病毒负性调节因子在内皮细胞稳定表达细胞株的建立

建立HIV-1的调节基因Nef基因在内皮细胞稳定表达的细胞株ECV304-Nef,为研究Nef对血管内皮细胞生物学活性的影响奠定试验基础。构建真核表达载体pcDNA3.1(+)-Nef,将其质粒和pcDNA3.1(+)质粒(阴性对照)分别转染血管内皮细胞ECV304,G418筛选。通过RT-PCR检测NefmRNA在细胞中的表达;细胞免疫荧光法检测Nef蛋白的表达及定位;Western blotting检测Nef蛋白的特异性表达,获得稳定表达的细胞株。构建的重组质粒pcDNA3.1(+)-Nef经BamHI和EcoRI双酶切鉴定,得到的片段大小与理论值相符,分别为载体的5400bp和目的基因的621bp。测序结果显示碱基序列与GenBank(登录号:K03455)序列相同。转染细胞经G418筛选后获得稳定表达Nef的ECV304细胞株,RT-PCR显示转染pcD-NA3.1(+)-Nef质粒的ECV304细胞出现621bp条带,对照组无目的条带出现;荧光显微镜下观察转染pcDNA3.1(+)-Nef质粒的ECV304细胞表达的Nef蛋白主要定位于细胞质中。Western blotting结果显示,转染pcDNA3.1(+)-Nef质粒的ECV304细胞约27kD处检测到目的条带,表明pcDNA3.1(+)-Nef表达正确。     

含人纤溶酶原K5基因的腺病毒载体制备和功能研究

应用PCR将人纤溶酶原信号肽序列引入K5 cDNA基因,与真核表达载体pcDNA3重组,形成重组质粒pcDNA3K5,与穿梭质粒pShuttle 重组得pShuttleK5,经与腺病毒DNA重组,PCR鉴定正确,即为pAdK5。脂质体法将其转染293细胞后,制备细胞裂解液;噬斑分析法测定病毒滴度为5×10⁸ pfu/mL。将病毒以不同的感染系数(MOI)感染人脐静脉内皮细胞株ECV304和人乳腺癌细胞株MDA-MB-231,MTT法检测两者的增殖情况：ECV304细胞增殖受抑制,而MDA-MB-231细胞增殖未受明显影响。将感染病毒的ECV304细胞接种于ECMatrix^TM胶,显示内皮细胞分化和毛细血管管腔形成受抑制。表明所构建的含人纤溶酶原K5基因的重组复制缺陷型腺病毒具有抑制ECV304细胞增殖、分化和管腔形成的作用而对MDA-MB-231细胞的生长则无影响。     

SDF—1α对单核细胞的黏附作用

目的探讨pcDNA3．1-基质细胞衍生因子1α重组质粒对单核细胞的黏附作用。方法将pcDNA3．1-2基质细胞衍生因子1α质粒用脂质体的方法导入人脐静脉内皮细胞株ECV304细胞中,G418筛选细胞克隆。通过逆转录聚合酶链反应法在转录水平检测基质细胞衍生因子1α在转基因ECV304细胞中的表达。在6孔板上种植转染基质细胞衍生因子1α基因的ECV304细胞,加THP-1细胞37℃孵育30min,磷酸缓冲液轻洗3次,去除未黏附细胞,倒置显微镜下计数上、下、左、右、中5个视野,取其平均值得到每个视野黏附单核细胞数。结果获得了稳定表达基质细胞衍生因子1α基因的ECV304细胞克隆,pcDNA3．1-基质细胞衍生因子1n重组质粒对单核细胞具有黏附作用。结论基质细胞衍生因子1α对单核细胞具有明显黏附作用。     

VEGF重组质粒pcDNA/V的构建及其在大鼠急性心肌缺血模型中的表达

构建人血管内皮细胞生长因子(VEGF165)真核表达载体,并研究其在细胞水平和大鼠急性心肌梗死动物模型中的表达。利用RT-PCR方法,从人扁桃体组织中扩增人VEGF165基因,构建真核表达载体pcDNA/V。应用脂质体介导的基因转移技术,将pcDNA/V转染至人胚肾细胞(293细胞)中,经G418筛选获得稳定表达的重组质粒细胞克隆。ELISA、Westernblot检测证实重组质粒pcDNA/V能在293细胞中高效表达外源VEGF基因,鸡胚绒毛尿囊膜血管生成实验证实表达产物具有促血管生成的活性。进一步的体内表达研究,建立大鼠急性心肌梗死模型,将重组质粒pcDNA/V、空质粒pcDNA3.1( )分三点注射于梗死交界处心肌内,四周后取材。经免疫组化染色检测,pcDNA/V组在梗死交界区有VEGF阳性表达;电镜观察显示,pcDNA/V组在梗死交界处心肌细胞间有大量毛细血管内皮细胞增生。实验结果表明成功克隆了人VEGF165基因,构建了其真核表达载体。体内、外表达研究证实重组质粒的表达产物具有促血管生成的生物学活性,为VEGF基因治疗缺血性心肌病的研究提供实验基础。     

水蛭素12肽与低分子量单链尿激酶融合基因的构建、表达及产物特性分析

用化学合成的方法合成了水蛭素12肽基因的编码序列,通过DNA重组技术将水蛭素12肽基因片段与低分子量单链尿激酶cDNA片段连接构建了融合基因。融合基因在大肠杆菌中获得表达。体外实验结果表明,表达的融合蛋白具有溶纤活性和抗凝活性。     

白细胞抑制因子和水蛭肽嵌合蛋白表达纯化及活性研究

[目的]采用大肠杆菌表达白细胞抑制因子和水蛭肽嵌合蛋白,表达纯化后进行抗细胞黏附活性及抑制凝血酶活性的功能研究.[方法]将编码白细胞抑制因子和水蛭肽嵌合蛋白的基因构建到pET28a载体上,转化菌株Rosetta(DE3)并经IPTG诱导表达后分别经镍柱和分子筛进一步纯化,使用实时细胞分析仪和血凝仪分别测定了白细胞抑制因...     

重组凝血酶敏感蛋白-1N端片段对CD36表达阴性的ECV304细胞增殖的抑制

目的：构建凝血酶敏感蛋白-1N端片段（TSF）的原核表达体系,测定重组蛋白的活性。方法： 从人脐静脉内皮细胞cDNA中扩增得到thbs1基因片段,克隆到pET32c(+)载体系统并转化BL21大肠杆菌,表达纯化得到TSF。MTT法体外测定目的蛋白对ECV304细胞增殖的抑制作用。免疫荧光标记流式细胞仪测定CD36的表达。结果： 获得了原核表达的重组TSF蛋白。目的蛋白对CD36阴性的ECV304细胞有增殖抑制作用。结论： 低剂量重组TSF蛋白是一个具有临床应用前景的抗肿瘤治疗的辅助方法。     

重组人VEGF可溶性受体的表达纯化及结合性质初探

目的:表达优化的血管内皮细胞生长因子(VEGF)受体1(VEGFR1)胞外区第2个类免疫球蛋白结构域(VEGFR1D2)和VEGF受体2(VEGFR2)胞外区第3个类免疫球蛋白结构域(VEGFR2D3)与人IgG1 Fc片段的融合产物VEGF-Trap2,探讨该产物与人源VEGF165(hVEGF165)之间的亲和力。方法:将优化的目的基因VEGFR1D2/R2D3连接到真核表达载体pIRES2-EGFP-Fc中,转染CHO-K1细胞并筛选高表达目的蛋白VEGF-Trap2的细胞系,亲和纯化VEGF-Trap2蛋白,通过非竞争性ELISA及生物膜干涉技术检测VEGF-Trap2与hVEGF165之间的亲和力。结果:DNA测序表明真核表达载体pIRES2-EGFP-VEGF-Trap2序列正确;获得表达VEGF-Trap2的细胞系;非竞争性ELISA实验中,VEGF-Trap2与hVEGF165功能性亲和常数达到1.86×107L/mol;生物膜干涉实验中,hVEGF165与VEGF-Trap2的平衡解离常数达到3.13×10-9mol/L。结论:构建了真核表达载体pIRES2-EGFP-VEGF-Trap2并在CHO-K1细胞中稳定表达,重组蛋白VEGF-Trap2与hVEGF165有较高的亲和力,提示其可用于阻断VEGF信号传导途径,为该蛋白进一步的体外及体内实验奠定了基础。     

汉滩病毒M和S基因不同拼接方式嵌合基因免疫效果的研究

本文在前期工作的基础上,构建了汉滩病毒76-118株M基因G2片段与S基因5'端0.7Kb片段的嵌合基因真核表达载体pcDNA3.1-G2S0.7及pcDNA3.1-S0.7G2;用该质粒免疫BALB/c小鼠,结果表明两种质粒免疫小鼠可同时诱导产生抗汉滩病毒核蛋白(NP)及糖蛋白(GP)特异性的抗体,且前者刺激产生的抗体效价明显高于后者.淋巴细胞增殖实验表明,pcDNA3.1-G2S0.7组免疫小鼠脾细胞时NP及GP的增殖指数均明显高于空载体对照组,而pcDNA3.1-S0.7G2组未检测到其淋巴细胞有明显的增殖.这说明汉滩病毒M基因G2片段及S基因0.7Kb片段的嵌合基因既可刺激机体产生特异的抗汉滩病毒体液免疫应答,也可刺激机体产生特异的细胞免疫应答.不同拼接方式对嵌合基因免疫效果有很大影响,嵌合基因G2S0.7这种拼接方式明显优于S0.7G2.     

含人纤溶酶原K5基因的腺病毒载体制备和功能研究

应用PCR将人纤溶酶原信号肽序列引入K5cDNA基因 ,与真核表达载体pcDNA3重组 ,形成重组质粒pcDNA3K5 ,与穿梭质粒pShuttle重组得pShuttleK5 ,经与腺病毒DNA重组 ,PCR鉴定正确 ,即为pAd K5。脂质体法将其转染 2 93细胞后 ,制备细胞裂解液 ;噬斑分析法测定病毒滴度为 5× 10 8pfu mL。将病毒以不同的感染系数 (MOI)感染人脐静脉内皮细胞株ECV30 4和人乳腺癌细胞株MDA MB 2 31,MTT法检测两者的增殖情况 :ECV30 4细胞增殖受抑制 ,而MDA MB 2 31细胞增殖未受明显影响。将感染病毒的ECV30 4细胞接种于ECMatrixTM胶 ,显示内皮细胞分化和毛细血管管腔形成受抑制。表明所构建的含人纤溶酶原K5基因的重组复制缺陷型腺病毒具有抑制ECV30 4细胞增殖、分化和管腔形成的作用而对MDA MB 2 31细胞的生长则无影响。     

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.     

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。     

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。     

Screening of human vascular endothelial growth factor (VEGF) receptor Flt-1 domain and study on its biological activity

Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placen-tal cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1-3, but also the smaller fragment loop 2-3 could bind to hVEGF165. Recombinant expression plasmids pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1 (2-3) were constructed and transformed to Pichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1 % methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1 (2-3) to hVEGF165 and its inhibiting effect on the proliferation of human um     

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.     

ZNF230/荧光蛋白融合基因表达载体的构建及其在Cos细胞中的表达与定位

为了用绿色荧光蛋白标记观察人类无精症相关基因ZNF230在Cos7细胞中的蛋白质表达及定位,用PCR方法扩增得到突变的人和小鼠mt ZNF230和mt znf230基因,使其3′端的终止密码TGA突变为TGG,并装入T 载体,双酶切后通过定向克隆将其与真核表达载体pEGFP N1的绿色荧光蛋白(greenfluorescenceprotein,GFP)基因融合,构建了ZNF230—荧光蛋白融合基因表达载体。然后经真核表达质粒-脂质体介导,导入Cos7细胞系。荧光显微镜观察显示:在空白载体pEGFP N1转染的Cos细胞中荧光布满整个细胞,而在转染阳性载体pEGFP ZNF230和pEGFP znf230的Cos细胞中荧光主要聚集在细胞核中。表明转染的Cos细胞系能高效表达人ZNF230和小鼠znf230蛋白,ZNF230基因表达的蛋白定位于细胞核内。     

人组织型纤溶酶原激活剂-水蛭素融合基因的构建及其在毕赤酵母中的表达

构建并表达兼有溶栓和抗凝活性、减少出血副作用的人组织型纤溶酶原激活剂(t-PA)和水蛭素(HV2)的融合蛋白。通过提取总RNA和RT-PCR获得t-PA基因,与HV2基因通过活化凝血因子X(Fxa)识别序列（IEGR）的对应碱基序列连接构成融合蛋白基因,融合蛋白基因经pGEM-T、pIC9克隆至表达载体pIC9K上,电转导入毕赤酵母（Pichia pastoris）GS115。转化子摇瓶内甲醇诱导表达。纤维蛋白平板溶圈法和纤维蛋白凝块法分别检测溶栓和抗凝活性。琼脂糖凝胶电泳结果显示克隆的t-PA基因片段大小为1700bp,序列测定结果表明其35位氨基酸由文献报道的精氨酸突变为色氨酸。限制性酶切和PCR鉴定结果均表明融合蛋白基因已克隆入表达载体和宿主菌。甲醇利用实验、G418抗性筛选获得多拷贝甲醇利用快型克隆。甲醇诱导表达产物具有纤溶活性并可被抗t-PA抗体抑制。完整融合蛋白无抗凝活性,但以Fxa裂解后可释放抗凝活性。同时,融合蛋白以单链和双链两种形式存在。融合蛋白在血栓部位特有的Fxa作用下靶向释放抗凝活性,具有溶栓抗凝双功能,有望降低临床出血副作用。     

High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli

Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.     

抗CEA单链抗体与链亲和素融合基因的表达

克隆分泌CEA杂交瘤细胞重链可变区（V_H）和轻链可变区（V_L）,以Linker连接V_H及V_L构建抗CEA单链抗体.同时以Spacer连接单链抗体和链亲和素,构建成功单链抗体和链亲和素融合基因,克隆该融合基因至原核表达载体,pET21a(+),经IPTG诱导表达出该双特异性融合蛋白.活性鉴定表明该融合蛋白具有结合CEA及生物素的双特异性.该融合蛋白在生物领域中有较广阔的应用前景.