含日本血吸虫脂肪酸结合蛋白（SjFABPc）基因DNA重组质粒 …

目的 观察裸ＤＮＡ重组质粒ｐＣＤ－ＳｊＦＡＢＰｃ在体外转染ＨｅｐＧ２细胞以及质粒ＤＮＡ免疫鼠肌组织中的表达，为进一步应用该质粒进行ＤＮＡ免疫实验奠定基础。方法 用脂质体介导ＤＮＡ转染法将ｐＣＤ－ＳｊＦＡＢＰｃ转染贴壁细胞ＨｅｐＧ２，Ｇ４１８加压筛选获得阳性克隆细胞并传代培养，ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ－ｂｌｏｔ鉴定目的基因在ＨｅｐＧ２细胞中的表达，将ｐＣＤ－ＳｊＦＡＢＰｃ质粒肌注免疫ＢＡＬＢ     

重组丙型肝炎病毒基因转染H9细胞模型的建立

目的：建立重组ＨＣＶ基因转染的Ｈ９细胞（人ＣＤ４，Ｔ细胞）模型。方法：以载体ＣＤＺ２（连接有１６９３ｂｐＨＣＶＤＮＡ，包括完整的ＨＣＶ结构区）为模板，通过ＰＣＲ获得高保真的１．７３ｋｂＤＮＡ片段（包括１６９３ｂｐＨＣＶｃＤＮＡ和部分载体ＤＮＡ序列），其特异性被ｓｏｕｔｈｅｒｎｂｌｏｔ证实。将ＨＣＶｃＤＮＡ片段插入载体ｐＣＤ－ＳＲα，经菌落原位杂交以及酶切分斤和ｓｏｕｔｈｅｒｎｂｏＩｔ筛选，获得重组ＨＣＶ（ｐＣＤ－ＨＣＶ），并与ｐＳＶ２－ｇｐｔ载体共转染Ｈ９细胞。结果：从选择性培养基中筛选口阳性克隆细胞，经ＲＴ－ＰＣＲ．ｄｏｔＥＬＩＳＡ和ｗｅｓｔｅｒｎｂｌｏｔ验证表明ｐＣＤ－ＨＣＶ在Ｈ９细胞中可进行复制、转录和表达，表达的约１．３×１０５大小蛋白具有ＨＣＶ抗原性。转染ｐＣＤ－ＨＣＶ的细胞培养至９０天时，仍可检测到ＨＣＶｍＲＮＡ和ＨＣＶ抗原。结论：建立ｐＣＤ－ＨＣＶ转染的Ｈ９细胞模型获得成功。     

HBsAg在不同真核表达载体中的表达

目的建立ＨＢｓＡｇ及其变异体的高效真核表达系统。方法从已构建的ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋－ＨＢｓ系列突变重组体获取ＨＢＶ－Ｓ基因片段,将其亚克隆到真核表达载体ｐＭＥＰ４,ｐＣＥＰ４,ｐＬＸＳＮ,ＰＸＴ１及ｐｃＤＮＡ３,从而构建了含ＨＢＶ－Ｓ基因及其突变型的一系列表达载体,并将其转染（或感染）人肝癌细胞系ＨｅｐＧｅ２。结果获得稳定分泌ＨＢｓＡｇＲ其突变体的抗性细胞系。结论各组真核表达载体皆能表达ＨＢｓＡｇ及其突变体,但在转染率、表达量及稳定性上存在差异。选择其中的高效、准确表达系统,将为ＨＢｓＡｇ变异株生物学性状的深入研究及进一步基因治疗、基因免疫的研究提供有用的工具。     

乙型病毒S基因单独和与白细胞介素—12基因联合免疫小鼠诱生的体液免疫应

以ＰＣＲ技术扩增得到乙肝病毒表面抗原的编码基因,构建Ｓ基因真核表达载体ｐｃＤＮＡ３．１（＋）－Ｓ。以ＰＣＲ方法扩增得到小鼠白细胞介素－１２的全长ｃＤＮＡ序列,构建真核表达载体ｐｃＤＮＡ３－ＩＬ－１２（以下简称ＩＬ－１２）。以Ｓ和Ｓ＋ＩＬ－１２经肌肉注射免疫ＢＡＬＢ／Ｃ小鼠,以空载体ｐｃＤＮＡ３．１（＋为阴性对照,血源ＨＢｓＡｇ蛋白为阳性对照,用ＥＬＩＳＡ方法检测下同时间免疫小鼠血清中的抗０－ＨＢｓ     

日本血吸虫（中国大陆株）磷酸丙糖异构酶DNA疫苗的研制及其保护性免疫的研

目的 研究日本血吸虫中国大陆株磷酸丙糖异构酶ＤＮＡ疫苗诱导Ｃ５７ＢＬ／６小 保护性免疫作用。方法 分别以ｐＵＣ１９－ＳｊＣＴＰＩ和ｐｃＤＮＡ１．１－ＩＬ－１２为模板设计引物。将ＰＣＲ扩增到的ＳｊＣＴＰＩ基因以及ＩＬ－１２的亚单位Ｐ３５和Ｐ４０基因克隆到真核表达载体ｐｃＤＮＡ．３．１中大量制备ｐｃＤ－ＮＡ３．１－ＳｊＣＴＰＩ和ｐｃＤＮＡ３．１－Ｐ３５、ｐｃＤＮＡ３．１－Ｐ４０的质粒ＤＮＡ,把４５只５～６周龄Ｃ５７ＢＬ／６小鼠分成３组。对照组（ｐｃＤＮＡ组）肌肉注射１００ｕｇ的ｐｃＤＮＡ３．１;实验组（ＴＰＩ组）肌肉注射１００ｕｇ的ｐｃＤＮＡ３．１－ＳｊＣＴＰＩ;加强组（ＴＰＩ＋ＩＬ－１２组）肌肉注射１００ｕｇ的ｐｃＤＮＡ３．１－ＳｊＣＴＰＩ及１００ｕｇ的ｐｃＤＮＡ３．１－Ｐ３５和ｐｃＤＮＡ３．１－Ｐ４０的混合物     

日本血吸虫Sj32DNA疫苗免疫效果的观察

目的：探讨血吸虫Ｓｊ３２ＤＮＡ疫苗抗血吸虫感染的免疫效果。方法：采用ＰＣＲ技术,人工合成引物,以重组ＤＮＡｐＳｊ３２为模板,扩增出编码３２ｋＤａ天冬酰胺肽链酶,即Ｓｊ３２全长ｃＤＮＡ和引入了起始密码子终止密码子的部分ｃＤＮＡ片段,将其分别克隆到ｐＫＢ－ＣＭＶ,构建真核表达载体ｐＢＫ－Ｓｊ３２－１和ｐＢＫ－Ｓｊ３２－２。采用脂质体介导的基因转移将两种重组ＤＮＡ导入ＮＩＨ３Ｔ３细胞,间接免疫荧光法证实Ｓｊ３２在真核细胞中表达。小批量制备纯化ＤＮＡ,进行动物免疫试验。将两种构建的真核表达载体分别直接ｉｍＢＡＬＢ／ｃ小鼠,共免疫３次,攻击感染后６ｗｋ剖杀小鼠。收集血清,ＥＬＩＳＡ法检测抗体水平,并计数成虫负荷及肝组织虫卵数。结果：ｐＢＫ－Ｓｊ３２－１组与ｐＢＫ－Ｓｊ３２－２组免疫小鼠后的减虫率分别为３８．６％和３０．９％,肝组织减卵率分别为５５．７％和５５．２％,与对照组比较,其间差别均具有极显著性意义（Ｐ＜０．０１）。各ＤＮＡ疫苗免疫组能诱导一定水平的抗血吸虫抗体。结论：Ｓｊ３２ＤＮＡ疫苗可诱导小鼠产生一定程度的保护性免疫力,有可能作为血吸虫疫苗候选抗原分子。     

中国人极低密度脂蛋白受体基因的克隆及其在中国仓鼠卵巢细胞中的表达

为了研究极低密度脂蛋白受体结构与功能的关系,本文利用反转录聚合酶链反应技术从中国人心肌组织中克隆出极低密度脂蛋白受体的全长ｃＤＮＡ后,将其插入真核表达载体ｐｃＤＮＡ３获得重组体ｐＣＤ－ＶＲ。测序结果发现,这一源于中国人的极低密度脂蛋白受体基因ｃＤＮＡ的碱基序列与文献报道基本相符,反转录聚合酶链反应检测证实,转染ｐＣＤ－ＶＲ的中国仓鼠卵巢细胞可有效表达极低密度脂蛋白受体ｍＲＮＡ。脂蛋白结合实验结果发     

日本血吸虫26kDa谷胱甘肽S-转移酶基因真核表达载体的构建及序列测定

目的 扩增日本血吸虫２６ｋＤａ谷胱甘肽Ｓ－转移酶（Ｓｊ２６ＧＳＴ）基因片段，构建其重组真核表达载体，以研制ＳｊＧＳＴ核酸疫苗。方法 根据已知基因序列设计一对引物，运用ＲＴ－ＰＣＲ技术扩增Ｓｊ２６ＧＳＴ目的基因片段，将其克隆到ｐｃＤＮＡ３质粒中，并经酶切、ＰＣＲ鉴定，测序证实。结果 获得ＧＳＴ－ｐｃＤＮＡ３重组克隆。结论：本实验获得Ｓｊ２６ＧＳＴ重组克隆，为进一步研制核酸疫苗创造了条件。     

HBV反义全基因真核细胞表达载体的构建及在肝癌细胞内的表达

１．材料与方法：乳哺动物细胞表达载体ｐＢＫ－ＣＭＶ,长约４．５ｋｂ,带有Ｔ_７公用序列。ｐＣＰ１０是ｐＢＲ３２２ＥｃｏＲＩ位点中插入双拷贝头尾相接的ＨＢＶａｙｗ亚型全基因ＨＢＶＤＮＡ重组质粒。ＰＣＲ引物ＸＩ１：１４２８－１４４８ｎｔ５'ＣＧＴ　ＣＣＣＧＴＣ　ＧＧＣＧＣＴＧＡＡＴＣＣ３',ＸＩ_２：１６７２－１６５３ｎｔ５'ＡＧＴＣＣＡＡＧＡＧＴＣＣＴＣＴＴＡＡＧ３'。人肝癌细胞系ＳＭＭＣ－７７２１,出上海细胞所提供。将ＥｃｏＲＩ酶切后回收的全基因ＨＢＶ片段。在Ｔ_４ＤＮＡ连接酶作用下,与ＥｃｏＲＩ酶切…     

反义乙肝病毒S基因真核载体构建及体外抗乙肝病毒作用

目的：探讨反义ＲＮＡ抗乙肝病毒（ＨＢＶ）作用。方法：构建了正，反义ＨＢＶＳ基因重组ＥＢ病毒载体ｐＭＥＰ４ｓ，ｐＭＥＰ４Ｓａｓ，ＤＮＡ－磷酸钙共沉淀法将重组载体ＤＮＡ转染２．２．１５细胞，潮霉素筛选１个月得到抗性细胞克隆，ＥＬＩＳＡ，斑点杂交法分别检测抗性细胞上清ＨＢｓＡｇ，ＨＢｅＡｇＨＢＶＤＮＡ水平，结果：转染后１，２月，反义载体ＨＢｓＡｇ，ＨＢｅＡｇ的抑制率分别达７５％，５１．６％，７０％，４６     

Feline Foamy Virus-Based Vectors: Advantages of an Authentic Animal Model

New-generation retroviral vectors have potential applications in vaccination and gene therapy. Foamy viruses are particularly interesting as vectors, because they are not associated to any disease. Vector research is mainly based on primate foamy viruses (PFV), but cats are an alternative animal model, due to their smaller size and the existence of a cognate feline foamy virus (FFV). The potential of replication-competent (RC) FFV vectors for vaccination and replication-deficient (RD) FFV-based vectors for gene delivery purposes has been studied over the past years. In this review, the key achievements and functional evaluation of the existing vectors from in vitro cell culture systems to out-bred cats will be described. The data presented here demonstrate the broad application spectrum of FFV-based vectors, especially in pathogen-specific prophylactic and therapeutic vaccination using RD vectors in cats and in classical gene delivery. In the cat-based system, FFV-based vectors provide an advantageous platform to evaluate and optimize the applicability, efficacy and safety of foamy virus (FV) vectors, especially the understudied aspect of FV cell and organ tropism.     

蛋白激酶C受体1过表达载体、干扰载体的构建及鉴定

目的：构建并鉴定蛋白激酶C受体1（ RACK1）过表达及干扰真核表达载体。方法采用逆转录聚合酶链反应（ RT-PCR）从人肝癌细胞系Huh-7．5．1中扩增RACK1全长cDNA系列,用巢式PCR扩增对应的CDS,并将其克隆到PIRES2-EGFP,PCR鉴定后进行测序分析。以人RACK1基因为靶基因,设计合成两对短发夹结构的互补DNA序列和一对阴性对照序列,退火后与酶切后的载体RNAi-Ready pSIREN-RetroQ-ZsGreen进行连接,并行酶切分析和测序鉴定。将构建好的过表达载体和干扰载体用脂质体转染HUVEC细胞系。结果两对引物PCR扩增的序列大小与预期结果一致,PIRES2-EGFP/RACK1载体954个碱基成功插入到预计位点,序列完全一致。 RNA干扰载体pRetroQ/RACK1-1、pRetroQ/RACK1-2和pRetroQ/HK插入序列均与预计相符。荧光显微镜观察,转染的HUVEC细胞均表达EGFP和GFP。结论成功构建RACK1过表达载体及其RNA干扰载体,并可被成功导入HU-VEC细胞系。     

A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans

Aims/hypothesis Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets. Materials and methods We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion. Results In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure. Conclusions/interpretation These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research. Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users     

人内皮抑素小环载体在真核细胞中的生物活性

目的通过对人内皮抑素小环载体在真核细胞中的表达和生物学效应的研究,探讨小环载体作为生物治疗载体的可行性。方法将mc-hES、pcDNA-hES分别转染人肝癌细胞HepG2后,通过ELISA、RT-PCR和Westernblot观察hES表达。MTT法检测其对人脐静脉内皮细胞（HUVEC）的增殖抑制作用。结果 mc-hES和pcDNA-hES均能表达hES,mc-hES能明显抑制HUVEC的生长,而对HepG2无明显作用（P〈0.05）。在相同条件下,小环较常规质粒在体外能快速介导转基因的表达,且较传统质粒高6～8.3倍。结论 mc-hES较普通质粒pcDNA-hES在肝癌细胞中能高效表达转基因,为小环载体进一步研究提供了很好的实验基础。     

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.     

利用融合PCR技术和T载体克隆技术构建结核分枝杆菌PhoPR双组份系统缺失突变载体

目的 基于融合PCR技术(SOE-PCR),T载体克隆技术,构建结核分枝杆菌(Mycobacterium tuberculosis,MTB)双组份系统缺失突变载体的方法.方法 利用SOE-PCR技术,将PhoP,PhoR和PhoPR基因上,下游的同源臂与卡那霉素抗性基因融合,构建PhoP,PhoR和PhoPR突变片段,然后将突变片段直接与T载体连接,将其转入E. coli DH5α感受态细胞并筛选抗性克隆,进而获得MTB PhoP,PhoR和PhoPR缺失突变载体.结果 成功构建MTB PhoPR双组份系统缺失突变载体,与传统方法相比,效率高,周期短.结论 基于融合PCR技术和T载体克隆技术成功构建MTB PhoPR双组份系统缺失突变载体,为下一步构建MTB突变株以及相关研究奠定基础.     

Transgenic rhesus monkeys produced by gene transfer into early-cleavage–stage embryos using a simian immunodeficiency virus-based vector

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage–stage embryos. We show that infection does not alter embryo development. Moreover, the timing of infection, either before or during embryonic genome activation, has no observable effect on the level and stability of transgene expression. Of 70 embryos injected with concentrated virus at the one- to two-cell stage or the four- to eight-cell stage and showing fluorescence, 30 were transferred to surrogate mothers. One transgenic fetus was obtained from a fraternal triple pregnancy. Four infant monkeys were produced from four singleton pregnancies, of which two expressed EGFP throughout the whole body. These results demonstrate the usefulness of SIV-based lentiviral vectors for the generation of transgenic monkeys and improve the efficiency of transgenic technology in nonhuman primates.     

Honey bees navigate according to a map-like spatial memory

By using harmonic radar, we report the complete flight paths of displaced bees. Test bees forage at a feeder or are recruited by a waggle dance indicating the feeder. The flights are recorded after the bees are captured when leaving the hive or the feeder and are released at an unexpected release site. A sequence of behavioral routines become apparent: (i) initial straight flights in which they fly the course that they were on when captured (foraging bees) or that they learned during dance communication (recruited bees); (ii) slow search flights with frequent changes of direction in which they attempt to "get their bearings"; and (iii) straight and rapid flights directed either to the hive or first to the feeding station and then to the hive. These straight homing flights start at locations all around the hive and at distances far out of the visual catchment area around the hive or the feeding station. Two essential criteria of a map-like spatial memory are met by these results: bees can set course at any arbitrary location in their familiar area, and they can choose between at least two goals. This finding suggests a rich, map-like organization of spatial memory in navigating honey bees.     

Electrocardiographic interpretation (1995): Can we do better?—Part II

An explanation of the Grant method and six steps the author of this paper considers essential to electrocardiographic interpretation are presented in Part I of the paper (Clin. Cardiol. 18, 433–439, 1995).