人vasostatin的克隆、表达、纯化及活性检测

从成人肝脏cDNA文库中,PCR扩增得到人vasostatin基因编码区序列,将此序列插入原核表达载体pQE30进行表达,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定表明产物以包涵体形式存在,表达量占菌体总蛋白量的50%以上.包涵体洗涤后溶于8 mol/L尿素溶液,在变性条件下通过镍-氨三乙酸(Ni-NTA)金属螯合亲和层析柱进行纯化后,再经透析进行复性.N端氨基酸序列、分子质量、等电点等理化指标的测定结果与理论值相符.用内皮细胞增殖试验、内皮细胞迁移试验以及鸡胚尿囊膜血管生成试验等方法进行活性检测,证实复性的表达产物具有抑制内皮细胞增殖和迁移、抑制鸡胚尿囊膜血管生成的功能.     

Combination of vasostatin and cyclophosphamide in the therapy of murine melanoma tumors

Growth of tumors is strongly dependent upon supply of nutrients and oxygen by de novo formed blood vessels. Inhibiting angiogenesis suppresses growth of primary tumors as well and affects development of metastases. We demonstrate that recombinant MBP/vasostatin fusion protein inhibits proliferation of endothelial cells in vitro. The therapeutic usefulness of such intratumorally delivered recombinant protein was then assessed by investigating its ability to inhibit growth of experimental murine melanomas. In the model of B16-F10 melanoma the MBP/vasostatin construct significantly delayed tumor growth and prolonged survival of treated mice. A combination therapy involving MBP/vasostatin construct and cyclophosphamide was even more effective and led to further inhibition of the tumor growth and extended survival. We show that such combination might be useful in the clinical setting, especially to treat tumors which have already formed microvessel networks.     

Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization

According to codon preference of Escherichia coli, the optimized coding sequence of human vasostatin120-180aa (VAS) was obtained by chemical synthesis and molecular cloning methods. Using PCR and enzyme digestion, the full encoding sequence for VAS was cloned into the E. coli expression vector pALEX and expressed as a GST fusion protein in BL21 (DE3) strain. GST-VAS protein approximately accounted for 45% of the total bacterial proteins. Most of target protein existed in inclusion body. To improve the solubility of GST-VAS, the contribution of low temperature and molecular chaperone co-expression to the solubility of GST-VAS was tested. The results showed that co-expression with chaperons, TF and GroES/GroEL, and low expression temperature cooperatively improved the solubility of GST-VAS from 10 to 85%, and the yield of soluble GST-VAS was sixfold increased. When purified by GST affinity chromatography, 50 mg GST-VAS was obtained with purity over 85% from 1 L culture. Intact VAS was released by enterokinase digestion and further purified by Sephadex G50 gel filtration chromatography. About 7.2 mg intact homogeneous VAS protein was finally produced from 1L bacterial culture. The identity of GST-VAS and VAS was validated by Western blotting analysis. Recombinant VAS protein displayed distinct inhibition of endothelial cell proliferation and anti-angiogenic activity by chick embryo chorioallantoic membrane assay.     

人血管生成抑制素抑瘤研究进展

肿瘤的发展和转移需要新生血管的形成。人血管生成抑制素是近年发现的能够专一性抑制血管内皮细胞的内皮抑制因子。大量研究表明,在体外用血管生成抑制素处理血管内皮细胞可以抑制新生血管的形成,在体内单独使用血管生成抑制素,或者将血管生成抑制素与其他物质如基质金属蛋白酶、尿激酶联合处理荷瘤小鼠,可以降低小鼠体内肿瘤组织新生血管密度,抑制肿瘤的生长和肿瘤细胞的迁移。简要综述了血管生成抑制素抑制肿瘤生长和转移及其作用机理。     

Combination of vasostatin gene therapy with cyclophosphamide inhibits growth of B16(F10) melanoma tumours

Angiogenesis, i.e. formation of new blood vessels out of pre-existing capillaries, is essential to the development of tumour vasculature. The discovery of specific antiangiogenic inhibitors has important therapeutic implications for the development of novel cancer treatments. Vasostatin, the N-terminal domain of calreticulin, is a potent endogenous inhibitor of angiogenesis and tumour growth. In our study, using B16(F10) murine melanoma model and electroporation we attempted intramuscular transfer of human vasostatin gene. The gene therapy was combined with antiangiogenic drug dosing schedule of a known chemotherapeutic (cyclophosphamide). The combination of vasostatin gene therapy and cyclophosphamide administration improved therapeutic effects in melanoma tumours. We observed both significant inhibition of tumour growth and extended survival of treated mice. To our knowledge, this is one of the first reports showing antitumour efficacy of electroporation-mediated vasostatin gene therapy combined with antiangiogenic chemotherapy.     

Chromogranin A and Its Fragments as Regulators of Small Intestinal Neuroendocrine Neoplasm Proliferation

Introduction

Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation.

Methods

Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines.

Results

Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05).

Conclusion

Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.     

Characterization of natural vasostatin-containing peptides in rat heart

Chromogranin A (CGA) is a protein that is stored and released together with neurotransmitters and hormones in the nervous, endocrine and diffuse neuroendocrine systems. As human vasostatins I and II [CGA(1-76) and CGA(1-113), respectively] have been reported to affect vessel motility and exert concentration-dependent cardiosuppressive effects on isolated whole heart preparations of eel, frog and rat (i.e. negative inotropism and antiadrenergic activity), we investigated the presence of vasostatin-containing peptides in rat heart. Rat heart extracts were purified by RP-HPLC, and the resulting fractions analyzed for the presence of CGA N-terminal fragments using dot-blot analysis. CGA-immunoreactive fractions were submitted to western blot and MS analysis using the TOF/TOF technique. Four endogenous N-terminal CGA-derived peptides [CGA(4-113), CGA(1-124), CGA(1-135) and CGA(1-199)] containing the vasostatin sequence were characterized. The following post-translational modifications of these fragments were identified: phosphorylation at Ser96, O-glycosylation (trisaccharide, NAcGal-Gal-NeuAc) at Thr126, and oxidation at three methionine residues. This first identification of CGA-derived peptides containing the vasostatin motif in rat heart supports their role in cardiac physiology by an autocrine/paracrine mechanism.     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

The vasoinhibitory activity of bovine chromogranin A fragment (vasostatin) and its independence of extracellular calcium in isolated segments of human blood vessels.

Endothelium-independent vasoconstrictor responses in isolated segments of human internal thoracic artery (ITA) and saphenous vein (SV) were used as a bioassay system for the vasoinhibitory activity of bovine chromogranin A (CGA). Preincubation with vasostatin (0.8 micrograms/ml), containing the N-terminal domain of CGA, (CGA1-76, CGA1-113 and CGA1-143ff), inhibited the contractile responses evoked by 80 mM K+, 2.6 microM noradrenaline (NA), or 65 nM endothelin-1 (ET-1) in Ca(2+)-free solution in SV but not in ITA. The results demonstrate a vasoinhibitory activity in vasostatin and show that there is a marked difference between the arterial and venous segments in the Ca2+ independent component of the inhibitory response. A vascular role for the N-terminal domain of CGA is indicated, presumably by inhibiting Ca2+ release from intracellular stores in the human vein but not the artery.     

Relaxation induced by N-terminal fragments of chromogranin A in mouse gastric preparations

A definitive role for chromogranin A (CGA)-derived fragments in the control of the gastrointestinal smooth muscle contractility has not been yet established. The purpose of the present study was to evaluate, in vitro, the effects of the recombinant vasostatin 1-78 (VS-1), CGA 7-57 and CGA 47-66 on the mouse gastric mechanical activity, recording the changes of intraluminal pressure. VS-1, CGA 7-57 and CGA 47-66 produced concentration-dependent relaxations. Mouse anti-vasostatin-1 monoclonal antibody 5A8, recognising the region 53-57, abolished the relaxation induced by VS-1, indicating the specificity of the effect. The relaxation was significantly reduced by tetrodotoxin (TTX), blocker of neuronal voltage-dependent Na(+) channels, l-NAME, inhibitor of nitric oxide (NO) synthase, or apamin, blocker of small conductance Ca(2+)-dependent K(+) channels. The joint application of TTX and l-NAME did not show any additive effects, whereas TTX plus apamin abolished the VS-1 response. The results suggest that the N-terminal CGA-derived peptides are able to relax mouse gastric muscle and, therefore, they point out an inhibitory role of vasostatin I in the gastrointestinal tract. The relaxation is mediated in part by neural mechanisms through NO production and in part by non-neural mechanisms involving the opening of small conductance Ca(2+)-dependent K(+) channels.     

铜绿假单胞菌LexA蛋白的纯化及免疫活性分析

目的：对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的LexA蛋白进行表达、纯化,并检测其免疫活性。方法： lexA基因片段插入表达载体pET32a(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,镍离子亲合柱层析为第一步纯化,Superdex 75凝胶过滤层析作为第二步精细纯化,HPLC测定蛋白的浓度,将纯化的LexA蛋白经注射途径免疫家兔,制备兔抗LexA血清,采用免疫双扩、ELISA及Western Blot分析LexA的免疫活性。结果：LexA以包涵体形式表达,经镍离子亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.97%,表达及纯化的LexA具有良好的免疫活性。     

GPI锚固型Met-RANTES真核表达载体的构建和表达

目的：在仓鼠卵巢细胞（CHO细胞）中高效表达糖基磷脂酰肌醇（GPI） 锚定修饰的Met-RANTES融合蛋白,以研制新型免疫抑制分子GPI锚固型 Met-RANTES。方法：构建真核表达载体PEF/GPI-Met-RANTES,利用电转化法转染CHO-DHFRˉ细胞,氨甲喋呤（MTX）筛选抗性克隆。用流式细胞仪、细胞免疫荧光和免疫金标记电镜检测细胞膜上 GPI 锚固型Met-RANTES融合蛋白的表达情况。结果：构建了阅读框完整的 GPI 锚固型Met-RANTES 嵌合分子,经测序是正确的,并在CHO-DHFRˉ细胞膜上稳定表达。结论：在CHO 细胞表面高效表达GPI修饰的Met-RANTES融合蛋白,GPI 锚固型Met-RANTES分子有可能作为新型的免疫抑制剂用于器官移植中,抑制移植排斥反应。     

Modification of a recombinant GPI-anchored metalloproteinase for secretion alters the protein glycosylation

The N-linked glycans of recombinant leishmanolysin (GP63) expressed as a glycosylphosphatidylinositol (GPI)-anchored membrane protein or modified for secretion in Chinese hamster ovary (CHO) cells were analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The glycans isolated from both membrane and secreted protein were predominantly complex biantennary structures. However other aspects of the glycan profiles showed striking differences. The degree of sialylation of the membrane form was greatly reduced and the core fucosylation of biantennary structures was increased compared to the secreted form. Glycans isolated from membrane expressed protein also contained a higher proportion of lactosamine repeats. Residence times in the secretory pathway were similar for both secreted and membrane protein. Glycosylation differences may therefore be due to differences in protein conformation and accessibility to glycosyltransferases or glycosidases. These differences in glycosylation represent an important factor when considering modifying membrane expressed proteins for secreted production.     

p65蛋白的诱导表达及电子显微镜观察其显微结构

目的:构建重组原核表达载体pGEX-5x-1-p65,诱导GST-p65融合蛋白的表达并观察其包涵体的显微结构.方法:应用PCR技术扩增得到p65全长序列,并亚克隆至带有GST标签的pGEX-5x-1载体中.经酶切、测序鉴定后,在原核细胞中诱导表达GST-p65融合蛋白并将诱导后的菌体制作透射电镜标本,观察菌体内部显微结构.结果:成功构建表达载体pGEX-5x-1-p65,原核细胞中诱导表达、凝胶电泳后未见可溶性融合蛋白的高效表达.透射电镜观察到在承载有重组载体的菌体内部出现大量高电子密度的包涵体.结论:成功构建了原核表达载体pGEX-5x-1-p65,电子显微镜观察并证实在原核细胞内p65蛋白诱导表达形成包涵体.     

庚型肝炎病毒NS5 cDNA片段的表达及其免疫原性的研究

一段长度为880 bp的庚型肝炎病毒cDNA在大肠杆菌BL21(DE3)菌株中得到表达。此cDNA被插入到表达质粒pGEX-5X-1中,位于编码日本血吸虫谷胱甘肽硫转移酶（GST）的DNA序列下游,并与GST处于同一阅读框。用乳糖在37℃下诱导表达出以包涵体形式存在的GST-NS53融合蛋白,并用脲溶法提取了该蛋白;在20℃诱导时,表达出的蛋白大部分可溶,用谷胱甘肽Sepharose-4B亲和层析柱对可溶性的融合蛋白进行了纯化。免疫印迹实验证明,此融合蛋白能被庚型肝炎病人的血清和自制的抗GST血清特异性地识别。用PCgene软件对NS53氨基酸序列的亲水性和抗原决定簇进行了分析。本研究为庚型肝炎ELISA诊断试剂研制打下了基础。     

汉坦病毒H8205部分核壳蛋白基因在E.coli中的表达

根据汉坦病毒Ｈ８２０５株ＮＰ基因的序列,设计一对引物,扩增ＮＰ前２９２个氨基酸多肽基因片段,克隆于表达载体ｐＧＥＸ３Ｘ,与载体中２６ｋＤ的谷胱苷肽巯基转移酶（ＧＳＴ）融合表达。ＳＤＳＰＡＧＥ显示表达产物（ＧＳＴＮＰ）主要以包涵体形式存在。Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ表明此融合蛋白有抗原性。包涵体经分离、洗涤、溶解后,Ｓｅｐｈａｒｏｓｅ６Ｂ层析纯化,用此融合蛋白作抗原,进行ＥＬＩＳＡ法检测临床ＨＦＲＳ病人标本的ＩｇＧ和ＩｇＭ,有很好的特异性和敏感性。有生物活性的汉坦病毒Ｈ８２０５ＮＰ的体外表达成功,为汉坦病毒基因工程抗原的大量制备奠定了基础,也为汉坦病毒的临床检测和流行病学调查提供了一种廉价、安全、可靠的抗原。     

表达血管抑制因子的腺相关病毒载体的构建及其体内外活性

血管抑制因子(Vasostatin,VAS),为集钙蛋白N-末端180个氨基酸大小的蛋白,是一种内源性血管生成抑制因子,对多种肿瘤的生长具有很强的抑制作用.近期有研究显示,VAS可以促进神经内分泌肿瘤的恶化,提醒研究人员在开发该抗肿瘤药物时必须非常谨慎.将VAS cDNA插入腺相关病毒-2表达质粒pAAV-2,采用无辅助病毒参与的三质粒共转染法制备rAAV-VAS病毒.体外分别转染小鼠胰内皮细胞MS1和结肠癌细胞HCT-116,MTT法测定对细胞生长的影响,Western blotting方法检测VAS的表述.采用小鼠皮下移植瘤模型,验证VAS的表达对肿瘤生长、新生血管密度、以及细胞增殖的作用.结果证明构建的rAAV-VAS病毒载体,能抑制小鼠胰内皮细胞的生长,转染HCT-116后能有效表达VAS蛋白,但HCT-116的体外生长不受影响.瘤体注射rAAV-VAS后,HCT-116移植瘤在小鼠体内的生长速度明显减缓,肿瘤新生血管密度明显降低.结果显示,rAAV-VAS可以抑制HCT-116移植瘤的新生血管形成,但对其细胞增殖无明显作用.     

拟南芥中一种AtBAG4蛋白的抗体制备和特异性检测

以拟南芥AtBAG4的全长cDNA,构建pET-51780重组质粒,转化大肠杆菌BL21(DE3),IPTG可诱导融合蛋白高效表达,表达产物以可溶形式存在;经Ni-NTA层析柱分离纯化,可得到分子质量约为49kDa的PAGE纯蛋白。用纯化的融合蛋白pET-51780免疫家兔,可得到抗AtBAG4抗体。Western blot结果显示该抗体能特异识别原核系统内表达的抗原以及拟南芥自身的抗原。     

Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.