新型血管内皮细胞抑制因子VEGI_(151)的基因克隆表达及其活性

血管内皮细胞生长抑制因子 (vascularendothelialcellgrowthinhibitor,VEGI)是近年发现的一类肿瘤坏死因子超家族成员 ,具有抑制内皮细胞增殖的作用。从人脐静脉内皮细胞株 (ECV30 4)克隆到其基因 ,构建N端缺失 2 3个氨基酸的表达载体 ,并通过原核表达系统进行表达 (命名为VEGI151) ,表达量为 2 5 .5 % ,纯化后纯度达92 .5 %。通过生物学效应检测 ,发现VEGI151可明显抑制无血清培养基中内皮细胞的增殖 ,2 4h时VEGI151对内皮细胞的IC50 为 10mg/L ,0 .6 13mg/L时使内皮细胞在 36h内完全凋亡。通过检测体外培养肿瘤细胞 (A5 49、HepG2、Hela等 )的存活率 ,未发现明显的增殖或抑制效应。提示VEGI是一种主要作用于血管内皮细胞 ,在新生血管性疾病及肿瘤的治疗中有潜在的应用前景。     

嵌合分子VEGI+的构建、表达及其抗血管生成和抗肿瘤活性

血管内皮细胞生长抑制因子VEGI是肿瘤坏死因子超家族新成员 ,通过诱导内皮细胞凋亡而抑制肿瘤生长。通过PCR方法将CTTHWGFTLC与VEGI2 3- 1 74氨基酸相连 ,构成嵌合分子VEGI 。原核表达的VEGI 经过纯化后 ,以非重折叠的沉淀形式进行活性实验 ,VEGI 能够抑制鸡胚尿囊膜血管新生 ,对于Lewis肺癌小鼠移植瘤 ,5mg L组抑瘤率 99.7% ,1 0mg L组抑瘤率96% ,2 5mg L组抑瘤率 83%。实验说明VEGI 通过抑制血管新生对移植瘤的早期发展起抑制作用。     

嵌合分子VEGI~+的构建、表达及其抗血管生成和抗肿瘤活性

血管内皮细胞生长抑制因子VEGI是肿瘤坏死因子超家族新成员,通过诱导内皮细胞凋亡而抑制肿瘤生长。通过PCR方法将CTTHWGFTLC与VEGI23-174氨基酸相连,构成嵌合分子VEGI+。原核表达的VEGI+经过纯化后,以非重折叠的沉淀形式进行活性实验,VEGI+能够抑制鸡胚尿囊膜血管新生,对于Lewis肺癌小鼠移植瘤,5mgL组抑瘤率99.7%,10mgL组抑瘤率96%,25mgL组抑瘤率83%。实验说明VEGI+通过抑制血管新生对移植瘤的早期发展起抑制作用。     

纤溶酶原K5抗血管增生活性依赖其完整Kringle结构域

根据K5蛋白(Pro451—Ala541)的结构特征和二硫键分布特点,设计K5的两个缺失突变体K5 mut1(Cys461—Cys540,保留K5 kringle环3个完整二硫键但去除N端和C端多余氨基酸)和K5 mut2 (Cys482—Cys535,打开kringle环,只保留2个二硫键).以野生型人纤溶酶原K5 cDNA为模板,用PCR方法得到编码缺失突变体的DNA片段,定向克隆入pET22b(+)质粒载体,重组体转化进大肠杆菌BL21（DE3）,诱导表达,产物经亲和层析和高浓度甘油透析纯化后进行鉴定和生物活性测定.K5 mut1蛋白特异性抑制人视网膜微血管内皮细胞增殖,且活性强度是完整的K5蛋白2倍;K5 mut2对人视网膜微血管内皮细胞无显著抑制作用.结果提示,完整的Kringle结构（包含3个二硫键）是维持人纤溶酶原K5抗血管增生活性的必需结构域,而K5分子中Kringle结构域外的N端和C端氨基酸臂则并非其活性所必需.     

钙网蛋白122～180片段基因克隆、表达和活性分析

钙网蛋白是高等动物细胞中普遍存在的一种钙结合蛋白,近年发现它及其N端1～180位氨基酸能抑制内皮细胞生长和血管生成.为了寻找高效和小分子质量的血管生成抑制因子,用PCR技术扩增出钙网蛋白N端122～180位氨基酸的DNA序列,克隆进原核表达载体pET-3c,转化大肠杆菌BL21(DE3), 经IPTG诱导后,该片段以包涵体形式表达,表达量约占菌体总蛋白的35.4%.包涵体经变性溶解、复性和初步纯化后,纯化产物可以抑制人脐静脉内皮细胞的生长,鸡胚绒毛尿囊膜的血管生成和小鼠原位黑色素瘤的生长.     

内皮抑素-血管内皮细胞抑制因子重组腺病毒对新生血管的抑制效应

内皮抑素与血管内皮细胞抑制因子均为内源性新生血管抑制分子。为了探讨两分子融合后的生物学功能,以重组腺病毒为载体介导融合基因在体内外表达。体外实验表明重组腺病毒Ad-hENDO-VEGI151能够在ECV304、HepG2、L929等细胞中表达41000大小的融合蛋白,对血管内皮细胞增殖有明显的特异性抑制作用,对HepG2和L929细胞无抑制作用(F=13112.13,P=0.0001)。体内实验表明Ad-hENDO-VEGI151重组腺病毒表达的融合蛋白可以显著抑制鸡胚绒毛尿囊膜血管的生成;与AdLacZ对照组相比,Ad-hENDO-VEGI151对兔炎症性角膜新生血管的抑制明显(F=1413.11P=0.0001),免疫组化结果显示,融合蛋白主要在角膜上皮层表达;治疗荷肝癌裸鼠,注射Ad-hENDO-VEGI151的肿瘤体积(487.7±241.2mm3)与AdLacZ对照组(4075.9±1849.9mm3)差异显著(F=14.80P=0.0085),抑瘤率达到88.03%,免疫组化结果显示,胞膜染色阳性,Ad-hENDO-VEGI151组肿瘤的微血管密度30.75±3.31%与AdLacZ组50.25±8.65%差异明显F=17.72P=0.0056抑制率为39%。结果提示:重组腺病毒Ad-hENDO-VEGI151能够表达有生物学活性的融合蛋白,并发挥特异性的新生血管抑制作用。     

短肽库中VEGF受体拮抗剂的筛选及生物学活性鉴定

为获得可溶性血管内皮细胞生长因子受体 (s VEGFR,soluble VEGF receptor)的拮抗剂 ,以固相化可溶性 VEGF受体 s Flt- 1和 s KDR通过生物淘选筛选噬菌体十二肽库 .采用 ELISA及竞争性 ELISA筛选阳性克隆 ,12 5I- VEGF竞争性放射免疫吸附实验进一步鉴定阳性克隆的体外结合活性 .经过 3～ 4轮生物淘选的短肽库有明显富集 .初筛后约 3%的噬菌体克隆可同时结合相应的可溶性受体及人脐静脉内皮细胞 .其中 6个克隆与内皮细胞的结合 ,可以部分地被变性复性处理的原核表达血管内皮细胞生长因子 (VEGF)竞争抑制 .4个噬菌体克隆可竞争性抑制 12 5I- VEGF与可溶性受体的体外结合反应 .两个 KDR阳性克隆 K2 37与 K93可在体外抑制内皮细胞的增殖 .克隆K2 37与 F90可抑制鸡胚绒毛尿囊膜血管形成 .阳性克隆可作为血管内皮细胞生长因子受体拮抗剂 ,具有良好的应用前景 .     

人内皮细胞抑制生长素在毕赤酵母中的高效表达

根据人胶原蛋白ⅩⅧNC结构域C末端编码内皮细胞抑制生长素 (Endostatin)的成熟多肽序列 ,人工合成了由毕赤酵母 (Pichiapastoris)中偏爱密码子组成的内皮细胞抑制生长素基因序列。该基因以N端融合的方式正确插入毕赤酵母诱导型表达载体pPICZαA中。通过电激将线性化的重组质粒转化到毕赤酵母SMD1 1 68细胞中 ,筛选获得表达具有生物活性的内皮细胞生长抑制素的高产菌株。在摇瓶发酵水平上 ,产量达到 80mg L。而利用生物反应器进行高密度发酵 ,Endostatin的产量可达 1 2 5mg L。纯化后的Endostatin具有抑制小鼠血管内皮细胞增殖的活性     

抗血管生成活性的重组人proEMAPⅡ/p43蛋白缺失突变体的构建和筛选

proEMAPⅡ(又称p43蛋白)是哺乳动物氨酰tRNA合成酶的辅助因子,近年来发现,p43具有细胞因子活性以及抗新生血管生成的作用,具有潜在的抗肿瘤疗效.p43的C端结构域即为EMAPⅡ,其结构和功能都已知,具有细胞因子和抗血管生成的双重功能.但是N端的结构还不清楚,研究表明全长的p43比EMAPⅡ具有更高的生物学活性,但是p43的结构和作用机制尚不明确.此外,作为蛋白质药物,更小的分子能够减少免疫原性和副作用,从而发挥更好的活性.本文利用生物信息学方法,对p43 N端的二级结构进行预测,并且在不破坏二级结构的前提下,构建了10个p43的缺失突变体,在体外对10个缺失突变体与全长的p43蛋白进行抗新生血管生成活性验证比较,最终我们获得了3个活性高的p43缺失突变体,并且发现N端的1~16,1~79位氨基酸和C端的264~312位氨基酸不是p43发挥抗血管生成功能所必需的,而且它们的删除使得活性位点更好地呈现并发挥活性.通过我们的研究,有助于揭示p43蛋白结构和功能的关系以及其作用机制,同时为临床筛选肿瘤治疗候选药物奠定基础.     

耐热碱性磷酸酯酶的功能结构域的定位

 为了确定耐热碱性磷酸酯酶 (TAPND2 7)发挥活性所必需的功能结构域 ,通过 PCR介导的诱变缺失 ,得到了 N端分别缺失 8、1 6、2 5个氨基酸的 3个缺失体 p TAPN8、p TAPN1 6和p TAPN2 5以及 C端分别缺失 1 0和 30个氨基酸的两个缺失体 p TAPC1 0和 p TAPC30 .经表达和活性测定 ,发现 p TAPN8和 TAPC1 0保持了较高的活性而其余 3个缺失体则失去酶活性 .据此 ,TAPND2 7的活性区域被定位在 8～ 465氨基酸之间 .在分离纯化的基础上测定了一些酶学性质 .发现 TAPN8和 TAPC1 0的比活没有大的改变 ,Tm 下降了 5.5℃ ;TAPN8的最适反应温度上升了1 0℃ .结果提示了 N端和 C端的这些氨基酸残基对热稳定性有一定的贡献 ,N端氨基酸残基还对酶的亲热性有贡献 .     

The endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces dendritic cell maturation

Angiogenesis is an essential component of chronic inflammation that is linked to carcinogenesis. In this study, we report that human vascular endothelial growth inhibitor (VEGI, TNF superfamily 15), an endothelial cell-produced antiangiogenic cytokine, induces mouse dendritic cell (DC) maturation, a critical event in inflammation-initiated immunity. VEGI-stimulated bone marrow-derived immature DCs display early activation of maturation signaling molecules NF-kappaB, STAT3, p38, and JNK, and cytoskeleton reorganization and dendrite formation. The activation signals are partially inhibited by using a neutralizing Ab against death domain-containing receptor-3 (DR3) or a truncated form of DR3 consisting of the extracellular domain, indicating an involvement of DR3 in the transmission of VEGI activity. A VEGI isoform, TL1A, does not induce similar activities under otherwise identical experimental conditions. Additionally, the cells reveal significantly enhanced expression of mature DC-specific marker CD83, secondary lymphoid tissue-directing chemokine receptor CCR7, the MHC class-II protein (MHC-II), and costimulatory molecules CD40, CD80, and CD86. Functionally, the cells exhibit decreased Ag endocytosis, increased cell surface distribution of MHC-II, and increased secretion of IL-12 and TNF. Moreover, VEGI-stimulated DCs are able to facilitate the differentiation of CD4+ naive T cells in cocultures. These findings suggest that the anticancer activity of VEGI arises from coupling the inhibition of endothelial cell growth with the promotion of the adaptive immune mechanisms through the stimulation of DC maturation.     

Sensitization of endothelial cells to VEGI-induced apoptosis by inhibiting the NF-κB pathway

Vascular endothelial growth inhibitor (VEGI) is an endogenous inhibitor of endothelial cell growth and a promising candidate for cancer therapy. VEGI is able to inhibit tumor growth by specifically targeting the tumor neovasculature. Increasing the anti-angiogenic potential of this cytokine is of great interest for its therapeutic potential. NF-κB is known to have an integral role in TNF superfamily signaling, acting as a pro-survival factor. A role of VEGI-induced NF-κB activation in endothelial cells has yet to be described. Here we show that suppression of the NF-κB pathway can increase the apoptotic potential of VEGI. We used siRNA to deplete NF-κB or its activator IKK2 from adult bovine aortic endothelial cells. The siRNA treatments diminished VEGI-induced NF-κB activation, evidenced from a reduced extent of NF-κB nuclear translocation and diminished expression of NF-κB-target genes such as interleukins-6 and -1β. The siRNA-treated endothelial cells when exposed to VEGI exhibited a marked decrease in cell viability and a significant increase in apoptosis. These results confirm that VEGI utilizes NF-κB as a pro-survival role factor in endothelial cells. We then examined whether a combination of VEGI with NF-κB inhibitors would constitute a more potential therapeutic regiment. We found that in the presence of the NF-κB inhibitors curcumin or BMS-345541 there was a marked increase in the apoptotic potential of VEGI on endothelial cells. These findings indicate that a combination therapy using VEGI and NF-κB inhibitors could be a potent approach for cancer treatment.     

Isoform of Vascular Endothelial Cell Growth Inhibitor （VEGI72-251） Increases Interleukin-2 Production by Activation of T Lymphocytes

The objective of this study is to investigate the characteristics of the recombinant variant ofhuman vascular endothelial cell growth inhibitor,VEGI_(72-251),and compare its biological activities with that ofits prototype VEGI_(24-174),The recombinant plasmid containing the variant VEGI_(72-251) gene was constructedand expressed in Escherichia coli.The effects of the expressed VEGI_(72-251) on cell proliferations were checkedin the human umbilical vein endothelial cell line and certain tumor cell lines (ECV304 and B 16).The inhibitionof VEGI_(72-251) on angiogenesis was detected in the chorioallantoic membrane of chick embryos.In comparisonwith VEGI_(24-174),the recombinant human VEGI_(72-251) seems to have no effect on the proliferation of endothelialcells and the angiogenesis of the chorioallantoic membrane in vitro.An enzyme-linked immunosorbent assay-based method was used for the measurement of interleukin-2 (IL-2) production by peripheral blood monocytes(PBMCs) treated with VEGI_(72-251).PBMCs were pretreated with VEGI_(72-251) (1.25-12.50μg/ml) for 24 h invitro,and the IL-2 concentration in PBMC medium was increased from 354 pg/ml to 1256 pg/ml.It can beconcluded that VEGI_(72-251) is able to increase the level of human IL-2 production by the activation of Tlymphocytes.Differing from VEGI_(24-174) on anti-angiogenesis,VEGI_(72-251) may serve as an anti-cancer factorthrough its activation of T lymphocytes.     

Identification and characterization of a novel protein inhibitor of type 1 protein phosphatase

We have isolated human cDNA for a novel type 1 protein phosphatase (PP1) inhibitory protein, named inhibitor-4 (I-4), from a cDNA library of germ cell tumors. I-4, composed of 202 amino acids, is 44% identical to a PP1 inhibitor, inhibitor-2 (I-2). I-4 conserves functionally important structure of I-2 and exhibited similar biochemical properties. I-4 inhibited activity of the catalytic subunit of PP1 (PP1C), specifically with an IC(50) of 0.2 nM, more potently than I-2 with an IC(50) of 2 nM. I-4 weakly inhibited the activity of myosin-associated phosphates (PP1M). However, the level of inhibition of PP1M was increased during preincubation of PP1M with I-4, suggesting that the inhibition is caused by interaction of I-4 with PP1C in such a manner that it competes with the M subunit of PP1M. Gel overlay experiments showed that I-4 binds PP1C directly. Three I-4 peptides containing the N-terminal residues 1-123, 1-131, and 1-142 all showed strong binding ability to PP1C but did not show PP1 inhibitory activity, whereas an I-2 peptide (residues 1-134), lacking the corresponding C-terminal residues, potently inhibited PP1C activity as previously reported. Removal of the 18 N-terminal amino acid residues from I-4 dramatically reduced the PP1 binding activity with a correlated loss of inhibitory activity, whereas removal of the 10 N-terminal residues had only a little effect. The two peptides GST-I-4(19-131) and GST-I-4(132-202) showed ability to bind to PP1C, albeit very weakly. These results strongly suggest a multiple-point interaction between I-4 and PP1C, which is thought to cause the inhibition of I-4 which is stronger than the inhibition of I-2.     

Mapping binding domains of kininogens on endothelial cell cytokeratin 1

Human cytokeratin 1 (CK1) in human umbilical vein endothelial cells (HUVEC) is expressed on their membranes and is able to bind high molecular weight kininogen (HK) (Hasan, A. A. K., Zisman, T., and Schmaier, A. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3615-3620). New investigations have been performed to demonstrate the HK binding domain on CK1. Four overlapping recombinant (r) CK1 proteins were produced in Escherichia coli by a glutathione S-transferase gene fusion system. Biotin-HK specifically bound to rCK128 and rCK131 in the presence of Zn2+ but not to Deleted1-6rCK131. Recombinant CK128 and rCK131 also inhibited biotin-HK binding to HUVEC with IC50 of 0.4 and 0.5 microM, respectively. Alternatively, rCK114 and Deleted1-6rCK131 did not inhibit binding at concentrations >/=1 microM. Seven sequential 20 amino acid peptides of CK1 were prepared to cover the protein coded by exons 1-3. Only the first peptide (GYG20) coded by exon 1 significantly inhibited HK binding to HUVEC with an IC50 of 35 microM. Fine mapping studies isolated two overlapping peptides also coded by exon 1 (GPV15 and PGG15) that inhibited binding to HUVEC with IC50 of 18 and 9 microM, respectively. A sequence scrambled peptide of PGG15 did not block binding to HUVEC and biotin-GPV20 specifically bound to HK. Peptides GPV15 and PGG15 also blocked prekallikrein activation on endothelial cells. However, inhibition of PK activation by peptide PGG15 occurred at 10-fold lower concentration (IC50 = 1 microM) than inhibition of biotin-HK binding to HUVEC (IC50 = 10 microM). These studies indicate that HK binds to a region of 20 amino acids coded by exon 1 on CK1 which is carboxyl-terminal to its glycine-rich amino-terminal globular domain. Furthermore, HK binding to CK1 modulates PK activation on HUVEC.     

玉米黑粉菌CYP51稀有密码子和mRNA二级结构分析及与杀菌剂戊唑醇分子对接

通过截短玉米黑粉菌CYP51(P450-14DM,UmCYP51)基因(去除编码跨膜区部分)和选取不同的表达载体,构建了9种重组表达质粒,在大肠杆菌中进行UmCYP51基因的表达,发现只有BL21(DE3)/pET32-Um-35重组表达工程菌获得了表达.对稀有密码子和mRNA翻译起始区二级结构进行分析,结果表明稀有密码子和mRNA翻译起始区二级结构对UmCYP51蛋白的表达都有影响.适用于稀有密码子表达的菌株Rosetta(DE3)不利于UmCYP51蛋白的表达;同时只有翻译起始区二级结构自由能值最低的重组载体pET32-Um-35可以表达.为了设计以UmCYP51为靶标的新型抗真菌抑制剂,基于最新解析的真核生物人类的CYP51晶体结构,利用同源模建的方法构建了UmCYP51的三维结构并进行了分子动力学模拟优化.通过与商品化杀菌剂戊唑醇进行分子对接获得了此类抑制剂与UmCYP51的理论结合方式,阐述了戊唑醇分子的杀菌机理,为开发新型的抗真菌抑制剂奠定了基础.