人肿瘤坏死因子可溶性受体ⅠcDNA的克隆有其在原核和真核细胞？…

以人的胎盘总ＲＮＡ为模板，用ＲＴ－ＰＣＲ的方法，获得人肿瘤坏死因子可溶性受体Ⅰ的ｃＤＮＡ，并对其进行全序列分析，在此基础上构建了原核及真核的表达载体，进行真核瞬间表达及活性测定和原核表达及产物初步纯化。     

抗人红细胞单链抗体基因的构建及表达

从分泌抗人红细胞单克隆抗体的杂交瘤细胞３Ｆ５中提取总ＲＮＡ,逆转录成ｃＤＮＡ,用合成的寡核苷酸引物通过ＰＣＲ扩增和克隆单抗的轻、重链可变区基因,用双脱氧核苷酸链终止法分析核苷酸序列,采用ＰＣＲ拼接法构建单链抗体基因,并插入原核表达载体ＰＥｔ－２８ａ,转化大砀杆菌ＢＬ２１（ＤＥ３）,经ＩＰＴＧ诱导后表达,序 分析表明所克隆的ＶＬ、ＶＨ分别属于鼠ＩｇＫａｐｐａ轻链ＩＶ亚组和Ｉｇ重链Ⅱ亚组,ＶＨ、ＶＬ基     

人胰岛素样生长因子—Ⅰ的基因克隆,融合表达及产物纯化

利用聚合酶链式反应（ＰＣＲ）方法,以ＩＧＦ－ＩｃＤＮＡ为模板,扩增ＩＧＦ－Ｉ的基因。在序列测定确证后,将其定向克隆到载体ｐＥｘＳｅｃＩ上,构建了ＩＧＦ－Ｉ融合表达载体ｐＥｘＩＧＦ,经ＩＰＴＧ诱导表达后,ＳＤＳ－ＰＡＧＥ分析表明：含有重组表达质粒的菌株可表达出约３２．５８％的２６ＫＤ融合蛋白,且主要以可溶性形式存在。经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ亲和层析一步纯化后,可获得纯度约９０％的融合蛋白,经Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析,免疫学活性呈阳性反应。     

人胰岛素样生长因子—I的基因克隆融合表达及产物纯化

利用聚合酶链式反应（ＰＣＲ）方法,以ＩＧＦ－ＩｃＤＮＡ为模板,扩增ＩＧＦ－Ｉ的基因,在序更测定确证后,将其定向克隆到载体ｐＥｘＳｅｃｌ上,构建了ＩＧＦ－Ｉ融合表达载体ｐＥｘＩＧＦ,经ＩＰＴＧ诱导表达后,ＳＤＳ－ＰＡＧＥ分析表明：含有重组表达质粒的菌株可表达出约３２．５８％的６ＫＤ融合蛋白,且主要以可溶性形式存在。经ＩｇＧ－Ｓｅｐｈａｒｏｓｅ亲和层析一步纯化后,可获得纯度的９０％的融合蛋白,经Ｗｅｓ     

牛泡沫病毒LTR的反式激活因子靶序列研究

牛泡沫病毒（ＢＦＶ）是反转录病毒科泡沫病毒属成员之一．其基因组除编码ｇａｇ,ｐｏｌ,ｅｎｖ三个结构基因外,在ｅｎｖ和３＇ＬＴＲ之间有２个ＯＲＦ（ＯＲＦ－１和ＯＲＦ－２）,编码自身的反式激活因子Ｔａｓ等调节蛋白．本研究利用我们实验室分离鉴定的ＢＦＶ３０２６中国毒株［１２］为材料,克隆Ｏｒｆ－１基因,构建ｐＢＦＶＯＲＦ－１表达质粒,通过带有ｌｕｃ基因的ＬＴＲ系列缺失质粒与ｐＢＦＶＯＲＦ－１共转染,瞬时表达分析结果将ＢＦＶＬＴＲ上Ｔａｓ应答元件（ＴＲＥ）定位于－９８３／－６６８（ＴＲＥＩ）,－４７０／－１４０（ＴＲＥＩ）和ＲＵ５区．其中ＴＲＥＩ、ＴＲＥＩＩ为正调控区域,ＲＵ５为负调控区域,并进一步证明ＲＵ５在异源启动子（ＢＩＶＬＴＲ）上具有抑制其下游基因表达的功能．这些结果表明ＢＦＶＴａｓ作用机理与慢病毒（Ｔａｔ）,致瘤病毒（Ｔａｘ）等均不相同     

STR对幼鲤生长和肝脏IGF—ImRNA表达的影响

通过以体质量６０￣７０ｇ的幼年鲤鱼,每ｋｇ体质量以２００ｍｇ的剂量体腔注射链脲佐菌素（ＳＴＲ）或载体溶液２００ｍｇ,观察鲤鱼生长、血糖、血清生长激素（ＧＨ）水平和肝组织胰岛素样生长因子－Ｉ（ＩＧＦ－Ｉ）ｍＲＮＡ水平的变化。注射ＳＴＲ的鱼生长速率比对照组显著减慢。在第１５天和第３０天实验组鲤鱼血糖水平以及血清ＧＨ水平皆无明显变化。在注射ＳＴＲ后的第１５天肝组织ＩＧＦ－ＩｍＲＮＡ的表达水平比对照组明显     

EIA RS-232-C/RS-485信号LonWorks节点

针对工业现场常用的ＥＩＡＲＳ－２３２－Ｃ／ＲＳ－４８５信号设计了一类基于Ｎｅｕｒｏｎ芯片的ＬｏｎＷｏｒｋｓ节点,该节点构成了ＥＩＡＲＳ－２３２－Ｃ／ＲＳ－４８５标准与ＬＯＮ的通讯协议之间的网关。     

重组人粒细胞集落刺激因子在Escherichia coli中的表达研究

根据人天然粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因序列设计出引物,通过ＲＴ－ＰＣＲ从人外周血单个核细胞ｍＲＮＡ获得Ｇ－ＣＳＦｃＤＮＡ片段,将该片段与原核表达载体ｐＴ７构建成重组体,导入大肠杆菌后发现天然Ｇ－ＣＳＦｃＤＮＡ表达量并不理想,这可能是由于天然ｈＧ－ＣＳＦ５’端Ｇ＋Ｃ的比例过高,使转录后的ｍＲＡ很容易形成二级结构而影响翻译起始,因此在大肠杆菌中很难获得高效表达,根据密码子简并性原则,在不改变编码氨基酸顺序的前提下,通过重新设计ＰＣＲ引物,将ｈＧ－ＣＳＦ的前３个氨基酸密码子中的几个ＧＣ碱基作了改动,获得了新的ｃＤＮＡ突变体,将其与ＰＴ７的重组导入大肠杆菌,获得了高效表达。     

胎肝及肝癌组织中IGF—IR的表达研究

应用ＲＴ－ＰＣＲ技术，从胎肝中克隆出ＩＧＦ－ＩＲβ严基的ｃＤＮＡ序列分析证明同已报道的人胎盘膜ＩＧＦ－ＩＲｃＤＮＡ顺序相同。提示ＩＧＦ－ＩＲ在胎肝的分化发育过程中具有生理意义。     

逆转录多聚合酶链反应检测排斥反应期移植肠基因的转录

排斥反应仍色是阻碍小肠移植成功的主要障碍,其机理、诊断及治疗均有待进一步研究。目前建立逆转录多聚合酶链反应方法,观察排斥反应期移植肠ＩＬ－２、ＩＦＮ－γ、穿孔素、粒细胞酶Ｂ的ｍＲＮＡ转录变化,取大鼠移植肠杆本提取总ＲＮＡ,并进行ＲＴ－ＰＣＲ检测ＩＬ－２、ＩＦＮ－γ穿孔素、粒细胞酶Ｂ的ｍＲＮＡ转录,ＰＣＲ产物以内参基因β－ａｃｔｉｎ为内对照,应用ＳＸ－１００生物影像分析系统进行半定量分析。结果显示Ｒ     

Expression of soluble human tumor necrosis factor receptor Ⅰ in Aspergillus niger

The cDNA of soluble human tumor necrosis factor receptorⅠ(sTNFRI) was inserted into fusion-protein expression plasmid pIGF of A. niger to construct fusion expression vector pHBC containing a KEX2 like protein processing site designed on the fusion position. Extracellular protease-deficient strain of A. niger 3.795-1-23 was transformed with pHBC. Positive clone was estimated by Southern hybridization. SDS-PAGE for protein produced by recombinant strain showed the distinctive expression band. Western blotting indicated that the secreted protein had immunoactivity of sTNFRI.     

Characterization of receptors for human tumour necrosis factor and their regulation by gamma-interferon

Tumour necrosis factors, TNF-alpha and TNF-beta (previously called lymphotoxin), are the products of activated monocytes and lymphocytes, respectively, and both have recently been purified, sequenced and cloned by recombinant DNA methods, revealing 35% identity and 50% homology in the amino-acid sequence. Both proteins have been found to be specifically toxic to many tumour cells. Furthermore, it has been reported that various interferons are synergistic with TNF for anti-tumour effects in vitro, while activities attributed to the two proteins have also been shown to necrotize various tumours in vivo. We have now prepared 125I-labelled highly purified recombinant human TNF-alpha to study in detail its binding to the human cervical carcinoma cell line ME-180. Our results indicate that there is a single class of specific high-affinity receptors for TNF on this cell line which has a Kd of about 0.2 nM and an average of 2,000 receptor sites per cell. The binding of labelled TNF-alpha to these cells can be inhibited by both TNF-alpha and TNF-beta but not by gamma-interferon (IFN-gamma). However, preincubation of cells with IFN-gamma increases the total number of TNF receptors two to threefold without any significant change in the affinity constant. This is the first report that TNF-alpha and -beta share a common receptor and that the receptors can be up-regulated by interferon. Our results may explain previous observations regarding similar biological activities observed for these two cytotoxic proteins and also their synergistic action with interferons.     

Biological function of H1V-1 transmembrane protein gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539–684) and gp41 immunosuppressive peptide (aa583–599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583–599 and 641–675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class I, II and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type I interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type I interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Cloning and expression in Escherichia coli of the gene for human tumour necrosis factor

Tumour necrosis factor (TNF) was found originally in mouse serum after intravenous injection of bacterial endotoxin into mice primed with viable Mycobacterium bovis, strain Bacillus Calmette-Guerin (BCG). TNF-containing serum from mice is cytotoxic or cytostatic to a number of mouse and human transformed cell lines, but less or not toxic to normal cells in vitro. It causes necrosis of transplantable tumours in mice. TNF also occurs in serum of rat, rabbit and guinea pig. Rabbit TNF has been purified recently to give a single band on SDS-polyacrylamide gel electrophoresis (PAGE). The purified TNF had a relative molecular mass (Mr) 40,000 +/- 5,000 measured by gel filtration, and 17,000 by SDS-PAGE. Its isoelectric point is 5.0 +/- 0.3. The necrotic activity in vivo and the cytotoxicity in vitro are produced by the same substance. The gene encoding TNF has been identified in a human genomic DNA library using as a probe a cloned cDNA encoding a portion of rabbit TNF. The regions of this gene encoding an amino-acid sequence corresponding to mature TNF have been expressed in Escherichia coli and the product of this expression isolated in pure form and shown to produce necrosis of murine tumours in vivo.     

人类扭转蛋白A的基因克隆、表达与蛋白质纯化(I)——DYT1基因克隆与原核表达

通过基因工程方法,用大肠杆菌原核表达系统表达人扭转蛋白A.从该蛋白编码序列中设计引物,以人肝cDNA文库作模板扩增到编码该蛋白的基因片段.将所得片段与pMD l8-T载体连接,转化到JM l09大肠杆菌中,从转化平板上挑出菌落,用碱裂解法提取质粒,通过PCR和酶切分析,筛选到阳性克隆,测序结果与文献报道结果一致.提取质粒,用BamHI和XhoI酶切,回收目的片段,分别克隆到原核表达载体pET28a( )和pGEX-6P-1中,转化JM l09受体菌,从JM l09受体菌中提出质粒,再转化到BL21(DE3)菌中,筛选出阳性克隆,构建了人扭转蛋白A原核表达载体.IPTG诱导该工程菌,培养并离心收集.SDS-PAGE结果表明该蛋白在两种载体中均得到了高效表达.     

人Delta-like4ext-26-217蛋白原核表达载体的构建及表达

构建人Delta-like4ext-26-217原核表达载体并表达。采用PCR方法扩增hDll4ext-26-217多核苷酸序列,克隆入pMD18T载体。测序正确后,将其亚克隆入pET32a( )原核表达载体,获得pET32a( )-hDll4ext-26-217载体。以该载体转化E.coli菌株BL21,IPTG诱导其表达。采用SDS-PAGE、Western Blot方法鉴定目的蛋白的表达。结果表明,采用SDS-PAGE,Western Blot等方法均可检测到目的蛋白TRX/hDll4ext -26-217,分子量约42.2 KD,主要以包涵体形式大量存在。以上结果为Detla-like4功能的进一步研究奠定了基础。     

水稻OsAAA1蛋白的原核表达载体构建及其可溶性表达研究

为使用外源表达载体表达并纯化有活性的水稻ATP酶蛋白Os AAA1,构建了p ET-32a-Os AAA1原核表达载体并进行体外IPTG诱导表达和Ni+柱亲和层析纯化.利用SDS-PAGE和Western Blot检测了目的蛋白.结果表明:将成功构建的p ET-32a-Os AAA1原核表达载体,转化到大肠杆菌Origami菌株后,在70~100 KD之间检测到可溶性目的蛋白带,并优化了诱导表达的较适温度、IPTG诱导浓度和诱导表达时间.故成功构建了p ET-32a-Os AAA1原核表达载体并获得了可溶性Os AAA1目的蛋白,为其后续研究奠定了基础.     

Biological function of HIV-1 transmembrane protein gp41──A study on a putative cellular receptor of gp41

Since 1992, the study of biological functions of HIV-1 gp41 has made great progress. Experimental evidence from several research groups demonstrated that gp41 has a putative cellular receptor. A recombinant soluble gp41 (aa539-684) and gp41 immunosuppressive peptide (aa583-599) could bind to human B lymphocytes and monocytes, but weakly bind to T lymphocytes. It was found that gp41 contains two cellular binding sites (aa583-599 and 641-675). GP41 could selectively inhibit cell proliferation of human T, B lymphocytes and monocytes, enhance human MHC class Ⅰ, Ⅱ and ICAM-1 molecule expression on cell surface. Gp41 binding proteins and a monoclonal antibody against the first binding site could inhibit this modulation effect. Amino acid sequence homology exists between gp41 and human type Ⅰ interferons, and the homologous region is located in the first binding site on gp41 and in the receptor binding site on type Ⅰ interferons. Studies in other groups indicate that both binding sites in gp41 may be associated with HIV infection of cells. Peptides containing two binding sites could respectively inhibit HIV infection of cells. A monoclonal antibody recognizing the second binding site could neutralize lab-strains and recently separated strains of HIV-1. Besides, antibodies against two regions (homologous with gp41 binding sites) of SIV transmembrane protein gp32 could protect macaques from SIV infection. These results suggest that the study of gp41 binding sites and cellular receptor could contribute to understanding the mechanism of HIV infection and to developing HIV vaccine and anti-HIV drugs.     

Cloning of murine BRI3 gene and study on its function for inducing cell death

To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.     

人RANKL胞外结构域原核表达载体的构建及表达条件优化

细胞核因子κB受体活化因子配体(Receptor activator of the NF-κB ligand,RANKL)是TNF超家族的重要成员之一,属于Ⅱ型跨膜蛋白,通过与其受体核因子κB受体活化因子(RANK)构建的信号通路参与乳腺癌的发生、肿瘤骨转移、骨质疏松、关节炎等病理过程。人RANKL胞外结构域基因片段与原核表达载体pET-21b融合并转化至表达菌Rosetta,并对其表达条件进行了优化。通过对诱导时机,诱导温度,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度,诱导时间及甘油浓度的条件优化表明,当IPTG的浓度为0.2 mmol/mL,20℃振荡诱导培养6 h时,甘油浓度为4%时,可在上清中获得高效表达的pET-21bRANKL融合蛋白,为该蛋白的进一步纯化及结构与功能研究打下了良好的基础。