Novel strategy for microsphere-mediated DNA transfection

A new approach for microsphere-mediated delivery of plasmid DNA has been developed and successfully evaluated. Basic molecular biology techniques were used to linearize and functionalize plasmid DNA by aminomodification, enabling efficient conjugation to carboxy-functionalized microspheres. A T cell hybridoma line was successfully transfected as determined by the efficient expression of a biologically relevant YFP fusion protein. Moreover, our data identified microsphere-mediated delivery of plasmid DNA as a noninvasive, nontoxic, and efficient gene delivery method with the potential to be applied to transfection-resistant, nondividing primary cells, including na?ve T cells.     

基于流式细胞术分选的高效表达外源基因细胞的筛选

目的：以增强型绿色荧光蛋白（EGFP）作为报告基因,用流式细胞术筛选高表达EGFP的细胞,从而获得外源基因高效表达细胞株。方法：构建在EGFPC端编码区融合新霉素（neomycin）抗性基因的融合基因EGFP-Neomycin,将其插入pcDNA3.1（＋）载体,构建EGFP-Neomycin融合基因表达载体pcDNAEN,转染CHO-K1细胞,G418加压筛选和倒置荧光显微镜观察证实所表达的EGFP-Neomycin融合蛋白具有新霉素抗性和激发EGFP荧光双功能;将编码组织型纤溶酶原激活剂（tPA）的cDNA插入pcDNAEN中CMV启动子下游,构建表达tPA的表达载体pcDNAEN/tPA。结果：流式细胞术分析和tPA纤维蛋白溶解活性测定表明,pcDNAEN/tPA转染CHO-K1细胞的EGFP相对荧光强度（RFT）的自然对数值与tPA表达水平呈明显的直线相关关系,相关系数为0.983;比较部分未经流式细胞仪分选的pcDNAEN/tPA转染阳性细胞克隆和RFT分布在100～1000的pcDNAEN/tPA转染阳性细胞克隆的tPA表达水平,经流式细胞术分选获得的细胞克隆的tPA平均表达水平和最高表达水平分别是未经分选获得的细胞克隆的3.9倍和4.1倍。结论：构建的EGFP-Neomycin融合基因具有双功能,建立了利用流式细胞术筛选外源基因高效表达物细胞株的方法。     

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.     

MOB2基因在真核细胞中的表达研究

通过RT-PCR从培养的HUVECs中扩增MOB2基因片段,将该片段克隆在真核表达载体pEGFP-C1中,转染NIH3T3细胞,经G418筛选,构建稳定表达细胞系,并用荧光显微镜和Western blot对其进行鉴定。经RT-PCR扩增出734 bp基因片段,经测序分析并与GenBank的DNA序列比对分析后,在NIH3T3细胞中表达。G418筛选后,细胞荧光信号较强,Western blot检测,细胞中的融合蛋白能够与抗MOB2的多抗结合。成功地扩增了HUVECs中的MOB2基因全长cDNA并进行真核表达与鉴定。     

Ad-sTNFRI-IgGFc腺病毒载体的构建及其转染人气道平滑肌细胞

TNF-a是一种具有广泛生物学活性的前炎症细胞因子, 参与哮喘整个病理生理过程。可溶性肿瘤坏死因子受体(sTNFR)可以拮抗肿瘤坏死因子活性, 已被用来治疗与TNF相关的炎性疾病。将sTNFRI-IgGFc基因插入腺病毒穿梭质粒pDC316, 与辅助质粒pBHGloxΔE1, 3Cre共转染HEK293细胞, 重组产生Ad-sTNFRI-IgGFc, PCR鉴定毒种正确后, 进行扩增、纯化和滴度测定, 转染人气道平滑肌细胞, 利用RT-PCR、免疫组化方法, 流式细胞仪, ELISA检测转染后细胞中sTNFRI-IgGFc的转录和表达。实验结果证明成功构建了Ad-sTNFRI-IgGFc腺病毒载体, 感染性滴度达3×1010 TCID50/mL, 200 moi转染气道平滑肌细胞阳性率达99.32%, 转染后气道平滑肌细胞在mRNA和蛋白水平均有sTNFRI-IgGFc表达。转染上清稀释64倍后仍对TNF有拮抗活性。为将表达sTNFRI-IgGFc腺病毒基因治疗哮喘的实验研究提供了基础。     

Maximal Expression of Recombinant cDNAs in COS Cells for Use in Expression Cloning

In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial β-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher β-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of β-galactosidase positive cells but considerably reduced the total β-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total β-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.     

绿色荧光蛋白与Wee1 Hu融合基因的构建及其真核表达

 构建Wee1Hu与增强型绿色荧光蛋白 (GFP)融合基因表达载体 ,并对其在真核细胞的表达及生物学效应进行研究 .应用基因工程技术构建重组载体 ,脂质体转染胰岛 β细胞株 ,流式细胞仪和免疫沉淀 Western印迹检测融合蛋白的表达 ,共聚焦显微镜分析融合蛋白在活细胞内的分布 ,3 D结构模建分析其结构特点 ,并用MTT法检测其生物学活性 .结果显示融合基因在瞬时或稳定转染的真核细胞中均获表达 ,融合蛋白主要分布在胞核区 ,融合蛋白中Wee1Hu的空间构象与天然Wee1Hu完全相同 ,表达融合蛋白的胰岛β细胞可避免其被细胞毒T淋巴细胞 (CTL)杀伤 .结果表明GFP Wee1Hu融合蛋白中 ,可发绿色荧光的分子标签GFP未能影响Wee1Hu的结构及其生物学活性 ;Wee1Hu可通过调控细胞周期而阻断CTL介导的细胞凋亡     

Uneven cellular expression of recombinant alpha2A-adrenoceptors in transfected CHO cells results in loss of response in adenylyl cyclase inhibition

Two populations of Chinese hamster ovary (CHO) cells expressing similar numbers of recombinant human alpha2A-adrenergic receptors (alpha2A-AR) showed different capacity to inhibit adenylyl cyclase (AC) activity. Cells transfected with an integrating vector exhibited agonist-dependent inhibition of forskolin-stimulated AC, whereas cells transfected with a non-integrating episomal vector showed no inhibition. Fluorescent microscopy and flow cytometry revealed a very uneven receptor distribution in the episomally transfected cell population. Monoclonal cell populations were expanded from this parent population. Most clones lacked significant amounts of receptors, while a few expressed receptors at high density; these exhibited efficient agonist-dependent inhibition of forskolin-stimulated AC activity. Thus, dense receptor expression in only a few cells is not sufficient to evoke a significant inhibitory response in a functional assay where AC is stimulated in all cells. Consequently, a false negative result was produced. Furthermore, the cell population transfected with an integrating vector showed loss of homogeneity with increasing passage number.     

绿色荧光报告基因在细胞免疫检测中的应用

将丙肝病毒C E1区基因插入绿色荧光报告基因pEGFP-N1中,构建真核表达重组质粒pEGFP-N1-HCV/C E1。转染小鼠骨髓瘤细胞SP2/0,在荧光显微镜下观察绿色荧光融合蛋白的表达情况。结果在细胞浆中出现了绿色荧光,表明目的基因得到表达,再通过G418筛选后大量培养用作细胞毒实验的靶细胞,结果表明以EGFP报告基因作筛选标记制备的靶细胞完全可以满足细胞毒实验要求。     

Protoplast-mediated gene transfer into human leukemia (K562) cells

We have examined the usefulness of a protoplast fusion technique as a tool to transfer cloned genes into hematopoietic cells. Protoplasts carrying cloned plasmids, which would express specific markers when successfully transfected into human cells, were prepared and fused with human leukemic cell line K562 cells using polyethylene glycol as a fusogenic factor. As a result, K562 cells fused with protoplasts containing a plasmid pSV2-cat constructed to code for chloramphenicol acetyltransferase (CAT) expressed CAT activity efficiently. K562 cells were also readily transformed to geneticin-(G418) resistant cells following fusion with protoplasts carrying a plasmid pSV2-neo-SV-gpt, which confers the resistance of mammalian cells to G418 and mycophenolic acid. It was also demonstrated that the plasmid genome was stably integrated into the chromosomal DNA of G418-resistant K562 cells. Our results proved that protoplast fusion could be used to study the specific expression and the biologic activities of cloned genes in human hematopoietic cells.     

The SDF-1/CXCR4 axis induces epithelial–mesenchymal transition in hepatocellular carcinoma

Targeted immunotherapy has become a popular research topic in cancer. The development and metastasis of cervical carcinoma are closely related to epidermal growth factor (EGF) and EGF-1 receptor (EGFR). We successfully constructed a single-chain human anti-EGFR antibody (scFv) and truncated protamine (tP) fusion protein (scFV/tP) expression vector using overlap extension PCR. Enzyme-linked immunosorbent assay and gel shift assay showed that the fusion protein retained the DNA and antigen-binding activity of the original antibody. Using the non-viral scFv/tP vector as a delivery tool, small interfering RNA (siRNA) of the human wings apart-like gene (hWAPL) was effectively transfected into cervical cancer HeLa cells. The hWAPL mRNA expression levels were reduced by 97.23 % in contrast with control cells, and the proliferation capability declined by 66.71 %, indicating significant inhibition. The present results provide a novel strategy for targeted gene therapy and siRNA therapy of EGFR-positive cervical cancers.     

鼠抗人纤维蛋白单链抗体-尿激酶杂合基因的构建及其在中国仓鼠卵巢细胞中的表达

采用 DNA重组技术构建了表达鼠抗人纤维蛋白单链抗体与低分子量尿激酶融合基因的真核表达载体。通过磷酸钙共沉淀法 ,将该表达载体转染到中国仓鼠卵巢细胞二氢叶酸还原酶基因缺陷株 ( CHO- dhfr-)中 ,利用选择培养基筛选出稳定表达的细胞株 ,溶解圈法测定融合蛋白的表达水平为每 1 0 6细胞每天 5 8IU。该融合蛋白保留了与纤维蛋白的结合活性和溶解纤维蛋白的溶纤活性。SDS- PAGE,Western印迹法分析证明融合蛋白的相对分子质量约为 70× 1 0 3     

p38L12 TAT融合肽对山梨糖醇引起的 ECV304细胞ATF2磷酸化的抑制作用

构建p38 Loop-12(L12)的TAT融合表达载体,纯化原核表达的p38L12融合蛋白并鉴定其在真核细胞内的功能.利用PCR方法分别扩增出p38L12及其“T-X-Y”双磷酸化位点的AF突变体p38L12（AF）片段,克隆入His标记的TAT-EGFP融合蛋白原核表达载体pHTE（pET14b-His-TAT-EGFP）,经酶切、测序鉴定正确后,将重组质粒转化原核表达菌,诱导表达纯化融合蛋白;将融合蛋白加入ECV304细胞后于荧光显微镜下观察并行Western印迹分析,检测融合蛋白的细胞内转导活性;通过检测内源性ATF2磷酸化水平,鉴定高渗刺激下p38L12对内源性p38活性的影响.成功构建了p38L12和p38L12(AF)片段与TAT的融合表达载体,并获得相应的融合蛋白.在ECV304细胞中可见导入的HTE-p38L12和THE-p38L12(AF)融合蛋白具有较高的细胞内转导活性和转导效率,并可竞争性抑制高渗刺激对内源性p38的活化.基于HIV-1 TAT细胞转导系统证实p38L12可竞争性抑制高渗刺激诱导的内源性p38对ATF2的活化,从而发挥对p38激活特异性抑制的功能.     

A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli

A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

表达分泌型脑源性神经营养因子的腺相关病毒载体的构建及其表达的检测

采用PCR和体外连接的方法构建了带有信号肽的脑源性神经营养因子(BDNF)的融合基因, 将此基因插入到腺相关病毒载体穿梭质粒pSNAV, 然后用重组质粒pSNAV-Ig-BDNF转染包装细胞, 筛选出永久细胞株后, 用携带腺相关病毒rep及cap基因的单纯疱疹病毒超感染包装细胞, 成功包装出含有目的基因的一型血清型腺相关病毒。经PCR鉴定该病毒含有目的基因片段, 被该病毒感染的细胞裂解液经Western blotting 证实有BDNF表达, 其培养液经ELISA检测证实含有高水平的BDNF蛋白。该病毒与小量的非复制型腺病毒Ad-null联合应用时, 其BDNF蛋白的表达水平显著提高。可弥补腺相关病毒起效慢、对细胞转导效率低的弱点, 为今后应用AAV作基因治疗提供了实验证据。     

人尿激酶型纤溶酶原激活因子截短型突变体与绿色荧光蛋白在真核细胞HEK293F中的融合表达

目的:构建人尿激酶型纤溶酶原激活因子(uPA)截短型突变体与绿色荧光蛋白(EGFP)分泌型融合表达载体并在真核细胞中表达。方法:采用PCR法,分别以质粒pIRES2-EGFP和重组质粒pcDNA3.1(+)/uPA为模板,扩增出带BamHⅠ和XbaⅠ酶切位点的EGFP及带NheⅠ和HindⅢ酶切位点的uPA截短体基因片段,先后将EGFP和截短型uPA基因片段克隆到真核表达载体pcDNA3.1(+)上,转入HEK293F细胞,用G418对转染细胞进行加压筛选,通过共聚焦显微镜观察和ELISA方法鉴定表达产物。结果:DNA测序结果显示,uPA不同截短型突变体基因片段与EGFP基因融合的真核表达载体构建成功,共聚焦显微镜观察发现HEK293F细胞中有绿色荧光且定位于细胞质中,ELISA检测到HEK293F细胞培养上清中分泌型融合蛋白的表达。结论:构建了uPA截短型突变体与EGFP分泌型融合表达载体并在真核细胞中表达,为后期研究uPA的相互作用蛋白及其生理功能奠定了基础。