人DC-SIGN真核表达载体的构建及表达

目的构建含有FLAG标签的人DC—SIGN真核表达载体,并在人胚肾293T细胞中进行表达。方法通过PCR方法获得含有FLAG标签的人DC—SIGN分子基因,将其插入到真核表达载体pIRES—neo中。构建的重组质粒pIRES—neO—FLAG—DC—SIGN转染293T细胞,利用流式细胞仪、免疫荧光及WesternBlot检测其表达情况。结果含FLAG标签的人DC—SIGN基因测序正确,PCR和酶切鉴定证明FLAG—DC—SIGN基因已成功连入真核表达载体pIRES—neo中;检测结果显示重组质粒pIRES—neo—FLAG—DC—SIGN在293T细胞中得到表达。结论成功构建了重组质粒pIRES—neo—FLAG—DC—SIGN,且在293T细胞中能有效表达,为后续DC靶向性疫苗研究奠定了基础。     

人二型大麻受体基因的pIRES2-EGFP质粒的构建及其真核表达

目的：构建含有人二型大麻素受体（CB2）基因的真核表达载体,并实现其在HEK293细胞的表达。方法：首先采用PCR方法从含有CB2基因的质粒pcDNA3.1（＋）-CB2中扩增获得人的CB2基因,再采用基因重组技术将CB2的DNA片段插入真核表达载体pIRES2-EGFP。将获得的pIRES2-EGFP-CB2重组子转染HEK293细胞,应用荧光显微镜观察EGFP的表达情况,RT-PCR和报告基因检测CB2的表达情况。结果：酶切和测序结果验证真核表达载体pIRES2-EGFP-GB2构建成功。可在荧光显微镜下观察到转染HEK293细胞表达的绿色荧光;RT-PCR和双荧光素酶报告基因法检测证明CB2蛋白在真核细胞中成功表达;CB2受体激动剂JWH-015能显著逆转腺苷酸环化酶激动剂forskolin诱发的荧光素酶活性增加。结论：成功构建了真核表达载体pIRES2-EGFP-CB2,为进一步研究CB2基因的生理学和病理生理学功能及调控机制奠定了实验基础。     

人G250真核表达载体的构建及稳定转染B16细胞系的建立

目的构建人G250真核表达载体,建立稳定表达人G250的小鼠黑色素瘤细胞系。方法采用PCR方法扩增出人G250基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo-sig中,并加入酶切位点和FLAG标签,得到重组表达质粒pIRES-neo-sig-FLAG-G250。利用阳离子脂质体介导法将其稳定转染入小鼠黑色素瘤B16细胞,经G418加压筛选出阳性克隆。RT-PCR及免疫荧光检测人G250在B16细胞中的表达。结果经限制性内切酶鉴定及序列分析,pIRES-neo-sig-FLAG-G250重组质粒构建正确,最终建立的表达人G250基因的B16细胞系阳性率接近100%。结论成功构建了真核表达载体pIRES-neo-sig-FLAG-G250,建立的稳定转染人G250小鼠黑色素瘤细胞系能够高效表达G250基因。该稳定转染细胞系的建立为G250在肿瘤免疫治疗中的应用奠定了研究基础。     

人白细胞介素12双亚基共表达pVAX1-IRES-hIL12载体的构建与表达

目的构建下游可以共表达人白细胞介素12(hIL12)双亚基的双顺反子真核表达载体pVAX1-IRES-hIL12。方法通过搭桥PCR获得人白细胞介素12P35及P40双亚基的融合基因P35-F2A-P40,插入DNA疫苗载体pVAX1-IRES的下游,瞬时转染293-T细胞,ELISA检测融合基因的表达。结果酶切鉴定和序列分析表明融合基因与设计完全一致,融合基因在体外细胞培养液检测中获得分泌表达。结论该载体的成功构建可以为肿瘤基因疫苗研制提供免疫增效载体。     

Hsa—miR-200a真核表达载体的构建及鉴定

目的：通过构建及鉴定miR-200a的真核表达载体pcDNA3．1（＋）-miR-200a,为进一步研究miR-200a的功能奠定实验基础。方法：RT-PCR扩增pre—miR-200a基因序列,连接于表达载体pcDNA3．1（＋）中,对重组质粒双酶切鉴定并测序验证。结果：pcDNA3．1（＋）-miR-200a真核表达载体经酶切及测序鉴定正确。结论：pcDNA3．1（＋）-miR-200a真核表达载体构建成功,为下一步深入研究miR-200a的生物学功能和验证miR-200a的靶基因提供有效工具。     

人miR-34a靶基因的生物信息学分析及载体构建

目的对人miR-34a(hsa-miR-34a)下游靶基因进行预测,并构建含自噬相关基因靶结合序列的GFP报告基因载体。方法使用TargetScan Release 5.1和RNA22软件分析hsa-miR-34a可能的靶基因,并利用Gominer软件对靶基因功能进行分析。根据预测结果,合成含自噬相关基因hsa-miR-34a靶序列的寡核苷酸,以pEGFPC2为载体构建系列质粒,并检测各载体的真核表达情况。结果预测结果显示hsa-miR-34a共有2904个下游靶基因,功能涵盖生物过程、细胞组分、分子功能三大类,其中与自噬作用直接相关的靶基因有beclin 1和sestrin 2。构建含beclin 1和sestrin 2作用位点的GFP载体共5个,质粒转染Hela细胞后在荧光显微镜下可见绿色荧光。结论成功构建hsa-miR-34a下游自噬相关基因靶位点序列GFP报告基因载体,为进一步研究hsa-miR-34a对自噬的调控作用奠定基础。     

实时荧光定量PCR检测人肝癌组织皮层肌动蛋白基因

目的建立实时荧光定量PCR检测皮层肌动蛋白基因表达水平的方法。方法提取人肝癌组织总RNA,RT—PCR扩增CTTN和GAPDH基因片段并分别构建两片段阳性表达载体。采用SYBR Green I实时荧光定量PCR绘制两基因拷贝数标准曲线,实测人肝癌标本并进行方法学评价。结果成功构建pMD18-T（＋）／CTTN和pMD18-T（＋）／GAPDH重组质粒。CTTN和GAPDH基因模板拷贝数分别在1．6×10^3-1．6×10^7和1．6×10^4-1．6×10^7之间时标准曲线有良好线性关系,R^2分别为0．996和0．985。由Ct值统计两基因组内变异系数分别为0．11％～2．25％和0．75％-3．63％;组间变异系数分别为0．83％～1．48％和0．47％～1．36％。组间无统计学差异（P〉0．05）。结论成功建立实时荧光定量PCR检测人肝癌组织CTTN基因的方法,为研究人肝癌组织CTTN基因表达水平奠定了方法学基础。     

shRNA对瘢痕疙瘩成纤维细胞Smad3及Ⅰ型胶原表达的影响

目的：构建抑制瘢痕成纤维细胞（keloid fibrobtast,KFB）Smad3基因表达的shRNA真核表达载体一研究Smad3 shRNA对KFBSmad3及Ⅰ型胶原（typ e I collagen,COLIA2）表达的影响：方法：用前期实验筛选出的1对最有效SiRNA重组构建Smad3shRNA表达质粒;通过脂质体介导的方法,将Smad3shRNA转染剑KFB,用RTPCR及Western blot的方法检测Smad3、COLIA2在不同时间点（0d至9d）表达的变化。结果：①重组质粒经酶切鉴定和测序、RT-PCR和westerll blot的结果均证实Smad3shRNA载体构建成功。②转染Smad3shRNA后,随着时间的延长,KFB中Smad3的mRNA蛋白表达都显著减低,到72h作用最强：光密度分析与对照组比较差异有统计学意义（P〈0．05）。COLIA2的mRNA与蛋白表达都明硅下降（P〈0．05）．结论：体外构建的shRNA-Smad3RNAi真核表达载体能显著抑制KFBSmad3的表达。并使其Ⅰ型胶原mRNA和蛋白水平表达出现相同的变化,提示Smad3 shRNA可能是改善皮肤创而愈合和抑制瘢痕增生一个新的治疗方向。     

XIAP表达水平对5-Fu诱导肝癌细胞凋亡的影响

目的研究XIAP mRNA及蛋白表达水平对不同剂量5-氟尿嘧啶（5-fluorouracil,5-Fu）诱导人肝癌细胞HepG2凋亡的影响。方法脂质体介导含XIAP基因全长的质粒pef-XIAP转染人肝癌细胞HepG2,采用逆转录聚合酶链反应（RT—PCR）及western blotting检测XIAP基因和蛋白表达水平。通过不同剂量5-Fu诱导细胞凋亡,使用CCK-8试剂通过比色法分析检测5-Fu对转染pef-XIAP的HepG2细胞生长活性的抑制作用。结果各组HepG2细胞在不同剂量的5-Fu诱导下发生凋亡、与普通HepG2细胞组比较．转染XIAP基因的HepG2细胞组的凋亡率显著降低。结论XIAP基因在人肝癌细胞HepG2中的高表达可以明显降低化疗药物5-Fu诱导的细胞凋亡,其可能在肝癌化疗耐药中起一定作用。     

中性纤维素酶β-葡萄糖苷酶基因的克隆表达及酶活测定

本实验以一株产胞外中性纤维素酶的菌株芽孢杆菌CY110为研究对象,以麸皮为诱导物发酵获得了纤维素酶的限速酶组分-β-葡萄糖苷酶,酶的总活力和比活力分别达到了10568U和557．4U／mg,将产物分离纯化后经SDS-聚丙烯酰胺凝胶电泳检测,在43kDa处得到了单一条带。利用RT-PCR方法从芽孢杆菌CYll0中克隆出β-葡萄糖苷酶基因,将此目的基因连接到pBS-T载体后导入大肠杆菌DH5α,测序得到的阳性克隆以载体pET-28a为表达质粒构建出重组质粒并转化到表达菌株BL21（DE3）中。IPTG诱导表达外源基因后测定酶活并对比原始发酵酶活。结果显示：目的基因大小为1398bp,该序列与GenBank中的β-葡萄糖苷酶基因ABQL01000004．1序列相比同源性达到99％,氨基酸序列同源性达到98％,将产物与pET-28a表达载体连接,经双酶切检验,证明原核表达载体构建成功。诱导表达后的β-葡萄糖苷酶比活力为370．21U／mg,高于原始发酵产生的β-葡萄糖苷酶比活力8％。     

Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells

Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.     

Chondroitin Sulfate‐Coated DNA‐Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection

Degradation of plasmid DNA (pDNA) in the endosome compartment and its release to the cytosol are the major hurdles for efficient gene transfection. This is generally addressed by using transfection reagents that can overcome these limitations. In this article, the first report is presented which suggests that controlling the release of pDNA from endosome is the key for achieving efficient transfection. In this study, chondroitin sulfate (CS)‐coated DNA‐nanoplexes are developed using a modular approach where CS is coated post‐pDNA/PEI nanoplex formation. To ensure good stability of the nanoplexes, imine/enamine chemistry is exploited by reacting aldehyde‐modified chondroitin sulfate (CS‐CHO) with free amines of pDNA/PEI complex. This supramolecular nanocarrier system displays efficient cellular uptake, and controlled endosomal pDNA release without eliciting any cytotoxicity. On the contrary, burst release of pDNA from endosome (using chloroqine) results in significant reduction in gene expression. Unlike pDNA/PEI‐based transfection, the nanoparticle design presented here shows exceptional stability and gene transfection efficiency in different cell lines such as human colorectal cancer cells (HCT116), human embryonic kidney cells (HEK293), and mouse skin‐derived mesenchymal stem cells (MSCs) using luciferase protein as a reporter gene. This new insight will be valuable in designing next generation of transfection reagents.     

Effects of intracellular Na+/K+ ratio on histone gene expression in ascitic cells of leukemia P-388 and Ehrlich cancer

The intracellular Na+/K+ ratio was studied for its effect on histone genes expression in Ehrlich carcinoma and leukemia P-388 ascites cells. After equilibration with certain Na+/K+ ratio buffers at 0-4 degrees C the cells were treated during 1 h at 37 degrees C with ouabain added to the corresponding medium. Then total RNAs were extracted and analyzed by the reaction of blot-hybridization with histone genes cluster in plasmid p604. The maximal level of histone genes expression in leukemia P-388 cells was observed under conditions when intracellular Na+/K+ ratio corresponded to those at S-phase of the mitotic cycle. In Ehrlich carcinoma ascites cells the expression of histone genes was not dependent on the Na+/K+ ratio. A conclusion is drawn that the intracellular ratio of monovalent cations is one of the possible mechanisms which regulates the gene expression during the mitotic cycle.     

Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction

A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.