小鼠BPI N端功能片段(muBPI_(25)蛋白)体外杀菌和中和内毒素作用研究

目的:建立胞内杀菌和体外中和内毒素实验模型,检测muBPI25目的蛋白对胞内寄生菌的抑杀作用和对内毒素的中和作用。方法:将pcDNA3.1(+)-muBPI36-259质粒导入RAW264.7细胞,用胞内寄生G+/G-菌感染上述细胞建立muBPI25目的蛋白胞内杀菌实验模型;将pSecTag2B-muBPI36-259与双荧光素酶报告基因质粒共转染RAW264.7细胞,建立体外检测muBPI25蛋白中和内毒素实验模型。结果:胞内杀菌实验模型证实muBPI25目的蛋白对G-伤寒杆菌具有抑杀作用;中和内毒素实验模型证实muBPI25目的蛋白对内毒素具有中和作用。结论:首次证实小鼠BPI N端功能片段,即muBPI25目的蛋白对G-菌具有抑杀作用,对其裂解产物内毒素具有中和作用。     

小鼠Ipr1基因与EGFP基因融合表达载体的构建及其在鼠巨噬细胞中的表达

目的: 构建小鼠胞内病原体抗性基因1(Ipr1)基因与EGFP基因融合表达载体, 观察小鼠Ipr1基因在小鼠巨噬细胞株RAW264.7中的表达.方法: 从C57BL/6J小鼠胸腺组织提取总RNA, 以RT-PCR法调取Ipr1基因编码序列, 克隆至pEGFP-C1载体中, 筛选阳性克隆作PCR、酶切及测序鉴定后得到pEGFP-Ipr1, 脂质体瞬时转染小鼠巨噬细胞株RAW264.7, 以空质粒pEGFP-C1转染组作为对照.采用RT-PCR法检测Ipr1的RNA水平表达及用激光共聚焦显微镜观察融合蛋白的表达及细胞内定位.结果: 扩增出小鼠Ipr1基因编码序列, 经PCR、酶切及测序鉴定证明得到正确的重组质粒pEGFP-Ipr1, 脂质体瞬时转染小鼠巨噬细胞株RAW264.7得到了成功表达, Ipr1基因表达产物定位于细胞核内.结论: 成功地调取小鼠Ipr1基因, 构建了小鼠Ipr1基因与EGFP基因融合表达载体并在小鼠巨噬细胞株RAW264.7得到了成功表达.Ipr1编码蛋白定位于细胞核内, 提示其是一种调节蛋白.     

小鼠巨噬细胞内表达结核分枝杆菌CFP10-ESAT6融合蛋白对细胞增殖和凋亡的影响

 目的：建立培养滤液蛋白10-早期分泌性抗原靶6（CFP10-ESAT6）真核表达质粒并转染RAW2647巨噬细胞,观察胞内表达CFP10-ESAT6对细胞活性及凋亡的影响,并初步探讨其机制。方法：将CFP10-ESAT6融合基因插入真核表达质粒pEGFP-N1,构建重组质粒,转染RAW264.7巨噬细胞。采用MTT的方法测定CFP10-ESAT6融合蛋白对RAW264.7巨噬细胞活性的影响,采用结核分枝杆菌19 kD脂蛋白和十字孢碱（staurosporine）处理RAW264.7巨噬细胞,并以流式细胞术检测细胞凋亡率以及Toll样受体2（TLR2）的表达。结果：成功构建了重组质粒pEGFP-N1/CFP10-ESAT6并转染至RAW264.7巨噬细胞;与对照组相比,胞内表达CFP10-ESAT6不能影响巨噬细胞的活性,但可以明显抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡(P＜0.05),并显著抑制了TLR2的表达(P＜0.05)。结论：巨噬细胞内表达的CFP10-ESAT6融合蛋白不具有细胞毒性作用,但可以通过下调TLR2的表达来抑制结核分枝杆菌19 kD脂蛋白所造成的凋亡。     

分枝杆菌噬菌体D29 LysinB/Holin真核表达载体的构建及杀菌试验

目的构建分枝杆菌噬菌体D29 LysinB/Holin融合蛋白真核表达载体, 通过细胞感染模型研究其在胞内对结核分枝杆菌的杀伤效果。方法构建原核重组质粒pET32a-LysinB并诱导表达, 纯化蛋白后制备多克隆抗体;构建真核重组质粒pcDNA3.1(+)-LysinB/Holin并转染进单核巨噬细胞RAW264.7, 经制备的LysinB多克隆抗体进行表达鉴定后, 建立细胞感染模型并通过抗酸染色和菌落计数检测LysinB/Holin融合蛋白的杀菌效果。结果成功制备LysinB的多克隆抗体。重组质粒pcDNA3.1(+)-LysinB/Holin在真核细胞中有效表达LysinB/Holin融合蛋白且对细胞无明显毒性。LysinB/Holin融合蛋白可有效杀伤胞内的结核分枝杆菌。结论重组质粒pcDNA3.1(+)-LysinB/Holin对胞内结核分枝杆菌有较好的杀伤效果, 且对细胞无明显毒性, 具有治疗结核病的潜能。     

人杀菌/通透性增强蛋白活性片段基因在CHO细胞中的表达

目的获得人杀菌/通透性增强蛋白(bactericidal/permeability-increasing protein,BPI)N端活性片段蛋白,为BPI结构和功能的研究提供有力的工具。方法提取人外周血多形核白细胞(PMN)总RNA进行逆转录反应,经套式PCR扩增出BPIN端基因片段,将其克隆入pUC19载体中并进行酶切鉴定和序列测定,然后亚克隆入质粒pcDNA3构建真核表达载体pcDNA-BPI;重组载体通过脂质体转染CHO细胞并筛选阳性克隆,免疫荧光法鉴定重组BPI的表达和免疫学活性。结果酶切鉴定和序列分析证实重组质粒含有包括信号肽在内的BPI活性片段编码序列,与文献报道的序列相比,有6个核苷酸差异。转染筛选的阳性CHO细胞克隆表达产物能够被抗BPI的单克隆抗体所识别。结论BPI活性片段表达载体的构建及真核表达成功,为BPI功能的研究奠定了基础。     

HSP72过度表达对脂多糖刺激的巨噬细胞中NF-κB活性的影响

目的研究过度表达的热休克蛋白72(heat shock protein,HSP72)对脂多糖(lipopolysaccha-ride,LPS)刺激的巨噬细胞中NF-κB活性的影响.方法构建pCD-hsp72重组质粒,转染小鼠巨噬细胞RAW264.7,G418筛选稳定转染hsp72基因的细胞,Western印迹检测转染细胞HSP72的表达水平及LPS刺激后RAW264.7细胞内HSP72的表达水平、LPS刺激后转染细胞中IκBα的含量和胞核中NF-κB的含量的变化.结果构建pCD-hsp72重组质粒并转入巨噬细胞,获得稳定转染hsp72基因的细胞株,LPS刺激细胞内HSP72表达增加;HSP72可通过减少LPS刺激的巨噬细胞中IκBα的降解而抑制NF-κB活性.结论HSP72能抑制LPS激活的巨噬细胞中NF-κB的活化.     

稳定表达RFP-GFP-LC3的RAW264.7细胞株的建立

目的建立能够稳定表达红色荧光蛋白-绿色荧光蛋白-微管相关蛋白轻链3(RFP-GFP-LC3)的小鼠RAW264.7巨噬细胞。方法构建含RFP-GFP-LC3基因的慢病毒重组质粒载体,将重组质粒与慢病毒包装质粒共转染HEK293T细胞,收集病毒上清后感染RAW264.7细胞。经嘌呤霉素筛选获得稳定表达RFP-GFP-LC3的RAW264.7细胞株,用荧光显微镜和流式细胞术分析感染效率。饥饿处理稳定感染细胞,荧光显微镜下观察RFP-GFP-LC3斑点。结果成功构建了慢病毒p LV-CMV-RFPGFP-LC3,经感染和嘌呤霉素筛选获得稳定表达RFP-GFP-LC3的RFP-GFP-LC3-RAW264.7细胞;荧光显微镜和流式细胞术显示RFP和GFP荧光率均达到100%,饥饿处理后自噬斑点显著增多。结论成功建立稳定表达RFP-GFP-LC3的RAW264.7细胞株,为自噬研究建立了可靠细胞平台。     

小鼠Ccr2基因真核表达载体的构建及稳定转染细胞株的建立

目的:构建小鼠Ccr2基因真核表达载体,转染RAW264.7,建立稳定转染Ccr2的RAW264.7细胞系。方法:应用RT-PCR从小鼠脾脏组织中扩增Ccr2mRNA全序列,插入真核表达载体pEGFP-N1中。用脂质体将重组质粒pEGFP-N1/Ccr2转染RAW264.7细胞,通过G418筛选建立Ccr2稳定转染的RAW264.7细胞系。经RT-PCR、Western blot检测、荧光显微镜和流式细胞仪FCM检测,了解Ccr2在RAW264.7细胞中的表达水平及细胞内定位。结果:成功构建了pEGFP-N1/Ccr2重组质粒,建立了稳定转染的RAW264.7细胞株。RT-PCR和Western blot检测证实,Ccr2基因在该细胞系中成功表达,荧光显微镜下观察到该基因定位于细胞膜上,FCM显示90%以上的细胞膜上均有CCR2的表达。结论:构建了Ccr2真核表达载体并建立了稳定转染的RAW264.7细胞株。为进一步研究MCP1/CCR2信号通路在结核杆菌诱导巨噬细胞凋亡中的作用奠定了基础。     

杀菌/通透性增加蛋白N端片段在原核细胞中的表达及其临床应用的初步探讨

目的：构建编码杀菌，通透性增加蛋白(BPI)N端片段基因原核表达载体，并在大肠杆菌中表达重组蛋白。方法：采用RT-PCR技术，从人外周血中性粒细胞的总RNA中扩增BPI蛋白N端片段cDNA编码区基因，DNA序列分析鉴定；将测序证实的BPI蛋白N端片段编码区基因PCR产物直接克隆于原核表达载体pBAD／Thio-TOPO中，再次测序鉴定；通过在大肠杆菌中以L-阿拉伯糖进行诱导表达，然后以SDS-PAGE、Westerm blot对表达的重组蛋白进行分析。重组蛋白经初步纯化处理后，进行生物学活性检测。结果：RT-PCR扩增出一个835bp的DNA片段，DNA测序表明为所需目的基因。限制性内切酶酶切和DNA序列分析表明，BPI蛋白N端片段正确克隆到大肠杆菌表达载体pBAD／Thio-TDPO中，SDS-PAGE和Wester blot结果表明在大肠杆菌中成功表达了BPI蛋白N端片段。对该蛋白进行初步活性测定显示其可与BPI单克隆抗体结合并具有杀灭耐药铜绿假单胞菌的活性。结论：成功构建了BPI蛋白N端片段编码区基因原核表达载体，并在大肠杆菌中进行了表达，表达的重组蛋白具有一定的生物活性。     

小鼠CD86基因启动子的构建及活性分析

为构建小鼠CD86启动子,并对其特异性调控能力进行鉴定。首先自C57BL/6J小鼠基因组中采用高保真PCR扩增小鼠CD86基因的启动子,经测序鉴定后,插入pCDNA3.1-EGFP重组质粒。然后分别转染小鼠来源细胞株:RAW264.7、NIH/3T3、P815、SP2/0、EL4细胞,观察各细胞内绿色荧光蛋白的表达。结果显示,小鼠CD86基因启动子的序列与GenBank报道一致;双酶切鉴定证实小鼠CD86启动子序列已经克隆入重组载体;小鼠CD86启动子能调控EGFP在巨噬细胞系RAW264.7细胞中表达,不能调控EGFP在NIH/3T3、P815、SP2/0和EL4非抗原提呈细胞内表达。说明成功构建小鼠CD86基因启动子,且该启动子具有特异性调控目的基因表达的活性。     

Nuclear inheritance of a gene affecting mitochondrial gene expression

Due to a deficiency in mitochondrial protein synthesis, Chinese hamster lung (CHL) cell mutant Gal⁻ 32 does not grow in galactose or fructose. This report examines the nuclear or cytoplasmic inheritance of this single, recessive mutation. In a control experiment, fusion of Gal⁺TG^sTK⁻ cells with Gal⁻32TG^RTK⁺ cells resulted in tetraploid hybrids (as verified by karyotyping) that were selected in hypoxanthine/aminopterin/thymidine medium. The majority (2/3) of the control hybrids grew in galactose as expected since Gal⁻ is dominant over Gal⁻. Fusion of Rhodamine 6-G treated Gal⁺TG^STK⁻ cells with Gal⁻32TG^RTK⁺ cells resulted in Rhodamine 6-G-tetraploid hybrids that were selected in hypoxanthine/aminopterin/thymidine medium. The majority (7/12) of the Rhodamine 6-G-hybrids grew in galactose as expected for a nuclearly encoded gene considering that Rhodamine 6-G interferes with transmission of mtDNA but not nuclear DNA. Therefore, these results are compelling in their demonstration of the nuclear origin of the Gal⁻ 32 mutation.     

Regulatable gene expression systems for gene therapy

It is feasible to restrict transgene expression to a tissue or region in need of therapy by using promoters that respond to focusable physical stimuli. The most extensively investigated promoters of this type are radiation-inducible promoters and heat shock protein gene promoters that can be activated by directed, transient heat. Temporal regulation of transgenes can be achieved by various two- or three-component gene switches that are triggered by an appropriate small molecule inducer. The most commonly considered gene switches that are reviewed herein are based on small molecule-responsive transactivators derived from bacterial tetracycline repressor, insect or mammalian steroid receptors, or mammalian FKBP12/FRAP. A new generation of gene switches combines a heat shock protein gene promoter and a small molecule-responsive gene switch and can provide for both spatial and temporal regulation of transgene activity.     

Cancer gene therapy by adenovirus-mediated gene transfer

Cancer arises as a direct result of genetic mutations. It therefore stands to reason that cancer should be well suited for the correction through gene therapy. Recent advances in the understanding of the molecular pathogenesis of cancer and the rapid development of recombinant DNA technology have made cancer gene therapy feasible in the clinical setting. The current efforts for cancer gene therapy mainly focus on immunogene therapy, chemogene therapy, restoration of tumor suppressor gene function, and oncolytic virus therapy. Central to all these therapies is the development of efficient vectors for gene delivery--this remains a work in progress. These vectors can be classified as viral and non-viral vectors. This paper will concentrate on viral vectors because of their practical advantages over non-viral vectors. Of the viral vectors, by far the most important are the human adenoviruses as is reflected by the enormous data and literature accumulated by studies relating to animal tumor models and clinical trials. In this review, we examine the recent progress in adenovirus-mediated cancer gene therapy with regard to cytokine gene, tumor suppressor gene, chemogene, and oncolytic adenovirus. We also discuss the current limitations of the adenoviral vector system and how they may be circumvented in future developments relating to targeted gene delivery.     

Characterization of a rotavirus rearranged gene 11 by gene reassortment

Summary.  The effect of replacement of gene 11 of rotavirus SA-11 by a gene carrying a head to tail duplication obtained from a swine rotavirus strain was studied. The swine rotavirus strain with a duplicated gene (CC86) exhibits both a phenotype that allows to overgrow other viral strains when coinfected and an increased plaque size when plated in both CV-1 and MA-104 monkey kidney cells. Using reassortment methods the duplicated gene of the swine rotavirus was introduced into the SA-11 virus, replacing the regular gene 11. The reassorted strain was characterized to find out the origin of each of the other viral gene segments. Based on electrophoretic mobilities segments 1, 2, 3, 5, 7, 8 and 10 were identified as of SA-11. The SA-11 origin of the segments 4, 6 and 9 was confirmed by neutralization with polyclonal and monoclonal antibodies and by ELISA. The results suggest that the new reassortant virus was a monoreassortant carrying SA-11 genes except the duplicated gene originated from the swine virus CC86. The ability to in vivo replicate and to synthesize viral proteins was identical in the reassorted virus and the parental strains. Sequence analysis indicates that the new phenotype does not originate in the duplication of gene 11 but possibly from mutations in the coding region of NSP5 gene that may result in different phosphorylation patterns of the protein. Received January 30, 1998 Accepted April 6, 1998     

Cancer gene therapy through autonomous parvovirus--mediated gene transfer

Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines/chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virus-free vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines/chemokines are presented.     

Adenoviral vector-mediated gene transfer for human gene therapy

Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.     

胱硫醚合酶基因及其在人群中的多态性

胱硫醚合酶的活性降低或丧失是心脑血管系统等系统疾病独立的危险因子或易感因素.目前研究发现使酶活性降低或丧失的胱硫醚合酶基因的变异在人群中存在多态性.     

Identification of theaciA gene controlled by theamdA regulatory gene inAspergillus nidulans

Summary Several strains of yeast used in the brewing industry have been transformed using a plasmid carrying the dominant gene for copper resistance, CUP1. This technique will make it possible to consider genetic improvement of commercially useful yeasts through the introduction of desirable genes one at a time without disturbing the other characteristics of the strain. Selection for copper resistance may make it possible to regulate plasmid copy-number and stability.