四个烙铁头蛇毒凝集素样蛋白基因的克隆与序列分析

从烙铁头蛇（Trimeresurus mucrosquamatus）的毒腺中提取mRNA，利用RT-PCR进行体外扩增，获得凝集素样蛋白基因，克隆至PMD18-T载体中，筛选出4种凝集素样蛋白基因（命名为TML-1、TML-2、TML-3和TML-4）。由基因序列推导出的氨基酸序列表明：TML-1，2，3，4序列中均有CRD结构。序列同源性比较和Cys位点分析推测：TML-1和TML-2可能分别是类似于flavocetin-A的蛇毒凝集素样蛋白的#alpha#亚基和#beta#亚基；TML-3可能类似于GPIb-bp的蛇毒凝集素样蛋白的#alpha#亚基，TML-4则可能是类似于IX/X-bp的蛇毒凝集素样蛋白的β亚基。

不同地区舟山种和孟加拉种眼镜蛇的神经毒素

先前的工作发现，眼镜蛇孟加拉种的神经毒分布存在明显的地理差异，但是导致这种现象的原因还不是很清楚。该工作首先采集夏季的眼镜蛇种，进行分类学的鉴定，以排除因为种属分类、季节和年龄而导致的不确定因素对蛇毒成分产生影响。再从鉴定好的成年浙江舟山种和云南孟加拉种眼镜蛇蛇毒中分离获得短链神经毒素;同时采用RT PCR方法得到相应 的基因。结果从浙江舟山种眼镜蛇蛇毒中分离到两个神经毒素;从云南孟加拉种蛇毒中得到三个神经毒素。这些神经毒素都是短链神经毒素。说明该两个蛇种与已经报道的台湾及浙江的舟山种眼镜蛇很相似，蛇毒中都以短链神经毒素为主。另外泰国孟加拉种眼镜蛇蛇毒中则以长链神经毒素为主。这种现象很难用种群发生的前后或者地理距离的长短来解释，而可能与地区间的气候、地理环境或者眼镜蛇的食物差别相关。实验统计表明在云南、浙江和台湾这些条件基本相似，而泰国就有很大的不同。

Temporal and Spatial Expression Patterns of Sox1 Gene in Xenopus laevis

克隆了非洲爪蟾的Sox1基因并研究了它在非洲爪蟾早期发育过程中的时空表达图式,比较了Sox1-3基因在发育的脑和眼中的表达图式.序列比对分析显示Sox1-3蛋白在其HMG框结构域具有高度的保守性.通过RT-PCR方法分析了Sox1基因在爪蟾早期不同发育时段的表达情况,结果显示Sox1基因从未受精卵到尾芽期均有表达,但表达强度有所差异.原位杂交结果显示,在早期卵裂阶段和囊胚期,Sox1基因主要在动物极表达;从神经板期开始,Sox1基因主要在中枢神经系统和眼原基中表达.在蝌蚪期,Sox1与Sox2、Sox3在脑部和眼睛的表达区域有所不同.对于爪蟾Sox1基因时空表达图式的研究将有助于阐明SoxB1基因家族在脊椎动物神经系统发生过程中的作用.

人、黑猩猩和叶猴FKN全基因的克隆及体外表达的比较研究

下载PDF阅读器目的:克隆人、黑猩猩和叶猴FKN全基因及体外表达,比较研究FKN在进化过程中基因组水平和蛋白表达水平的差异.方法:应用基因重叠延伸拼接PCR法(Gene splicing by overlapping extension PCR,SOE-PCR)将FKN的3个外显子编码序列依次进行前后拼接,然后插入pcDNA3.1/myc-His(-)A真核表达载体中,经酶切、测序鉴定后转染CHO细胞体外表达,RT-PCR、SDS-PAGE和Western blot检测其表达产物.结果:酶切、测序鉴定证实插入的基因片段为完整的FKN,RT-PCR可从转染的CHO细胞中扩增出一条与目的基因大小一致的DNA片段,其表达蛋白能分泌至胞外,SDS-PAGE显示其分子量约为95 000,抗c-myc抗体可与载体上的c-myc蛋白特异性结合.测序显示人、黑猩猩和叶猴相比,FKN基因除了有散在的点突变外,还发现有一明显的30 bp的缺失,但此缺失对FKN蛋白的表达并不影响.结论:成功克隆人、黑猩猩和叶猴FKN全基因,基因组水平和蛋白表达水平的比较研究为后续探讨FKN在高级灵长类物种进化过程中免疫学功能的演变奠定基础.

恒河猴CD1d基因的克隆及其组织表达差异性分析

目的:克隆非人灵长类疾病动物模型-恒河猴的CD1d基因并检测其在不同组织中的表达差异情况.方法:提取恒河猴血液及各组织的RNA, 以反转录得到的cDNA为模板, 用特异性引物扩增CD1d;以管家基因GAPDH为内参, 用半定量RT-PCR的方法对CD1d的组织表达差异性进行分析.结果:成功扩增了恒河猴CD1d基因的编码区序列;首次用半定量RT-PCR的方法检测了CD1d mRNA在恒河猴部分组织中的表达情况, 发现其表达水平存在明显的组织差异性, 其中, 在肝脏、脾脏和心脏中的表达水平较高, 在血液、小肠和肺中次之, 在脑中表达最少.结论:研究结果为今后表达恒河猴CD1d蛋白并制备其四聚体进而研究恒河猴NKT细胞在众多疾病中的作用奠定了基础;组织表达差异的研究结果提示其在相关疾病的发生发展过程中可能扮演着重要的角色, 具体作用还有待深入研究.

稳定表达HIV-1辅助蛋白Rev的THP-1细胞模型的建立和鉴定

Human immunodeficiency virus-1(HIV-1)辅助蛋白在其感染和艾滋病发病过程中起着非常重要的作用.Regulator of expression of virion proteins(Rev)作为HIV-1辅助蛋白之一,可以调节病毒结构蛋白mRNA出核转运和蛋白表达,对于病毒的复制至关重要.为研究Rev蛋白对靶细胞表犁和功能的影响,本实验采用电穿孔的方法,将HIV-1的rev基因导入THP-1细胞,通过流式分选结合G418筛选的方法建立稳定表达Rev蛋白的细胞模型;并通过RT-PCR、荧光观察及流式检测的方法,在mRNA和蛋白两个水平对所建立的细胞模犁进行鉴定.结果证实rev基凶成功导入了THP-1细胞并稳定表达,为后续rev基因产物与细胞相互作用的研究提供了平台.

人胎脑NT-4基因的体外构建

目的 体外构建人神经生长因子-4(neumtrophin-4,NT-4)与质粒载体pEGFP-N1(卡那霉素抗性)的重组体,为后续的转基因移植治疗脊髓损伤创造条件.方法 用Trizol法提取人胎脑组织总RNA,经RT-PCR扩增,EorR I和BamH I核酸内切酶酶切电泳纯化获得人神经营养因子-4(NT-4)DNA.用含质粒载体pEGFP-N1DNA的大肠杆菌接种经划板,挑选菌落、振荡培养,并用碱裂解法提取质粒载体pEGFP-N1DNA,经酶切、电泳纯化后,通过连接、转化感受态细胞,DNA小量提取法获得NT-4目的基因与质粒载体pEGFP-N1 DNA的重组体,经DNA测序证实.结果 用上述方法成功获得NT-4-pEGFP-N1重组体.结论 此方法是一种体外构建人胎脑NT-4基因的成熟、稳定而有效的方法,可为后续的神经干细胞转染NT-4基因移植治疗神经系统损伤创造条件.

恒河猴TRIM5α的组织分布及不同刺激促进PBMC中TRIM5α转录水平的上调

TRIM5α(tripartite motif protein 5-alpha)蛋白是恒河猴体内一种非常重要的限制因子,能抑制人免疫缺陷病毒(HIV-1,human immunodeficiency virus type 1)、马感染性贫血病毒(EIAV, equine infectious anemia virus)和猫免疫缺陷病毒(FIV, feline immunodeficiencyvirus)等逆转录病毒的复制.恒河猴TRIM5α的组织分布以及在受到外界刺激时TRIM5α mRNA表达量的变化研究还未见报道.本研究从中国恒河猴的各组织中提取总RNA,以β-actin基因作为内参照,通过半定量RT-PCR检测各组织中TRIMSα mRNA的表达.选择HIV-GFP-VSVG假病毒感染外周血单核细胞(peripheral blood mononuclear cell,PBMC),非特异性刺激剂--佛波脂(Phorbol myfismte acetate,PMA)+离子霉素(ionomycin,Ion)及CD28抗体+CD49d抗体分别共刺激恒河猴PBMC,研究不同刺激对恒河猴TRIM5α mRNA表达水平的影响.结果表明:TRIM5α mRNA表达于所研究的恒河猴21种组织中,免疫系统和泌尿生殖系统组织中表达量最高,而神经系统组织,如大脑、脊髓中表达较少,其他组织中未见明显的表达差异;HIV-GFP-VSVG感染和用PMA+Ion、CD28抗体+CD49d抗体分别共刺激PBMC能促进PBMC中TRIM5α mRNA的转录水平的上调.

槐花提取化合物K3体外抗HIV-1活性的研究

目的:研究槐花提取化合物K3的体外抗HIV-1活性,并对其抗HIV-1机制进行初步探讨.方法:采用MTT比色法检测化合物对各种细胞的毒性.用合胞体形成计数法,p24抗原捕获ELISA法及RT-PCR等多种方法研究化合物体外抗HIV-1活性.结论:槐花提取化合物K3体外有较好的抗HIV-1活性,能够抑制病毒实验株(HIV-1ⅢB,耐药株(HIV-1 74v)和临床分离株(HIV-1KM018)等多种病毒株的复制,且其作用机制是多靶点的,不仅可以抑制病毒的进入,还可以抑制HIV-1逆转录酶活性.

Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

HIV-1感染对T和B细胞核内UNG2 mRNA表达的影响

目的:探讨HIV-1感染是否影响细胞中UNG2的表达.方法:采用四步法SYBR green Ⅰ实时定量RT-PCR,对HIV-1感染者的T和B淋巴细胞,以及HIV-1感染的C8166细胞核内UNG2 mRNA的表达进行测定.结果:UNG2 mRNA的表达在HIV-1感染者的T细胞和HIV-1感染的C8166细胞中被明显上调,分别是对照的8.76倍和8.14倍,而在HIV-1感染者的B细胞中却没有被上调.结论:HIV-1感染导致的UNG2表达上调,可能通过减少TCR的多样性削弱Th的功能,另一方面可能有利于病毒对UNG2的包装.

树鼩CXCR4cDNA的克隆和序列分析

　目的　获得树　CXCR4 的cDNA 序列,探讨其是否可以支持HIV-1 病毒和细胞的结合。方法　设计 相应的引物, 用RT- PCR ,基因克隆,DNA 序列分析技术。结果　获得了全长为1059bp 树　CXCR4 (tsCXCR4) 基因 的cDNA。发现其核苷酸序列与人的CXCR4 (hCXCR4) 基因的cDNA 有9218 % 的相似性,由此推导出的氨基酸序列 有9619 % 相似性。与hCXCR4 功能相关的关键位点完全相同,tsCXCR4 的N 端第7 和12 位点为酪氨酸,第14、15 和 32 位点为谷氨酸,胞外环第183 ,188 为精氨酸, 第193、262 位点以及跨膜区97 位点为天冬氨酸。结论　树　的CX2 CR4 很可能会作为HIV-1 的辅助受体。　

树鼩细胞周期蛋白T1 cDNA的克隆和序列分析

树鼩细胞不能感染 HIV-1, 但支持 HIV-1进入靶细胞后的转录, 可能是因为树鼩细胞周期蛋白T1(tsCycT1)能结合HIV-1的 Tat 蛋白. 通过设计引物, 用 RT-PCR 技术, 获得全长为2175bpts CycT1 基因的cD NA. 其核苷酸序列与人的 CycT1(hCycT1) 基因的 cDNA 有92.6%的相似性; 由此推导出的氨基酸序列有94.1%相似性. 其中, hCycT1 和 tsCycT1 氨基端的1～272个氨基酸的相似性高达98.8%, 氨基酸第261位点为半胱氨酸. 这些结果提示, tsCycT1 会和 HIV-1 的 Tat 结合, 形成高亲和的、锌依赖的复合物, 支持 HIV-1转录。

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析

芳香化酶(P450arom)是雌激素合成过程中的关键酶,在性别分化中起重要作用。鱼类存在卵巢性和脑型两种芳香化酶,分别由Cyp19a和Cyp19b编码。稀有鮈鲫作为我国特有的实验动物,尚无其芳香化酶的有关资料,其性别分化机制亦不清楚。本研究采用RT-PCR的方法以简并引物扩增了稀有鮈鲫脑型芳香化酶基因Cyp19b的部分序列,其长度为1070bp,编码357个氨基酸残基,具有典型的芳香化酶结构域。RT-PCR分析发现该基因主要在稀有鮈鲫的脑中表达,在性腺、肠、肾中也有表达;其在雌、雄脑中的表达差异不显著。在