抑癌基因PTEN载体构建及在LOVO细胞中的表达

目的:构建抑癌基因PTEN的表达载体并在LOVO细胞中表达。方法:从人外周血中提取总RNA,通过RT-PCR扩增出PTEN基因片段,将PTEN基因连入pLenti6/V5载体。测序鉴定后,经脂质体转染入LOVO细胞,对细胞株进行RT-PCR和Western blot检测。结果:DNA测序证实pLenti6/V5-PTEN表达载体为阳性克隆;该表达载体转染LOVO细胞后上调了PTEN mRNA和蛋白的表达。结论:成功构建了pLen-ti6/V5-PTEN表达载体,为进一步研究PTEN在LOVO细胞中的功能奠定了基础。     

Lumican 基因真核表达载体的构建及其对人子宫内膜癌细胞 HEC -1A 增殖的影响

目的：克隆人Lumican基因及构建Lumican基因真核表达载体并研究其对人子宫内膜癌细胞HEC-1A增殖的影响。方法：取人新鲜正常子宫内膜组织,提取总RNA,采用逆转录-聚合酶链反应（RT-PCR）扩增Lumican基因,将该基因定向克隆到真核表达载体pEGFP-N1中,构建真核细胞表达载体pEGFP-N1-Lumican,用限制性内切酶酶切分析,DNA序列分析鉴定重组质粒;将测序正确的Lumican基因通过脂质体介导下转染人子宫内膜癌细胞HEC-1A;逆转录-聚合酶链反应（RT-PCR）和Western Blot 检测转染细胞HEC-1A的Lumican mRNA和蛋白表达。采用MTT法观察转染Lumican基因的人子宫内膜癌细胞HEC-1A的增殖能力。结果：重组质粒pEGFP-N1-Lumican经HindⅢ和BamHⅠ酶切,获得8311 bp片段和865 bp插入片段,序列分析表明插入的片段与Gene Bank发布的序列一致;荧光显微镜下可见转染的HEC-1A细胞有绿色荧光蛋白的表达;转染细胞HEC-1A的Lumican mRNA表达上调,Lumican蛋白相对上调率为74．62％（P＜0．05）。与对照组细胞比较,转染Lumican 基因的人子宫内膜癌细胞HEC-1A的抑制率为21．35％±2．78％。结论：成功构建了真核表达载体pEGFP-N1-Lumican,该载体在体外能有效抑制人子宫内膜癌细胞HEC-1A的增殖能力,为以Lumican基因为靶点研究其对人子宫内膜癌细胞HEC-1A的恶性生物学特性提供实验基础。     

小鼠Bmi1基因的克隆及其在支持细胞中的表达

目的从昆明鼠睾丸中克隆Bmi1基因,构建真核表达载体,并转染支持细胞,以便用作培养精原干细胞（SSCs）的滋养层.方法以5日龄昆明鼠为材料,提取小鼠睾丸组织中总RNA后,以RT-PCR技术克隆小鼠睾丸Bmi1基因,构建真核表达载体,并转染TM4细胞（睾丸支持细胞株）,在转染后40 h进行免疫荧光鉴定.结果成功克隆小鼠睾丸Bmi1基因的cDNA,测序正确;免疫荧光细胞染色显示,转染后的支持细胞中有Bmi1蛋白表达.结论本研究为以转染了Bmi1基因的支持细胞作饲养层培养SSCs奠定了基础.     

人鼻咽鳞癌细胞CNE1/R PECAM-1真核表达载体的构建

通过TA克隆技术及二步克隆的方法构建人鼻咽鳞癌细胞CNE1/R PECAM-1的真核表达载体。将已克隆至Pmd19-T Simple载体的PECAM-1基因片段使用Not/HindⅢ酶切,亚克隆至pcDNA3.1/myc-His(-)A载体并双酶切及测序鉴定。成功构建了人鼻咽鳞癌细胞CNE1/R PECAM-1的真核表达载体,为下一步建立人鼻咽鳞癌PECAM1基因过表达细胞系奠定了基础。     

日本七鳃鳗pcna基因克隆、生物信息学分析及真核表达

增殖细胞核抗原(proliferating cell nuclear antigen,PCNA),也称周期蛋白或DNA聚合酶的辅助蛋白,是真核细胞合成所必需的核蛋白,在DNA复制中起重要作用。前期实验发现日本七鳃鳗肝脏cDNA文库的表达序列标签(Expressed Sequence Tag,EST)中存在与高等脊椎动物pcna基因同源的序列。提取日本七鳃鳗(Lampetra japonica)肝脏组织RNA,通过RT-PCR方法扩增七鳃鳗pcna基因,对其进行生物信息学分析,并将Lj-pcna基因成功构建到pGFP-N2真核表达载体上,重组质粒PGFP-N2-Lj-pcna转染人Hela细胞,荧光显微镜下观察有荧光蛋白的表达。日本七鳃鳗pcna基因的真核表达载体成功构建和转染,为探讨七鳃鳗pcna基因功能研究及其它七鳃鳗相关研究提供条件。     

Bmi-1-siRNA对肺腺癌A549细胞迁移和转移的影响

观察Bmi-1基因沉默对A549细胞体外迁移和体内转移的影响。设计针对Bmi-1的小干扰RNA(siRNA)序列作为靶序列构建重组逆转录病毒siRNA表达载体并将其转染入肺腺癌A549细胞;应用RT-PCR和Western Blot方法检测对Bmi-1基因的沉默效果;应用划痕修复和Transwell方法检测Bmi-1-siRNA对A549细胞体外迁移的影响;通过裸鼠尾输入各组细胞,观察Bmi-1-siRNA对A549细胞在裸鼠体内转移能力的影响。结果显示:沉默Bmi-1基因的表达能够抑制A549细胞的体内外迁移能力,同时降低VEGF的分泌。Bmi-1-siRNA能抑制肺腺癌A549细胞的转移能力,VEGF可能参与其中。     

青蒿异分支酸合酶基因的克隆及功能分析（英文）

目的:研究青蒿异分支酸合酶的表达模式,评价其对青蒿素含量的影响。创新点:该研究首次克隆了青蒿异分支酸合酶基因(AaICS1),并发现AaICS1影响青蒿素的合成,为更有效地开发利用青蒿提供了新思路。方法:根据青蒿转录组数据,利用聚合酶链式反应(PCR)克隆AaICS1基因和启动子,并进行多重序列分析和启动子作用元件预测。通过实时定量PCR(q RT-PCR)对AaICS1进行表达分析,用Southern杂交分析AaICS1的拷贝数。构建AaICS1过表达载体和干扰表达载体,转化青蒿获得转基因植株,用高效液相色谱法(HPLC)分析青蒿素含量。结论:AaICS1含一个总长为1710 bp的完整阅读框,编码570个氨基酸,与其它植物的ICS基因具有较高的相似性。Southern杂交结果表明AaICS1为单拷贝(图4),q RT-PCR结果显示该基因能够响应伤害、干旱、盐胁迫和水杨酸的处理,处理后基因表达量提高(图6),和启动子作用元件预测相符。q RT-PCR结果显示过表达转基因青蒿中AaICS1表达量提高,干扰转基因青蒿中该基因表达量降低(图7)。HPLC显示过表达AaICS1转基因植株中青蒿素含量提升,最高可达对照的1.9倍(图8)。     

苹果ABP2基因克隆及其表达载体构建

以红富士苹果为材料,从苹果枝条形成层组织中提取总RNA,以随机引物及oligodT反转录成cD-NA。根据已知序列设计PCR引物并对其进行修饰,运用PCR技术扩增出苹果ABP基因片段和全长,经测序分析得知从红富士苹果中克隆到的基因ABP2(GenBank:HQ610832)为ABP(生长素结合蛋白)的突变体,同时完成了ABP2基因表达载体的构建。文章在苹果RNA提取、全长基因克隆、ABP2基因表达载体的构建方面积累了经验,并为进一步的转基因研究奠定了基础。     

RNA干扰T细胞mTOR表达后诱导T细胞分化对小肠移植耐受的影响

目的:探讨RNA干扰T细胞mTOR表达后诱导T细胞分化对小肠移植免疫耐受的影响。方法:通过shRNA干扰T细胞mTOR表达后,诱导T细胞向Foxp3+Tregs细胞分化。对60只雄性SD大鼠施行30次异位节段小肠移植,随机分为A组(mTOR-shRNA转染组)、B组(mTOR抑制剂RAD001干预)和C组(移植对照组、注射生理盐水)。观察小肠移植后受体大鼠的体重变化、生存时间及移植小肠病理切片评估排斥反应程度。结果:构建的mTOR-shRNA质粒表达载体在体外能有效地抑制大鼠骨髓细胞mTOR基因mRNA和蛋白的表达。RNA干扰大鼠T细胞mTOR基因可调节T细胞的定向分化,Tregs细胞增多,而Th17细胞分化减少。抑制mTOR基因可诱导大鼠异位小肠移植的免疫耐受,减轻受体抗移植物的排斥反应,显著延长移植物的存活时间。结论:RNA干扰T细胞mTOR表达后诱导T细胞分化对小肠移植耐受的研究为mTOR抑制剂(RAD001)在移植后的临床应用提供理论和实验依据。     

水生植物凤眼莲Ca2＋-ATPase的原核表达与蛋白纯化

从水生植物凤眼莲叶片中提取总 RNA,经 RT-PCR扩增出Ca2＋-ATPase基因片段,经限制性内切酶(Sma I,Not I)酶切后按正确的读码框顺序插入到pGEX-4T-2表达载体上,重组质粒转化大肠杆菌,经菌落PCR和质粒双酶切鉴定、序列测定确认,证实成功地构建了Ca2＋-ATPase基因融合表达载体,．转化菌经IPTG诱导表达,获得了大小约48 kD的可溶性目的蛋白,与预期相吻合．利用谷胱甘肽琼脂糖凝胶4B(Gluta-thione Sepharose 4B)亲和介质对重组蛋白进行纯化,获得了高纯度的目的蛋白．     

Construction of a eukaryotic expression plasmid of Humanin

Objective: To construct a eukaryotic expression plasmid pcDNA3.1 (-)-Humanin. Methods: The recombinant plasmid pGEMEX-1-Humanin was digested with restriction endonucleases BamH I and Hind III and the Humanin gene fragments, about 100 bp length, were obtained. Then the Humanin gene fragments were inserted into eukaryotic expression vector pcDNA3.1 (-) and the recombinant plasmids pcDNA3, l(-)-Humanin were identified by sequencing. Results: Recombinant plasmid DNA successfully produced a band which had the same size as that of the Humanin positive control. The sequence of recombinant plasmids accorded with the Humnain gene sequence. Conclusions: A eukaryotic expression plasmid of Humanin was successfully constructed.     

Effect of serum concentration on adhesion of monocytic THP-1 cells onto cultured EC monolayer and EC-SMC co-culture

Background:The adhesion of monocytes to the endothelium following accumulation oflow-density lipoprotein(LDL) in subendothelial spaces is an important step in the development of intimal hyperplasia in arterially implanted vein grafts and atherosclerosis in both animals and humans.However.it iS not well known how serum factors affect the adhesion of monocytes.Methods:We have studied the efrect of fetal calf serum(FCS).which we considered a source of LDL.on the adhesion of monocytes to endothelial cells(Ecs)by using human monocytic THP-1 cells and both a monolayer of cultured bovine aortic endothelial cells(EC monoculture)and a co-culture with bovine aortic smooth muscle cells(EC-SMC co-culture).Results:It was found that the addition of FCS to the medium greatly affected the adhesion of THP-1 cells,and the higher the concentration of FCS in the medium,the greater the adhesion of THP-1 cells to endothelial cells.Adhesion of THP-1 cells to an EC-SMC co-culture Was approximately twofold greater than that to an EC monoculture,and after adhering to endothelial cells,many THP-1 cells transmigrated into the layer of smooth muscle cells.Conclusion:The results suggest that the elevation of the LDL(cholesterol)level in blood provides a favorable condition for the development of intimal hyperplasia and atherosclerosis by promoting the adhesion of monocytcs to the endothelium and their subsequent migration into subendothelial spaces.     

Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken     

ICA69基因工程融合抗原的制备

基于基因工程技术 ,用PCR法扩增出编码ICA6 9的cDNA片段 ,直接克隆到pSPORT 1质粒上 ,经DNA序列测定 ,插入到GST融合蛋白表达载体 pGEX 2T ,构成重组质粒 p2T ICA6 9,得到的表达产物GST ICA6 9融合蛋白用间接ELISA法检测其免疫原性 .测序结果表明 ,所获PCR产物已正确重组到PGEX 2T表达型质粒中 .重组质粒在原核细胞中表达的融合蛋白具有免疫原性 ,并能应用于Ⅰ型糖尿病病人血清中抗ICA6 9抗体的检测 .所获得的表达产物为重组ICA6 9融合抗原 ,有助于提高Ⅰ型糖尿病的预报率和确诊率 .     

Expression of a bee venom phospholipase A2 from Apis cerana cerana in the baculovirus-insect cell

Bee venom phospholipase A2(BvPLA2) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids.In this work,a new BvPLA2(AccPLA2) gene from the Chinese honeybee(Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector.Tn-5B-4(Tn) cells were transfected with the recombinant bacmid DNA for expression.Sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa.Products of hexahistidine AccPLA2 fusion protein accumulated up to 5.32% of the total cellular proteins.The AccPLA2 fusion protein was cross reactive with the anti-AmPLA2(BvPLA2 of the European honeybee,Apis mellifera) polyclonal serum.The reaction resulted in a double glycosylation band,which agrees with the band generated by the native AmPLA2 in Western blot analysis.The PLA2 activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 μmol/(min·mg).In summary,the recombinant AccPLA2 protein,a native BvPLA2-like structure with corresponding biological activities,can be glycosylated in Tn cells.These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA2 in the pharmaceutical industry.     

Receptor-mediated gene delivery using polyethylenimine （PEI） coupled with polypeptides targeting FGF receptors on cells surface

Objective： To construct a novel kind of nonviral gene delivery vector based on polyethylenimine （PEI） conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. Methods： The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. Results： The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR 16 could be blocked by excess free polypeptides. Conclusion： The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.