噬菌体抗体库技术筛选抗VEGF165单克隆抗体

目的:获得抗血管内皮生长因子165(VEGF165)单克隆抗体,并对其功能进行初步验证。方法:利用噬菌体抗体库展示技术筛选与VEGF165结合的噬菌体克隆并测序,以测序正确的阳性克隆质粒为模板,PCR扩增抗体的轻重链可变区基因,并克隆至哺乳动物细胞表达载体中,构建全抗体表达载体;将全抗体表达载体转染293E细胞,收取培养细胞上清,利用ProteinA亲和纯化抗体;通过结合ELISA、表面等离子共振检测抗体的亲和力,以人脐静脉内皮细胞(HUVEC)为模型验证抗体功能。结果:经过噬菌体抗体库展示技术筛出1个与VEGF165特异性结合的抗体序列VG2;293E细胞表达了VG2全抗体蛋白,SDS-PAGE显示VG2抗体纯度较高;BIAcore检测结果表明该抗体具有较高亲和力(KD=0.56nmol/L),竞争抑制ELISA结果表明VG2抗体能抑制VEGF与VEGF受体(VEGFR)的结合(IC50为1.470μg/mL),进一步实验结果表明VG2能够抑制VEGF引起的HUVEC增殖。结论:制备了靶向VEGF165的全人源单克隆抗体VG2,该抗体具有较高的亲和力,能阻断VEGF165/VEGFR2的结合,并抑制HUVEC的增殖,可以作为潜在药物应用于肿瘤治疗。     

利用慢病毒载体构建稳定过表达人VEGF_(165)的小鼠巨噬细胞系

目的:通过构建人血管内皮生长因子165(humanVEGF_(165),hVEGF_(165)的慢病毒载体,感染小鼠单核巨噬细胞RAW264.7,建立稳定高表达人VEGF165的小鼠巨噬细胞系。方法:将聚合酶链反应(PCR)扩增得到的hVEGF_(165)和慢病毒载体pLVX-IRES-ZsGreen1双酶切后连接,构建慢病毒表达载体pLVX-hVEGF_(165)-IRES-ZsGreen1。再经双酶切和测序鉴定后,进行病毒包装及浓缩。将该慢病毒载体感染RAW264.7细胞,利用绿色荧光蛋白ZsGreen1进行2次流式分选。用Realtime-PCR、WesternBlot分别检测各组细胞中hVEGF165的mRNA和蛋白表达;ELISA分别检测细胞上清中人VEGF和小鼠VEGF的含量。结果:酶切及测序结果示慢病毒表达载体pLVX-hVEGF165-IRES-ZsGreen1构建正确;流式分选后得到高纯度的ZsGreen1-hVEGF_(165)-RAW264.7细胞。Realtime-PCR、WesternBlot显示该细胞特异高表达hVEGF_(165)基因和蛋白(P均0.01)。ELISA显示该细胞分泌人和小鼠VEGF均显著增加(P均0.01)。结论:成功构建hVEGF_(165)慢病毒表达载体,并建立稳定高表达hVEGF_(165)的小鼠巨噬细胞系。为深入研究该细胞的功能、机制及应用提供充足稳定的细胞来源。     

瞬时基因表达可溶性的VEGFR2: I-IV

通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。     

瞬时基因表达可溶性的VEGFR2: I-IV

通过RT-PCR的方法从三个月的流产绒毛组织中克隆目的基因VEGFR2 (Vascular endothelial growth factor receptor 2, 血管内皮细胞生长因子受体2) 胞外I-IV区, 连接到真核表达载体上构建了重组表达载体。首先在无血清悬浮培养的HEK293细胞中, 使用报告基因GFP(Green fluorescence protein, 绿色荧光蛋白)优化转染条件, 发现在转染时DNA: PEI=1:2 (W/W)、1.5 mg DNA/106 cells及开始转染4 h内使用无血清、摇床(120 r/min)时可以达到最佳的转染效率和细胞数量。在确定转染条件之后, 将构建的表达载体分别在HEK293细胞、COS-7细胞和CHO-K1细胞中进行瞬时转染表达, 结果发现仅在CHO-K1细胞的培养上清中检测到目的蛋白的表达。瞬时转染CHO-K1细胞至总体积约为1.5 L, 由于目的蛋白的羧基端有8-His标签, 通过Ni2+-IDA柱纯化得到5 mg左右的目的蛋白。     

真核表达载体pIRES2-EGFP-Nurr1的构建及鉴定

目的:构建带有增强型绿色荧光蛋白报告基因EGFP及目的基因Nurr1的真核表达载体pIRES2-EGFP-Nurr1,并检测其在293T细胞中的表达。方法:采用反转录-聚合酶链式反应(RT-PCR)方法从大鼠黑质中获取Nurrl基因,连接T载体测序正确后与真核空载体pIRES2-EGFP一起,经Nhe1和Xho1双酶切,T4 DNA连接酶连接,构建pIRES2-EGFP-Nurr1;真核表达载体pIRES2-EGFP-Nurr1测序正确后采用脂质体法将其转染293T细胞,倒置荧光显微镜下观察转染效率,PCR检测Nurr1基因mRNA水平的表达情况,免疫印迹试验(Western Blot)检测Nurr1蛋白的表达水平。结果:酶切及测序鉴定证实成功构建了重组真核表达载体pIRES2-EGFP-Nurr1;293T细胞转染pIRES2-EGFP-Nurr1后可以高度表达绿色荧光,有效转录Nurr1基因并正确的高表达Nurr1蛋白。结论:成功构建Nurr1真核表达载体且在293T细胞中高水平表达,为进一步转染大鼠骨髓间充质干细胞(BMSCs),基因治疗帕金森病奠定基础。     

抗 CD20融合蛋白在 CHO 细胞中的表达及其纯化与鉴定

目的：构建CD20胞外区与Igβ胞外区和人IgG1 Fc融合基因的表达载体,并在CHO细胞中表达。方法与结果：根据已知的IgM的CH2区域和Igp胞外区序列,分别设计PCR引物并进行PCR扩增,然后用重叠PCR法扩增得到900bp的Igp-CH2序列,插入本实验室构建的pIRIS-EGFP-Fc载体,转化大肠杆菌,得到pIRIS-Igβ-CH2-Fc重组质粒,将其转染CHO-K1细胞,在G418抗性培养基中培养,荧光显微镜观察结合ELISA法筛选高表达细胞系,细胞系扩大培养后,通过亲和层析rProteinA柱纯化得到纯度融合蛋白,SDS-PAGE显示目的蛋白去糖基化后,相对分子质量约为42×10^3。结论：得到了Igβ-CH2-Fc重链抗体样分子,并鉴定为糖蛋白;须进一步对所得蛋白的生物学活性进行检测,验证其是否能够通过胞外区蛋白定位于B细胞淋巴瘤细胞表面对B淋巴瘤细胞进行杀伤。     

rhVEGF165在中国仓鼠卵巢细胞中的稳定表达、纯化及其生物学活性

利用哺乳动物细胞表达系统,稳定表达和纯化高生物学活性的人重组血管内皮生长因子 (VEGF165) 蛋白。将VEGF165克隆于表达载体pCDNA4.0,与T-GS载体共同转染CHO-S (中国仓鼠卵巢细胞) 细胞,MSX (Methionine sulphoximine) 加压筛选高表达细胞株,5 L发酵罐培养,细胞培养上清液通过三步纯化得到rhVEGF165蛋白,通过Western blotting、Biacore和人脐静脉内皮细胞增殖实验等对表达蛋白的特异性、亲和力及生物学活性等进行检测。所建立的细胞     

炭疽毒素受体与人IgG1的Fc段融合蛋白ATR_Fc在CHO细胞中的表达

ATR-Fc是人炭疽毒素受体（ATR）的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1-227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3-1的HindⅢ和NotⅠ位点得到表达ATR-Fc融合蛋白的真核表达载体pcDNA31/ATR9Fc,并用脂质体方法将该载体转染至CHO-K1细胞中,用G418筛选并获得ATR-Fc表达水平为10～15μg/(106cells·d)的基因工程CHO细胞系ATR-Fc-1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR-Fc与PA的亲和性,表明ATR-Fc可与PA特异性结合。     

hVEGF165 和嵌合水蛭肽融合基因的构建、表达和活性检测

根据抗 PTCA 或支架后再狭窄的基因治疗需要多基因治疗的特点,用基因重组技术构建了 hVEGF165 和嵌合水蛭肽 (fused hirudin , FH) 融合基因,并克隆到真核表达载体 pcDNA3.0 中,通过脂质体介导将 pcDNA3.0/hVEGF165 - FH 转染到人内皮细胞株 (ECV304) 中, RT-PCR 及蛋白质印迹证明融合基因 hVEGF165 - FH 在 ECV304 细胞中得到表达 ( 分子质量为 24 ku 左右 ). 通过体外活性检测——— MTT 法检测 hVEGF165 - FH 对 ECV304 细胞增殖的影响,通过体外血管生成分析 hVEGF165 - FH 对内皮细胞株 ECV304 增殖的影响 . 通过体外抗栓活性检测,表明表达产物具有促进内皮细胞株增殖及加快血管生成的作用,同时显著抑制了 ADP 诱导的血小板聚集率 (P < 0.05) 并显著延长 APTT 和 TT (P < 0.05) . 实验结果表明,融合基因在内皮细胞株中得到表达,表达的融合蛋白具有 hVEGF165 和嵌合水蛭肽 (FH) 的双重活性,这为以后的融合基因治疗再狭窄的动物实验打下了良好基础 .     

猪PBD-1基因乳腺特异性表达载体的构建

为构建猪PBD-1基因乳腺特异性表达载体,采用PCR方法从质粒pcDNA3.1-BLG-HNP-1中扩增出山羊乳球蛋白(BLG)基因,插入到真核表达载体pIRES2-EGFP-PBD-1构建成乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1.经PCR鉴定、限制性内切酶酶切分析和克隆片段序列测定、比较,鉴定.结果成功构建了乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1.乳腺特异性表达载体pIRES2-EGFP-BLG-PBD-1的成功构建,为进一步研究PBD-1蛋白的抗菌活性、抗菌机理及将进一步该载体应用于动物乳腺生物反应器的研究奠定基础.     

人血管内皮生长因子受体Flt-1胞外配体结合域的筛选及其结构分析

 血管内皮生长因子受体 Flt- 1胞外区具有 7个免疫球蛋白样的袢 (Ig- like loop) ,氨基端 3个loop负责与其配体 VEGF的结合 .为了寻求能与配体结合的更小的 Flt- 1片段 ,在对 Flt- 1胞外前3个 loop氨基酸组成和晶体结构分析的基础上 ,应用酵母双杂交系统对 Flt- 1的配体结合域进行筛选 .利用 PCR技术 ,从人胎盘 c DNA文库扩增出 4个截短的 Flt- 1 c DNA,分别含胞外第 2 ,1 - 2 ,2 - 3和 1 - 3个 loop,构建酵母双杂交系统融合表达质粒 ,并将 p GBT9/h VEGF165与 p GAD42 4 /Flt- 1 s两两配对转化酵母菌 SFY52 6,采用滤纸法和液体培养定量检测法对阳性克隆进行β-半乳糖苷酶活性分析 .结果显示 ,Flt- 1胞外 loop 2 - 3与 loop 1 - 3的配体结合能力相差不大 ,loop 1 - 2的结合力较弱 ,单独第 2个 loop无配体结合能力 .     

人VEGF165基因转染成人成肌细胞的实验研究

目的：将人血管内皮生长因子165（hVEGF165）导人原代离体成肌细胞,观察该细胞hVEGF分泌情况,探讨成人自体转基因成肌细胞移植的可行性。方法：采用两步消化法对成人骨骼肌组织消化获取相对较纯的成肌细胞,通过差速贴壁法进行进一步的纯化。以脂质体转染法将pcDNA3．1-hVEGF165导入成肌细胞,通过RT—PCR、ELISA和Western-blot进行hVEGF165定量检测,MTT测定和Mile’s实验检测VEGF165的生物学活性。结果：转基因细胞经RT—PCR扩增出一条VEGF的特异性泳带,ELISA显示转基因细胞培养上清VEGF浓度分别达到18．92±1．77rig／mL、19．04±2．15ng／mL,Western blot检测转基因成肌细胞上清均检测到VEGF蛋白特异性的杂交带,MTT显示转基因细胞上清明显促内皮细胞增殖,Mile’s实验显示转基因细胞上清明显增加毛细血管通透性。结论：质粒pcDNA3．1-hVEGF165能成功转入成人成肌细胞,转基因细胞能分泌有生物活性的VEGF165蛋白。     

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC)

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.     

Construction of recombinant adenovirus vector containing hBMP2 and hVEGF165 genes and its expression in rabbit Bone marrow mesenchymal stem cells

To construct an adenovirus vector co-expressing human bone morphogenetic protein (hBMP2) and human vascular endothelial growth factor (hVEGF165) as well as green fluorescence protein (GFP) as a marker, with which the intracellular expression of the inserted genes could be identified in Bone marrow mesenchymal stem cells (BM-MSCs). BMP2 and VEGF165 genes were PCR amplified from a cDNA library and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. The virus solution (Ad-BMP2-VEGF165) was generated by co-transfecting HEK293 cells with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ (delta) E1, 3Cre. The virus solution was further purified and virus titer was determined accordingly. The expression of the target genes was subsequently detected and quantified in rabbit BM-MSCs by using real time PCR, ELISA and Western blotting. The recombinant adenovirus vector containing BMP2 and VEGF165 (Ad-BMP2-VEGF165) was successfully constructed, which was confirmed by Sanger sequencing, colony PCR, as well as visually detection of GFP, and the titer of the adenovirus was 1 × 10¹⁰ PFU/mL, and the proteins level of BMP2 and VEGF165 secreted in the supernatant are significantly higher than the control. Recombinant adenovirus vector containing hBMP2 and hVEGF165 genes was successfully constructed. The transfection rate of BM-MSCs by the adenovirus was high (95% at 100 MOI) and the BMP2 and VEGF165 genes was highly expressed in the cells. The present study provides a method to efficiently express the target genes in BM-MSCs and an vector for further research of bone defect repair using dual genes of BMP2 and VEGF165.     

The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1

Neuropilin-1 (NP-1) was first identified as a semaphorin receptor involved in neuron guidance. Subsequent studies demonstrated that NP-1 also binds an isoform of vascular endothelial growth factor (VEGF) as well as several VEGF homologs, suggesting that NP-1 may also function in angiogenesis. Here we report in vitro binding experiments that shed light on the interaction between VEGF165 and NP-1, as well as a previously unknown interaction between NP-1 and one of the VEGF receptor tyrosine kinases, VEGFR1 or Flt-1. BIAcore analysis demonstrated that, with the extracellular domain (ECD) of NP-1 immobilized at low density, VEGF165 bound with low affinity (K(d) = 2 microm) and fast kinetics. The interaction was dependent on the heparin-binding domain of VEGF165 and increased the affinity of VEGF165 for its signaling receptor VEGFR2 or kinase insert domain-containing receptor. The affinity of VEGF165 for the NP-1 ECD was greatly enhanced either by increasing the density of immobilized NP-1 (K(d) = 113 nm) or by the addition of heparin (K(d) = 25 nm). We attribute these affinity enhancements to avidity effects mediated by the bivalent VEGF165 homodimer or multivalent heparin. We also show that the NP-1 ECD binds with high affinity (K(d) = 1.8 nm) to domains 3 and 4 of Flt-1 and that this interaction inhibits the binding of NP-1 to VEGF165. Based on these results, we propose that NP-1 acts as a coreceptor for various ligands and that these functions are dependent on the density of NP-1 on the cell membrane. Furthermore, Flt-1 may function as a negative regulator of angiogenesis by competing for NP-1.     

Hypoxic response elements control expression of human vascular endothelial growth factor(165) genes transferred to ischemia myocardium in vivo and in vitro

BACKGROUND: Vascular endothelial growth factor (VEGF) gene transfer with recombinant adeno-associated viral (rAAV) vector for ischemia heart disease therapy is being increasingly studied. However, uncontrolled long-term expression of VEGF may cause some side effects. Therefore, an attempt to develop an effective gene control system for safeguarding against such side effects should be made. Pathphysiologically, an ideal control system for VEGF gene expression is letting it respond to hypoxia. We used nine copies of hypoxic response element (HRE) to regulate expression of hVEGF(165) in the myocardium, and tried to elucidate the feasibility and safety of the application of the HIF-1-HRE system. METHODS: Cardiomyocytes of neonatal Sprague Dawley rats were cultured and incubated with rAAV-9HRE-hVEGF(165), and pig ischemic heart models were established and rAAV-9HRE-hVEGF(165) was injected into ischemia myocardium. RT-PCR, Western blot, ELISA, and immunohistochemistry were used to determine hVEGF(165) expressions of cultured cardiomyocytes and myocardium under hypoxic and reoxygenation conditions. RESULTS: The results of RT-PCR and ELISA determinations revealed that, in cultured cardiomyocytes, expressions of hVEGF(165)mRNA and protein were up-regulated under hypoxic conditions. After 4 h of reoxygenation, hVEGF(165)mNRA expression was decreased, and disappeared following 8 to 12 h of reoxygenation (P < 0.01). RT-PCR and Western blot also showed that, under myocardial ischemia, hVEGF(165) expression was increased significantly (P < 0.01). Following myocardial reperfusion, both hVEGF(165)mRNA and protein expressions were inhibited (P < 0.01). The new vessels in the reperfusion condition were decreased. CONCLUSIONS: This study suggested that 9HRE can effectively control hVEGF(165) gene expression in vivo and in vitro. It has feasibility for using the HIF-1-HRE system for regulation of angiogenic factor expression in ischemia heart.     

Specific blockade of VEGF and HER2 pathways results in greater growth inhibition of breast cancer xenografts that overexpress HER2

We have previously reported that breast cancer cells which overexpress HER2 produce higher levels of VEGF than cells with low levels of HER2. This study tested the hypothesis that dual targeting of the VEGF (with VEGF-Trap) and HER2 (with trastuzumab) pathways would result in greater growth inhibition of HER2-overexpressing breast cancer xenografts than either agent alone. In this study we found that human and murine endothelial cells expressed high levels of VEGF receptors (VEGFR1, VEGFR2, & VEGFR3). VEGF-Trap decreased levels of secreted VEGF derived from both human and murine cells and effectively blocked VEGF-induced tyrosine phosphorylation of VEGFR2. VEGF-Trap as a single treatment inhibited tumor microvessel density (MVD), tumor vasculature, cell proliferation, and tumor growth of BT474 xenografts in a dose-dependent manner from 2.5 mg/kg to 25 mg/kg. VEGF-Trap decreased levels of both human VEGF and PlGF protein in vivo. Trastuzumab as a single agent effectively inhibited BT474 tumor growth in a dose-dependent manner, associated with a decrease in human VEGF, tumor MVD and tumor cell proliferation. Treatment with a combination of VEGF-Trap (2.5-10 mg/kg) and trastuzumab (1 mg/kg) produced significantly greater inhibition of BT474tumor growth than either individual agent, associated with greater inhibition of tumor MVD and tumor cell proliferation. Thus, VEGF-Trap in combination with trastuzumab produces superior growth inhibition of tumor xenografts which overexpress HER2, which may result from inhibition of both tumor angiogenesis and proliferation. Similar mechanisms may contribute to the clinical anti-tumor activity of trastuzumab in combination with inhibitors of VEGF signaling pathway in women with breast cancers which overexpress HER2.     

血管内皮生长因子全长抗体在毕赤酵母中的表达与活性鉴定

目的:构建血管内皮生长因子(VEGF)全长抗体表达载体pPICZαA-VH-CH-VL-CL,评价其在毕赤酵母中的表达产物与抗原的结合特性,及其抑制细胞增殖的活性。方法:利用基因合成分别获得VEGF抗体CL和CH序列,分别构建pPICZαA-CH和pPICZαA-CL重组质粒,再利用同尾酶特性构建双启动子表达盒的重组pPICZαA-CH-CL载体,用Westen印迹对其进行表达鉴定后将VH和VL序列插入该载体,获得VEGF的全长抗体表达载体pPICZαA-VH-CH-VL-CL;通过膜筛和ELISA进行菌株筛选,并对VEGF抗体表达阳性菌株进行小量表达和纯化,采用CCK-8法对其抑制人脐静脉内皮细胞(HUVEC)增殖的活性进行初步评价。结果:获得表达轻、重链的VEGF抗体表达载体,ELISA实验证明pPICZαA-VH-CH-VL-CL具有一定的VEGF抗原结合特性;体外增殖实验表明,该抗体可以以剂量依赖性抑制HUVEC增殖。结论:在毕赤酵母中表达、纯化了具有一定功能活性的VEGF全长抗体,为后续比较研究酵母糖基化改造对VEGF抗体的药效学和药代学的影响提供了基础。