)对肿瘤生长的抑制作用。结果表明：与CY单独给药相比,Tα1-TP5联合CY明显降低肿瘤体积及肿瘤重量,药物治疗2周后肿瘤重量减少了将近88.1%。Tα1-TP5也能逆转CY导致的外周血中白细胞数量减少,治疗2周后外周血中白细胞数量增加至正常水平。流式细胞术检测肿瘤组织中组织相容性复合体I(MHC I)的表达,发现CY组MHC I的表达率只有11.65%,而Tα1-TP5能显著增加肿瘤组织中MHC I的表达,与CY组比较差异具有显著性(28.71%vs.11.65%,p<0.05)。免疫组化结果显示Tα1-TP5较TP5及Tα1更能促进肿瘤组织中CD8+T细胞的浸润及CD86的表达。可见Tα1-TP5具有弱的直接杀伤B16细胞的功能,主要通过激活免疫系统而发挥辅助抗肿瘤作用。
4Tα1-TP5与TLR2结合活性研究
采用激光共聚焦显微镜观察考察Tα1-TP5与骨髓来源的巨噬细胞(BMDMs)的结合情况。将Tα1-TP5、TP5及Tα1分别用异硫氰酸荧光素(FITC)标记,并分别与分离的BMDMs孵育2h,再用Hoechst33342及DiI分别标记细胞核及细胞膜,激光共聚焦显微镜观察药物结合情况。结果发现,Tα1-TP5、P5及Tα1均能结合在BMDMs的细胞膜上。
采用SPR技术检测Tα1-TP5与存在于BMDMs上的TLR2(?)的结合。将TLR2作为配体以氨基偶联的方式偶联到CM5芯片上,Tα1-TP5、TP5及Tα1作为待分析物流过芯片表面,考察三种多肽与TLR2的结合能力及亲和力大小。结果显示：SPR技术能够准确反映三种多肽与TLR2(?)的结合水平。Tα1-TP5、TP5及Tα1与TLR2的解离平衡常数(KD)分别为6.84×10-6mol/L、1.57×10-6mol/L及35.4×10-6mol/L。 Tα1-TP5与TLR2的结合能力显著强于Tα1,但与TP5相比没有显著性差异。到目前为止,三种多肽可能的受体首次被确定。
本研究取得的主要成果和结论如下：
(1)首次构建了表达Tα1-TP5的大肠杆菌表达系统,对重组蛋白质成功进行了表达,并利用内含肽介导的纯化系统分别从细菌裂解上清及包涵体中分离纯化了Tα1-TP5, Superdex30进一步纯化得到了高纯度的目的多肽。获得的Tal-TP5C(?)(?)只含有一个多余的甘氨酸(Gly)残基。此研究提供了一种高效率、低成本的纯化策略,适合Tα1-TP5的大规模生产。
(2)纯化的Tα1-TP5能明显促进小鼠脾淋巴细胞的增殖、CD69及IFN-γ的表达,展示了较好的体外免疫调节活性。
(3)首次进行了Tα1-TP5的体内免疫调节活性研究。Tα1-TP5能抵抗HC诱导的免疫抑制,且效果较TP5及Tα1强。
(4)首次进行了Tα1-TP5的辅助抗肿瘤作用研究。Tα1-TP5联合CY展现了很好的抗肿瘤活性,同时Tα1-TP5激活了被CY抑制的免疫系统,增强对肿瘤的杀伤。Tα1-TP5(?)的辅助抗肿瘤活性较TP5及Tα1强。
(5)首次研究了Tα1-TP5的受体,Tal-TP5能与TLR2结合,提示Tα1-TP5介导免疫应答可能是通过TLR2信号通路。
Malignant tumors, which have high mortality, are one kind of the major diseases that seriously affect human health and threaten human life. Currently, the commenly used methods in the treatment of tumors in clinic are chemotherapy and radiotherapy, but, these remedies result in the severe damage on the immune function of patients. Therefore, searching for immunomodulators which can improve the immune function in cancer therapy has significant social and economic significance. Thymopentin (TP5) and thymosin alpha1(Tα1) are two thymic peptides with similar immunoregulatory activities and are being used in clinic for the treatment of some malignancies. They can improve the immune response and tolerance of patients to chemotherapy or radiotherapy, inhibit or reduce the progression of the diseases and improve the patient's quality of life. However, the in vivo half-life (t1/2) of TP5is very short, which is only30s. So. frequent administration is required to maintain its efficacy, which brings great inconvenience to patients. Tα1is mainly synthesized and purified by the method of solid phase synthesis, which is very expensive. The high price of Tal is a burdern for the majority of patients.
In order to prolong the t1/2and enhance the activity of TP5. a Tα1-TP5fusion peptide was designed and expressed in Pichia pastor is by our research team. Previous study showed that Tα1-TP5had higher activity than TP5in inducing T-lymphocyte proliferation in vitro. In in vivo carbon clearance test, the abilities of Tα1-TP5in promoting phagocytic capability of macrophages as well as the secretion of IL-2in peripheral blood of mice were also higher than those of TP5and Tα1. Besides. Tal-TP5had longer in vitro plasma t1/2than TP5and Tα1. However, the amount of Tα1-TP5secreted from Pichia pastor is was small and could not meet the demand of further in vivo study. Besides, the thrombin was needed to remove the fusion tag to obtain Tα1-TP5, which resulted in the high purification costs and complex purification steps. After thrombin cutting, two extra amino acids existed in the N-terminal of Tal-TP5and four extra amino acids existed in the C-terminal of Tα1-TP5, which might increase immunogenicity.
To obtain large quantity of active Tα1-TP5with low cost, a new intein-fused expression and purification strategy was used for the production of Tα1-TP5fusion peptide from the soluble fraction and inclusion bodies. The in vivo immune regulating activity and synergistic anti-tumor activity of Tα1-TP5were also investigated. Besides, the potential receptor of Tα1-TP5was investigated by surface plasmon resonance (SPR) binding studies. In sum, the research may provide important basis for developing Tα1-TP5as a novel immunomodulatory agent.
The main contents and results obtained were summarized as follows:
1Expression and purification of Tα1-TP5in Escherichia coli
Tα1-TP5was expressed in Escherichia coli using pTYB2as the expression vector. The gene of Tα1-TP5was inserted into the N-terminal of the gene of Sce intein-Chitin binding domain (CBD). Then the recombinant plasmid pTYB2-Tα1-TP5-Sce intein-CBD was transformed into Escherichia coli ER2566and recombinant protein Tα1-TP5-Sce intein-CBD was expressed in ER2566. After sonication, SDS-PAGE was used to confirm whether the recombinant protein was soluble. The influences of different induction temperature and the concentration of isopropyl-β-D-galactoside (IPTG) on the amount of soluble recombinant protein were examined. Affinity chromatography was used to purify the recombinant protein. The target peptide was released from the chitin beads via self-cleaving of intein induced by dithiothreitol (DTT). In order to increase production, Tal-TP5was also obtained from inclusion bodies. The condition of washing inclusion body was investated. Subsequently, the inclusion body was renatured by direct dilution method. Besides, the effect of redox ratios, L-Arg and final urea concentration on refolding efficiency was evaluated. The refolded protein was applied to affinity chromatography columns, and Tα1-TP5was released from the chitin beads via self-cleaving of intein induced by DTT. Further, Tα1-TP5was purified and desalted by Superdex30. The correctness of its molecular weight was identified by ESI-MS and the purity of Tα1-TP5was evaluated by Tricine-SDS-PAGE.
Tα1-TP5was successfully expressed in E.coli ER2566. After sonication, the fusion protein was partially in the form of inclusion bodies. As the induction temperature decreased from37℃to16℃, the amount of soluble protein increased from15.9%to18.9%of total bacterial protein. Change of IPTG concentration from1mmol/L to0.lmmol/L showed no effect on soluble expression level. When redox ratio (reduced:oxidized) was1:22.5and the final urea concentration was0.8mol/L, the fusion protein has the highest refolding efficiency. L-Arg showed no effect on the refolding efficiency. After further purified and desalted by Superdex30,4.6mg Tal-TP5was obtained from the supernatant and3.0mg Tal-TP5was obtained from inclusion bodies per liter medium. The purity was up to95%determined by16.5%Tricine-SDS-PAGE. Finally, this approach reported here allowed the production of approximately7.6mg of Tα1-TP5from1L shaking flask culture of E. coli.
2Study on the in vitro and in vivo immunomodulatory activity of Tal-TP5
The ability of Tal-TP5on promoting mouse spleen lymphocytes proliferation was detected by CCK-8assay. The effect of Tα1-TP5on promoting the expression of CD69on mouse spleen lymphocytes was investigated by flow cytometry. Then the effect of Tal-TP5on promoting the expression of IFN-γ was detected by RT-PCR. The result showed that Tal-TP5purified from the supernatant and inclusion bodies could promote the proliferation of mouse spleen lymphocytes in a concentration-dependent manner. When the concentration of Tα1-TP5was40μg/mL. the proliferation rate was26.21%and21.84%, respectively. Compared to negetive control, the difference was significant (p<0.01, p<0.01). Tα1-TP5could promote the expression of CD69and compared to negative control, the difference was significant (21.51%vs.9.5%, p<0.05). The ability of Tα1-TP5in promoting CD69expression was also higher than that of TP5(16.06%) and Tα1(16.65%). RT-PCR results showed that Tα1-TP5could promote IFN-γ expression at the gene level, and the ability was higher than that of TP5and Tα1. These all indicated that purified Tal-TP5possessed stronger immune regulating activity than TP5and Tal.
The immunosuppressive mice model was established by intraperitoneal injection of hydrocortisone (HC) at a dose of250mg/kg for two consecutive days. Tα1-TP5was subcutaneously injected for two consecutive weeks to evaluate the resistance of Tal-TP5to immunosuppression induced by HC. The results showed that, Tal-TP5could significantly enhance the thymus index of immunesuppressed mice, and compared with the model group, the difference was significant (2.253±0.427vs0.444±0.407,p<0.05). Compared with the TP5and Tal alone group, the difference was also significant (p<0.05). However, the effect of Tα1-TP5on spleen index was little. HE staining indicated that Tal-TP5could antagonize thymic atrophy induced by HC and increase the number of thymocytes in the thymus tissue. In addition, Tal-TP5made CD4+CD8+thymocytes returned to normal levels. Compared with model group, Tal-TP5significantly increased the concentration of IFN-γ in the peripheral blood. In sum, Tal-TP5has the ability to alleviate the immunosuppression induced by HC, and the activity was stronger than that of TP5and Tα1.
3Study on the synergistic anti-tumor activity of Tal-TP5
The inhibitory effect of Tal-TP5on breast cancer cell MDA-MB-231, lung cancer cell A549, hepatoma carcinoma cell HepG2and melanoma cell B16was evaluated by CCK-8assay. Tal-TP5could inhibit the proliferation of these four tumor cells in a time-dependent manner and Tal-TP5showed the highest inhibitory effect on MDA-MB-231. After72h incubation, cell viability was only64.6%of that of the negetive control, and compared to TP5group, the difference was significant (64.6%vs.90.8%, p<0.05), but compared to Tal group, there was no significant difference (64.6%vs.70.2%, p>0.05). The inhibitory effect of Tal-TP5on HepG2cell and A549cell was weaker than on MDA-MB-231cell, and cell viabilities were73.9%and69.9%of that of the negative group after72h incubation, which was stronger than that of TP5but had no significant difference from that of Tα1. Tα1-TP5also could inhibit the proliferation of B16cells, and after72h incubation, the cell viability was82.5%of that of the negative group. Compared to TP5and Tal group. there were no significant differences.
C57BL/6mice were inoculated subcutaneously with2×105of B16melanoma cells/mouse in the forelimb armpit. The effect of Tα1-TP5in combination with cyclophosphamide (CY) on inhibition of tumor growth was investigated. Compared to CY alone, Tα1-TP5combined with CY significantly reduced tumor volume and tumor weight, and the tumor weight was reduced by nearly88.1%after two weeks treatment. Tal-TP5also increased leukocyte number lowered by CY treatment, and leukocyte number in peripheral blood increased to normal levels after two weeks treatment of Tα1-TP5. In CY group, MHC I expression rate was only11.65%detected by flow cytometry, however, Tal-TP5significantly increased MHC I expression in the tumor tissue, and compared to CY group, the difference was statistically significant (28.71%vs.11.65%, p<0.05). Immunohistochemistry results showed that Tal-TP5promoted more CD8+T cell infiltration and CD86expression in tumor tissue than TP5and Tα1. From all the results above it was concluded that Tal-TP5alone had weak killing effect on tumor cells and mainly activated the immune system in combination therapy.
4Study on the binding character of Tal-TP5to TLR2
Confocal microscope was used to observe the binding character of Tα1-TP5to bone marrow-derived macrophages (BMDMs). Tα1-TP5. TP5and Tα1were labeled with fluorescein isothiocyanate (FITC) and incubated with cultured BMDMs for2h. Then, the cell nucleus and cell membrane were stained by Hoechst33342and Dil. respectively. The results showed that all the three peptides were located on the cell membrane of BMDMs.
SPR was used to detect the binding of Tα1-TP5to TLR2. which exist on the cell membrane of BMDMs. TLR2was immobilized by standard amine coupling using an amine coupling kit. The peptides were dissolved in running buffer, and a flow rate of30μL/min was employed for association and dissociation at a constant. SPR technology could accurately reflect the level of the binding of the three polypeptides with TLR2. The KD of Tal-TP5, TP5and Tal were6.84×10-6mol/L,1.57×10-6mol/L and35.4×10-6mol/L, respectively. The KD of Tal-TP5was much smaller than that of Tα1, indicating that Tα1-TP5had higher affinity to TLR2than Tα1. Compared with TP5, the affinity of Tal-TP5to TLR2was slightly low but there was no statistical significance. Thus, the potential receptor of the three peptides was first confirmed as TLR2.
The main results and conclusions are as follows:
(1) The expression strain of Tal-TP5was firstly constructed. The fusion protein was successfully expressed in E.coli and Tal-TP5was purified from the supernatant and inclusion bodes with intein-mediated purification systems. High purity of Tal-TP5was obtained by further purification with Superdex30. There was only a Gly residue at the end of Tal-TP5, which might greatly reduce the immunogenicity. The approach described here is a low-cost, convenient and potential way for obtaining a large amount of Tα1-TP5.
(2) Purified Tal-TP5can significantly promote the proliferation of mouse spleen lymphocytes, as well as the expression of CD69and IFN-y. Tal-TP5showed good in vitro immunoregulatory activity.
(3) The in vivo immunomodulatory activity of Tal-TP5was evaluated for the first time. Tα1-TP5can antagonize immune suppression induced by HC, and the effect was stronger than TP5and Tα1.
(4) The synergistic anti-tumor activity of Tal-TP5was evaluated for the first time. Tα1-TP5combined with CY exhibited good anti-tumor activity. Moreover, Tα1-TP5activated the immune system, which was responsible for killing tumor cells. The synergistic anti-tumor activity of Tα1-TP5was stronger than that of TP5and Tal.
(5) The receptor of Tα1-TP5was first studied. Tα1-TP5could bind to TLR2, suggesting that Ta-TP5may mediate immune response through TLR2signaling pathway.
引文
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