O型口蹄疫病毒VP1基因与大肠杆菌不耐热肠毒素LTB 基因的融合表达及表达产物的免疫原性分析

以质粒pMDTLT为模板、用PCR的方法扩增出LTB基因,然后将其插入到pETVP1质粒中VP1基因的上游,构建了含有融合基因LTBVP1的表达质粒pETLTBVP1。转化宿主菌BL21(DE3)LysS后进行诱导表达,诱导菌经SDS-PAGE显示重组蛋白以包涵体的形式表达,分子量约为39kD;Western blotting分析表明,重组蛋白能与FMDV阳性血清及兔抗霍乱毒素(CT)血清反应,说明融合蛋白保持了LTB和VP1各自的免疫学活性。小鼠免疫实验表明:该融合蛋白通过腹腔接种小鼠能诱导产生较强的免疫应答反应,免疫鼠产生的血清抗体水平高于试验中商品口蹄疫疫苗免疫组。     

淋球菌porB和大肠杆菌ltB融合基因的构建、表达及其免疫活性

【目的】构建融合基因原核表达载体pET-30a/ltB-porB,并表达重组融合蛋白LTB-PorB,鼻饲途径免疫雌性BALB/c小鼠,分析重组融合蛋白的免疫活性,为研制抗淋病蛋白疫苗提供实验依据。【方法】构建大肠杆菌不耐热肠毒素B亚单位(LTB)与淋球菌外膜孔蛋白B(PorB)融合基因及LTB、PorB单基因pET-30a原核表达载体,在大肠杆菌BL21中表达重组蛋白;鼻饲途径免疫雌性BALB/c小鼠,检测体液免疫和细胞免疫水平。【结果】在大肠杆菌BL21中获得高效表达的重组蛋白;经鼻饲免疫小鼠后,重组融合蛋白LTB-PorB组生殖道黏膜产生的PorB特异性sIgA水平随免疫时间呈上升趋势,第42天A450值达0.66,明显高于对照组(P0.01),效价高达1∶1280;血清中产生的PorB特异性IgG第28天达最高,A450值为0.60,明显高于LTB和蛋白溶解液(Solution Buffer)对照组(P0.01),效价高达1:2560,但与PorB对照组血清IgG水平(A450:0.57)无明显差异(P0.05)。LTB-PorB组脾淋巴细胞刺激指数明显高于LTB和SolutionBuffer对照组(P0.05),但脾淋巴细胞诱生的IFN-γ水平与对照组无明显差异(P0.05)。【结论】重组融合蛋白LTB-PorB通过鼻饲途径免疫雌性BALB/c小鼠后,能诱导产生高水平的体液免疫和一定水平的细胞免疫。首次证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道粘膜免疫。     

O型口蹄疫病毒VP1 T细胞、B细胞表位双拷贝基因与大肠杆菌LTB、STⅠ肠毒素基因融合表达产物的免疫应答

合成O型口蹄疫病毒VP1蛋白中与细胞免疫(21～40表位肽)及体液免疫(141～160表位肽)相关的基因序列2020VP1,运用基因工程技术构建了含有肠毒素大肠杆菌LTB、STⅠ基因及双拷贝2020VP1的融合表达载体r2020-B-2020-STⅠ,转化宿主菌 BL21(DE3) RIL后的表达产物经SDS-PAGE分析,结果显示重组融合蛋白的分子量约为45 kDa,表达量较高.ELISA实验结果显示,融合蛋白能与霍乱毒素(cholera toxin)CTB抗体特异结合.动物实验表明,融合蛋白能够诱发兔体产生较强的FMDV中和抗体,免疫豚鼠在低浓度FMDV刺激下能够产生特异性T淋巴细胞增殖反应,说明融合蛋白能诱导机体产生FMDV特异性细胞及体液免疫反应;同时,融合蛋白免疫雌鼠能够抵抗大肠杆菌强毒株攻击,免疫兔体能够产生STⅠ中和抗体,且融合蛋白不具STⅠ毒性,证明融合蛋白具有良好的LTB、STⅠ免疫原性.实验结果表明,此融合蛋白具有开发成为口蹄疫及肠毒素腹泻联合疫苗的应用价值.     

猪水泡病病毒VP1基因的克隆和表达

以猪水泡病病毒RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了849bp的VP1基因,通过T-A克隆技术,将VP1基因片段克隆至pMD18-T克隆载体质粒中,构建SVDVVP1基因克隆重组质粒,进行核苷酸序列分析。然后亚克隆插入pBAD/ThioTOPO表达载体,经测序鉴定,筛选获得VP1基因正向插入、有正确读码框的阳性克隆,成功构建了猪水泡病病毒VP1基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP1蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002%的L-阿拉伯醛糖进行诱导,5h后表达可达到高峰,表达蛋白为融合蛋白,质量约47.13kDa,表达产量约占菌体总蛋白的16%。Westernblotting检测表明,诱导的蛋白能与猪水泡病阳性血清发生特异性反应。融合蛋白中含有猪水泡病病毒VP1蛋白抗原,为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础。     

猪水泡病病毒VP1基因的克隆和表达

以猪水泡病病毒RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了849bp的VP1基因,通过T-A克隆技术,将VP1基因片段克隆至pMD18-T克隆载体质粒中,构建SVDV VP1基因克隆重组质粒,进行核苷酸序列分析.然后亚克隆插入pBAD/Thio TOPO表达载体,经测序鉴定,筛选获得VP1基因正向插入、有正确读码框的阳性克隆,成功构建了猪水泡病病毒VP1基因重组表达载体.经L-Arabinose诱导表达,可稳定、高效地表达VP1蛋白抗原.SDS-PAGE结果表明,以终浓度为0.002%的L-阿拉伯醛糖进行诱导,5h后表达可达到高峰,表达蛋白为融合蛋白,质量约47.13kDa,表达产量约占菌体总蛋白的16%.Western blotting检测表明,诱导的蛋白能与猪水泡病阳性血清发生特异性反应.融合蛋白中含有猪水泡病病毒VP1蛋白抗原,为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础.     

O型口蹄疫病毒VP1 T细胞、B细胞表位双拷贝基因与大肠杆菌LTB、STI肠毒素基因融合表达产物的免疫应答

合成O型口蹄疫病毒VP1蛋白中与细胞免疫（21～40表位肽）及体液免疫（141～160表位肽）相关的基因序列2020VP1,运用基因工程技术构建了含有肠毒素大肠杆菌LTB、STI基因及双拷贝2020VP1的融合表达载体r2020-B-2020-STI,转化宿主菌BL21(DE3)RIL后的表达产物经SDS-PAGE分析,结果显示重组融合蛋白的分子量约为45kDa,表达量较高。ELISA实验结果显示,融合蛋白能与霍乱毒素（choleratoxin）CTB抗体特异结合。动物实验表明,融合蛋白能够诱发兔体产生较强的FMDV中和抗体,免疫豚鼠在低浓度FMDV刺激下能够产生特异性T淋巴细胞增殖反应,说明融合蛋白能诱导机体产生FMDV特异性细胞及体液免疫反应;同时,融合蛋白免疫雌鼠能够抵抗大肠杆菌强毒株攻击,免疫兔体能够产生STI中和抗体,且融合蛋白不具STI毒性,证明融合蛋白具有良好的LTB、STI免疫原性。实验结果表明,此融合蛋白具有开发成为口蹄疫及肠毒素腹泻联合疫苗的应用价值。     

猪水泡病病毒VP1基因抗原区的原核表达

利用RTPCR和nested PCR(nPCR)技术扩增出猪水泡病病毒VP1基因的抗原区，将其克隆到表达载体pProEXHTb中，获得重组质粒，经PCR、酶切和序列分析鉴定表明，目的基因插入的位置、大小和读码框均正确。将重组质粒导入BL21（DE3），经IPTG诱导表达后SDSPAGE检测表明，重组菌能表达猪水泡病病毒VP1抗原区蛋白；Western blot检测表明，诱导表达的抗原区蛋白能与猪水泡病阳性血清发生特异性反应。     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

大肠杆菌肠毒素ST1、LTB基因融合及其免疫原性研究

目的:构建大肠杆菌肠毒素ST1-LTB融合基因,并研究其表达产物的免疫原性。方法:采用PCR和基因突变技术,从大肠杆菌C83902质粒中扩增ST1突变基因和LTB基因,通过基因分离、纯化、内切酶酶切、连接和转化,构建含ST1-LTB融合基因表达载体的重组菌株,并用酶切和DNA序列分析鉴定重组质粒,同时用ST1-LTB融合蛋白粗提物免疫小鼠,观察免疫攻毒保护效果。结果:构建了含ST1-LTB融合基因表达载体的重组菌株BL21(DE3)(pXSL1),ST1-LTB融合基因的序列和阅读框架均正确,其表达的ST1-LTB融合蛋白能够被ST1单抗和LTB抗体识别,且该融合蛋白已丧失天然ST1肠毒素的活性。ST1-LTB融合蛋白能够诱发小鼠产生抗体,该抗体具有中和天然ST1肠毒素的毒性作用。结论:构建的重组菌株BL21(DE3)(pXSL1)可以高效表达ST1-LTB融合蛋白,其表达产物ST1-LTB融合蛋白具有良好的免疫原性,为更有效地预防仔猪黄痢提供了一种新型基因工程菌苗候选菌株。     

鸭坦布苏病毒E蛋白的原核表达及多克隆抗体的制备

目的用RT-PCR技术扩增鸭坦布苏病毒(DTMUV)AH-F10株E基因,并克隆至pET32a(+)载体,构建重组表达质粒pET32a-E,表达E蛋白。方法重组表达质粒转化感受态细胞BL21(DE3),经IPTG诱导后,获得含6个His标签的融合蛋白,大小约54kDa。表达的蛋白以包涵体形式存在。对目的蛋白进行纯化,用纯化的E蛋白免疫Balb/c小鼠,制备多克隆抗体血清。结果SDS-PAGE和Western blot试验结果表明E基因在大肠埃希菌中成功表达,并能与抗DTMUV多克隆抗体产生特异性反应,具有良好的反应原性。间接免疫荧光试验表明免疫小鼠后获得的多克隆抗体能与DTMUV反应。结论本研究为DTMUV新型疫苗和诊断试剂盒的进一步研究奠定了基础。     

转录后水平沉默与基因表达

基因沉默是1个非常复杂和普遍的现象。转录后水平的基因沉默是指转基因在细胞核里能稳定转录,细胞质里却无相应的稳定态mRNA存在的现象。它往往被称为共抑制、静息作用或RNA干预等。本文介绍了转录后水平的基因沉默现象的发现、分子机理和应用等方面的进展。提出了克服转录后水平基因沉默的一些对策。     

The genetics of gene expression and gene mapping

A recent publication has shown that a significant portion of gene expression levels are under genetic control in different organisms, that there are hotspot regions in the genome that control the expression of many other genes, and how gene expression data can be used to localize genes that affect clinical traits.     

Heterochromatin,gene position effect and gene silencing

Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.     

Multifunctional nanocomplexes for gene transfer and gene therapy

DNA formulated into aggregates with polycationic reagents are referred to by a variety of terms including non-viral vectors, synthetic vectors, lipoplexes, polyplexes and more recently nanoparticles. The capacity for delivery of multiple genes, genomic-sized constructs and siRNA delivery, with a diversity of possible formulations, as well as the possibilities of improved efficiency of in vivo gene deliveries, means that nanoparticles, or nanocomplexes to reflect self-assembling systems, will be investigated with increasing vigour in the coming years. This review briefly outlines the applications and challenges for nanoparticle technologies in the field of gene therapy then focuses on the development of a specific kind of formulation, receptor-targeted nanocomplex (RTN), that we have found to be particularly useful in our gene therapy research. An overriding guiding concept that has emerged in the development of synthetic nanodelivery systems is the idea to develop formulations and structures that mimic viruses, whilst retaining the safety elements of synthetic, non-viral systems. RTNs have been optimised and developed for airway epithelial transfection, leading towards gene therapy for cystic fibrosis and for vascular transfection in vein grafts used in bypass surgery. The modular design of the RTN platform further allows for the testing of specific hypotheses relating to the structure and functional role of components in the formation of stable particles and in the transfection pathway, leading to their ultimate disassembly in the nucleus.     

金黄色葡萄球菌耐消毒剂基因及抗生素耐药相关基因检测

目的 了解温州地区临床分离的金黄色葡萄球菌(SA)的耐药特点,探讨SA中耐β-内酰胺类、氨基糖苷类、四环素类药物耐药基因及耐消毒剂基因(qacA)的存在情况.方法 采用聚合酶链式反应(PCR)法对SA进行β-内酰胺酶基因、氨基糖苷类修饰酶基因、四环素类基因和耐消毒剂基因检测.结果 PCR结果显示94株SA中耐药相关基因检出率mecA 53.2％、aac(6’)/aph(2")68.1％、aph(3’)-Ⅲ 37.2％、tetM 53.2％和qacA 7.4％,其中59株MRSA的耐药相关基因检出率分别为mecA 83.1％、aac(6’)/aph(2")86.4％、aph(3 ′)-Ⅲ 42.4％、tetM 76.4％和qacA 8.5％.结论 多数SA菌株存在耐β-内酰胺类、氨基糖苷类、四环素类等多种抗生素耐药基因,具有多重耐药特征,但尚未出现明显耐消毒剂状况.     

Zinc finger gene clusters and tandem gene duplication.

Zinc finger genes in mammalian genomes are frequently found to occur in clusters with cluster members appearing in a tandem array on the chromosome. It has been suggested that in situ gene duplication events are primarily responsible for the evolution of such clusters. The problem of inferring the series of duplication events responsible for producing clustered families is different from the standard phylogeny problem. In this paper, we study this inference problem using a graph called duplication model that captures the series of duplication events while taking into account the observed order of the genes on the chromosome. We provide algorithms to reconstruct a duplication model for a given data set. We use our method to hypothesize the series of duplication events that may have produced the ZNF45 family that appears on human chromosome 19.