兔抗人重组生长激素rhGH多克隆抗体的制备、纯化及鉴定

目的制备抗人生长激素(growth hormone,GH)的多克隆抗体并鉴定其性能。方法真核细胞表达质粒pcDNA3.0-GHcDNA免疫家兔,制备rhGH多克隆抗体;ELISA法检测抗体效价。亲和层析法纯化后,进行Western blot、免疫组织化学和激光共聚焦显微镜检测,鉴定抗体的特异性与适用范围。结果得到兔抗人重组人生长激素rhGH多克隆抗体,效价达1:40000。纯化后的抗体可特异识别超表达的hGH和人内源性GH,可用于免疫组织化学实验、Western blot及激光共聚焦。结论用质粒免疫家兔制备的抗GH抗体具有较高的效价以及特异性,为hGH的功能性研究提供了有力的支持。     

Description and plasmid characterization of the qnrD determinant in Proteeae in Wenzhou,Southern China

Background/Purpose

Only limited information is available about the detailed characteristics of qnrD, a plasmid-mediated quinolone resistance (PMQR) gene. This study aimed to understand the distribution of qnrD and the characterization of qnrD-carrying plasmids in Proteeae.

miR-570对胃癌细胞B7-H1蛋白表达的抑制作用

B7-H1是一种重要的负性共刺激分子,在肿瘤免疫逃逸中发挥重要作用,而微小RNA具有直接的转录后调控作用。本文研究miR-570对B7-H1表达的调控作用。首先将miR-570及其抑制剂anti-miR-570分别转染B7-H1表达阳性的人胃癌细胞SGC-7901和B7-H1表达阴性的人乳腺癌细胞MDA-MB-435,以流式细胞术检测B7-H1分子表达情况;然后构建pcDNA/B7-H1表达质粒与miR-570共转染CHO细胞,以流式细胞术检测CHO细胞上B7-H1分子的表达情况;最后分别构建含B7-H1基因3-UTR片段和含miR-570作用靶点序列的荧光素酶表达载体与miR-570共转染CHO细胞,用双荧光素酶报告系统检测荧光素酶活性。结果显示miR-570能显著抑制SGC-7901细胞和B7-H1基因转染细胞膜上B7-H1蛋白的表达,并能显著抑制荧光素酶表达载体表达的荧光素酶蛋白,而且anti-miR-570能上调MDA-MB-435细胞上B7-H1表达。本研究证明miR-570能显著抑制B7-H1蛋白表达,为通过抑制B7-H1信号通路以增强机体抗肿瘤免疫力的治疗途径奠定了基础。     

菌落PCR和质粒PCR对转化菌的筛选

为建立简便,可靠转化菌的筛选方法。方法应用菌落ＰＣＲ和重组质粒ＰＣＲ方法对转化菌进行筛选和ＤＮＡ序列分析。克隆载体和表达载体筛选及鉴定采用与载体多克隆插入位眯序列互补的通用引物或免疫球蛋白家族特异性引物。结果菌落ＰＣＲ和质粒ＰＣＲ技术可作为基因克隆筛选和鉴定的方法。结论菌落ＰＤＲ和质粒ＰＣＲ方向简便,快速,可靠,其产可作为模板直接用于序列分析。     

A circular mitochondrial plasmid incites hypovirulence in some strains of Cryphonectria parasitica

In the chestnut-blight fungus Cryphonectria parasitica, a plasmid, pCRY1, occurs in the mitochondria of several strains isolated at various locations in the northeastern United States and Canada. The monomer of this plasmid is a 4.2-kb circular double-stranded DNA that has no detectable sequence homology with the 160–kb mitochondrial DNA of Ep155, a standard virulent laboratory strain of C. parasitica. The circular nature and oligomeric characteristics of the plasmid were deduced from the heterogeneous size of plasmid DNA molecules as detected by one- and two-dimensional gel-electrophoresis, the nature and alignment of restriction fragments, and the lack of detectable termini in the nucleotide sequence. The cytoplasmic location of the plasmid was deduced from its co-purification with mitochondria, uniparental (maternal) transmission in sexual crosses, dissociation from the nuclei of the donor strain during its horizontal transfer between vegetatively compatible strains through hyphal anastomoses, and mitochondrial codon usage (UGA=Try). The pCRY1 plasmid contains a long open reading frame that is transcribed and potentially encodes a unique 1214 amino-acid, B-family DNA polymerase similar to those encoded by the LaBelle and Fiji circular mitochondrial plasmids of Neurospora. In this subgroup of proteins, the DTD motif characteristic of B-family DNA polymerases is replaced by TTD. Amino-acid motifs related to those that are characteristic of the 3′→5′ exonuclease domains of B-family DNA polymerases have been located in the amino-terminal portion of the proteins. A comparison of isogenic plasmid-free and plasmid-containing cultures indicates that pCRY1 is an infectious agent that effects a reduction in the pathogenicity of some, but not all, strains of C. parasitica. Received: 12 August / 9 December 1999     

Electron microscopic investigation of mitochondrial DNA fromChenopodium album (L.)

DNA molecules from mitochondria of whole plants and a suspension culture ofChenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0.3–183 kb represented 23–26% of all mtDNA molecules in the preparations from the suspension culture and 13–15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rollingcircle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.     

蛋白激酶C在红细胞生成素介导的早期信号转导中的作用

利用pfosluc 2质粒转染对Epo刺激有反应的ELM-I-1细胞系细胞,测定c-fos表达活性的研究方法观察蛋白激酶C在Epo介导的早期信号转导过程中的作用,c-fos表达活性以光子量表示。pfosluc 2质粒转染的细胞加PMA 50 nmol/L刺激,光子量由2 501±126/1×10~5细胞升高到4272±242/1×10~5细胞(n=3,P<0.001);PMA刺激诱导的c-fos活性表达呈剂量依赖性。以Epo 2 U/ml刺激细胞同时以 PMA 50 nmol/L刺激时,光子增加量明显比单独用Epo和PMA刺激时的光子增加量之和增多,结果提示PMA不仅本身能诱导c-fos表达活性,同时能增强Epo刺激诱导的c-fos表达活性。另一方面蛋白激酶C抑制剂H7明显抑制Epo和PMA诱导的c-fos表达活性,Epo诱导的c-fos表达活性下降57.8％。本研究结果提示蛋白激酶C在Epo介导的早期信号转导过程中具有重要的正性调节作用。     

Pulmonary delivery of siRNA against acute lung injury/acute respiratory distress syndrome

The use of small interfering RNAs (siRNAs) has been under investigation for the treatment of several unmet medical needs, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) wherein siRNA may be implemented to modify the expression of pro-inflammatory cytokines and chemokines at the mRNA level. The properties such as clear anatomy, accessibility, and relatively low enzyme activity make the lung a good target for local siRNA therapy. However, the translation of siRNA is restricted by the inefficient delivery of siRNA therapeutics to the target cells due to the properties of naked siRNA. Thus, this review will focus on the various delivery systems that can be used and the different barriers that need to be surmounted for the development of stable inhalable siRNA formulations for human use before siRNA therapeutics for ALI/ARDS become available in the clinic.     

Growth inhibitory effects of THY1 gene on epithelial ovarian cancer SKOV3 cells

Objective： The aim of this study was to construct THY1 eukaryotic expression plasmid ,and study its effects on ovarian cancer SKOV3 cells. Methods： The gene fragment coding for THY1 was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1 （＋） to construct the recombinant plas- mid pcDNA3.1（＋）-THY1, which was transfected into SKOV3 cells. The experimental cells were classified into three groups： SKOV3-THY1, SKOV3-Null and SKOV3. The expression of gene was measured using RT-PCR and Western blotting. The percentage of apoptotic cells and cell cycle analysis and cell proliferation were assessed by flow cytometry and MTT assay. Both SKOV3-THY1 and SKOV3-null cells were inoculated subcutaneously into nude mice to determine in vivo tumorigenicity. Results： The gene fragment of THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1 （＋） and veri- fied by PCR, restriction endonucleases digestion and DNA sequencing and the plasmid of pcDNA3.1（＋）-THY1 （THY1 gene overexpression） has been stably transfected into SKOV3 cells. The analysis of flow cytometry indicated that the pcDNA3.1（＋）- THY1 transfected ceils in G1 phase were significantly elevated, but in S phase were decreased. The growth of transfected cells was suppressed, and more apoptosis cells were identified in pcDNA3.1（＋）-THY1 transfectants compared with vector vehicle transfectants. The tumor suppressing activity of THY1 in SKOV3 cells was associated with inhibition of SKOV3 cellular proliferation, in vivo tumorigenesis in nude mice. Conclusion： THY1 transfection can inhibit the growth of SKOV-3 cells in vitro and in vivo. THY1 gene may play an important role in generation and development of ovarian cancers.     

外源表达Nkx2-5基因诱导P19细胞向心肌分化

目的:构建人Nkx2-5基因融合绿色荧光蛋白真核表达质粒pEGFP-N1-Nkx2-5,探讨外源性Nkx2-5基因诱导P19细胞向心肌分化的作用。方法:以真核表达质粒pEFSA-HA-Nkx2-5为模板,PCR扩增得到人Nkx2-5基因,将目的片段亚克隆到pEGFP-N1载体,鉴定正确。将重组质粒pEGFP-N1-Nkx2-5以脂质体法转染P19细胞,G418筛选2周,聚集4d,贴壁培养16d。以免疫细胞化学检测desmin、α-sarcomeric actin和cardiac Troponin T(cTnT)的表达。结果:将Nkx2-5基因正确插入pEGFP-N1载体,外源表达Nkx2-5基因可使P19细胞在无诱导剂、聚集条件下表达desmin、α-sarcomeric actin和cTnT蛋白。结论:成功构建真核表达质粒pEGFP-N1-Nkx2-5,外源表达Nkx2-5基因诱导P19细胞向心肌分化。