G-蛋白 Rab3a 对神经生长抑制因子 (GIF) 抑制神经元细胞生长作用的影响

为了深入研究 Rab3a 和神经生长抑制因子 (GIF) 的相互作用,在大鼠海马神经元细胞原代培养体系中,用 MTT 还原法定量研究了 Rab3a 对 GIF 神经生长活性的影响,发现 Rab3a 可以替代脑提取物而使 GIF 发挥神经生长抑制活性 . 接着又利用多克隆抗体方法研究了 GIF 与 Rab3a 的相互作用,结果说明 Rab3a 是 GIF 发挥神经生长抑制活性所必需的蛋白质,并且它们的相互作用受空间位置的因素影响较大 . 在随后对它们相互作用的机理和可能的生物学意义进行了讨论 .     

生长抑制因子(GIF)与G蛋白Rab3a直接相互作用

生长抑制因子(growth inhibitory factor, GIF), 又称金属硫蛋白-3, 为68个氨基酸组成的脑特异性金属硫蛋白, 具有广泛的生理功能; GIF可能与阿尔茨海默氏症(Alzheimer's)病理相关, 在Alzheimer's脑提取物存在下, 还对神经细胞具有特异的生长抑制活性.然而, 对其发挥生长抑制作用的分子机制并不清楚.运用酵母双杂交系统从人脑cDNA文库中筛选与GIF相互作用因子,从4.1×10⁶个人脑cDNA文库转化子中,首次筛选到Ras家族G蛋白Rab3a C端,包含87个氨基酸的片段能与GIF相互作用;用PCR自人胎盘总cDNA中获得包含完整Rab3a编码序列的cDNA;通过酵母双杂交实验表明,全长Rab3a蛋白亦能与GIF相互作用.免疫共沉淀和蛋白质印迹实验进一步验证了GIF与Rab3a在哺乳动物细胞中可以相互作用; 而且, Rab3a是以GTP结合形式(GTP-Rab3a)与GIF发生相互作用.     

神经生长抑制因子的功能结构域双体

神经生长抑制因子 (neuronalgrowthinhibitoryfactor,GIF)又名金属硫蛋白 III (metallothionein III,MT III) ,是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白质 ,β结构域为其功能结构域。为深入系统地研究GIF及其结构域的结构与功能 ,构建了神经生长抑制因子功能结构域双体 (GIFβ β)。PCR扩增得到N端 β结构域和C端 β结构域的cDNA ,酶切后克隆入原核表达载体pGEX 4T 1,经发酵、诱导表达、亲和层析、凝血酶切和进一步纯化 ,每升菌液约可得重组GIFβ β蛋白 6 0mg。测其电泳行为、氨基酸组成、质谱、金属巯基含量等 ,证明得到了目的蛋白质。圆二色性图谱显示 ,GIFβ β拥有金属硫蛋白家族成员的特征———金属巯基簇结构域。MTT还原法测定神经元抑制活性大小为 :GIF >GIFβ β>GIFβ结构域     

神经生长抑制因子研究进展

神经生长抑制因子(neuronal growth inhibitory factor, GIF) 又名金属硫蛋白-Ⅲ (metallothionein-Ⅲ,MT-Ⅲ),特异分布于中枢神经系统(CNS),是神经系统中第一个被鉴定的具有神经元生长抑制功能的蛋白. GIF一级序列、高级结构、金属结合特性类似于其他MTs,基因结构也与其他MTs高度同源,但表达调控途径相异. GIF可能以其β结构域的CPCP区,与脑组织提取物中的相关因子结合,进而表现其生物学功能. 有研究认为GIF与阿尔茨海默等脑相关疾病均有密切关系.     

重组泛素连接酶SH2-U—box／RING的构建与表达

目的构建重组泛素连接酶SH2-U—box、SH2-RING,并克隆进入pFlag—CMV4真核表达载体,为研究靶向降解慢性粒细胞白血病（chronic myelocytic leukemia,CML）患者瘤细胞中过度活化的BCR／ABL,抑制肿瘤细胞的生长提供基础。方法设计引物,扩增接头分子Grb2的SH2结构域以及E3泛素连接酶CHIP的U—box、Cb1的RING结构域,通过重组PCR,将SH2分别与U—box、RING进行融合,融合片段双酶切之后插入真核表达载体pFlag—CMV4,经过酶切鉴定及测序后,转染HEK293T细胞,Western印迹验证重组质粒的表达。结果PCR结果提示SH2-U—box条带大小888bp,SH2一RING大小为633bp,重组质粒酶切鉴定和测序结果均正确,转染后可见融合蛋白的表达。结论成功构建真核重组表达载体pFlag—CMV4-SH2-U—box和pFlag—CMV4-SH2-RING,转染HEK293T细胞后能够正确表达,为后续研究奠定了基础。     

神经生长抑制因子相互作用蛋白的分子克隆、鉴定及其生物学特性

为了探讨神经生长抑制因子(Neuronal growth inhibitory factor,GIF)与Alzheimer’s病(Alzheimer’s disease,AD)的关系,将GIF的cDNA全基因克隆到载体pHyblex中,运用酵母双杂交系统从Alzheimer’s病人脑cDNA文库中筛选出与GIF相互作用蛋白的cDNA克隆。免疫共沉淀和蛋白质印迹实验进一步验证了该蛋白在体内与GIF相互作用的特异性。克隆并鉴定了其中1个与GIF特异性结合的蛋白,与人细胞核dUTP焦磷酸酶(DUT)同源。进一步构建了重组表达质粒pGEX-4T-1／DUT,转化大肠杆菌BL21,经谷胱甘肽-Sepharose 4B亲和层析、凝血酶酶切和Sephacryl S100纯化,得到纯度95％以上的dUTPase蛋白。体外生物学活性检测表明,表达的dUTPase蛋白可以与GIF共同作用嗜铬细胞瘤株(pheochromocytoma)PC12,对细胞的生长产生抑制作用。     

丙型肝炎病毒NS2蛋白在Huh-7细胞中抑制NF-κB活性

将丙型肝炎病毒(HCV)非结构蛋白NS2基因的全长序列插入到真核表达载体pCDNA3.1(-)CMV启动子下游,构建成重组质粒pCNS2.用脂质体LipoVecTM转染Huh-7细胞,转染细胞内可检出NS2的mRNA和蛋白质,表明构建的pCNS2可在Huh-7细胞内成功表达;把不同剂量的pCNS2质粒DNA与报告质粒pNF-κ B-Luc共转染Huh-7细胞,48h后检测荧光素酶活性,结果显示与pCNS2共转染的细胞中pNF-κ B-Luc表达出的荧光素酶的活性比对照细胞降低了约2～4倍,并呈明显的剂量相关性.表明HCV NS2对NF-κ B激活转录活性有明显的抑制作用.这可能与HCV慢性持续性感染的致病性有一定的相关性.     

乙型肝炎病毒HBx蛋白抑制TTRAP启动子活性的体外研究

旨在探讨体外培养条件下HBx蛋白对TTRAP(TRAF and TNF receptor-associated protein)基因转录水平表达的影响。用RT-PCR及Real-time PCR检测TTRAP在HepG2细胞和HepG2.2.15细胞中的表达;构建TTRAP启动子虫荧光素酶报告质粒;分别与HBV、HBs、HBp、HBc、HBx表达质粒共转染HepG2细胞,比较虫荧光素酶活性。RT-PCR和Real-time PCR结果显示,TTRAP在HepG2.2.15细胞中的表达量分别是其在HepG2细胞中表达量的44.9%和27.8%(P0.05)。TTRAP启动子虫荧光素酶报告质粒与HBV表达质粒共转染组的相对荧光素酶活性,与对照组相比下降了43.8%。转染HBx表达质粒组的相对荧光素酶活性与其对照组相比下降了35%,而转染HBc、HBs及HBp表达质粒组对相对荧光素酶活性没有影响。因此证实HBx蛋白能抑制TTRAP启动子活性。     

抑癌基因p16对人肝癌细胞生长抑制的机制研究

目的：探讨抑癌基因p16对肝癌细胞生长的抑制作用及其机制。方法：将p16cDNA亚克隆至pcDNA3．1真核表达载体上,并经脂质体介导转染至人肝癌细胞株SMMC-7721：用MTT法和Western blot分析转染细胞的生长情况。结果：成功构建重组表达质粒pcDNA3．1-p16,转染pcDNA3．1-p16的SMMC-7721细胞生长速度受到明显抑制;转染后有外源p16蛋白的表达,且伴随Bax上调,Bcl-2和cIAP2的下调。结论：重组pcDNA3．1-p16质粒能在人肝癌细胞SMMC-7721内表达,且能抑制SMMC-7721的生长,其机理与诱导肿瘤细胞凋亡相关。     

禽流感病毒HA基因真核表达质粒的构建与表达

血凝素蛋白（HA）基因是禽流感病毒（AIV）重要的保护性抗原基因.为了研究 HA基因疫苗,用PCR扩增H5亚型AIV HA基因,将其克隆到质粒pcDNA4/HisMax和pRc/CMV上得到真核表达质粒pC4H5和pCMVH5.采用Tfx^TM-20、Superfect转染试剂和电转染法转染HeLa细胞,转染后的HeLa细胞经蛋白质印迹和血凝试验检测HA蛋白及其活性.结果表明,Superfect转染和电转染均能正确表达HA蛋白并具有生物学活性,蛋白质印迹检测到HA和HA裂解的HA1和HA2,与AIV 的HA、HA1、HA2蛋白的分子质量一致.从血凝试验结果看,Superfect和电转染表达的HA均具有血凝活性,而经Superfect转染的pC4H5的表达量是pCMVH5的8倍,表明pC4H5是一高效的真核表达质粒.     

The role of Thr5 in human neuron growth inhibitory factor

GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer’s disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.Bin Cai and Qi Zheng contributed equally to this work.     

金属硫蛋白-3对dUTPase调节dUTP细胞毒性作用的影响

金属硫蛋白-3（MT-3）,又称神经生长抑制因子,是一种脑特异性金属硫蛋白。人细胞核dUTP焦磷酸酶（dUTP pyrophosphatase, dUTPase）是最近在脑中研究发现的能与人金属硫蛋白-3（human metallothionein-3,hMT-3）相互作用的一个蛋白,两者共同作用具有神经元生长抑制活性。为了探讨hMT-3对dUTPase调节dUTP引起的细胞毒性作用的影响,通过基因转染HEK293细胞观察细胞在dUTP中的生长情况,发现共转染hMT-3和dUTPase基因的细胞比单转染dUTPase或hMT-3基因的细胞具有更强的对dUTP细胞毒性的耐受能力;同时在大肠杆菌BL21中表达重组蛋白,测定hMT-3在dUTPase水解dUTP中的作用,结果显示重组蛋白hMT-3可以促进dUTPase对dUTP的水解。结果均初步证实了hMT-3对dUTPase抵抗dUTP引起的正常细胞死亡有一定的协同作用,为进一步研究dUTPase及其相互作用蛋白hMT-3在化疗中的应用提供理论依据。     

Effect of α-domain substitution on the structure, property and function of human neuronal growth inhibitory factor

Human metallothionein-3 (hMT3), also named human neuronal growth inhibitory factor (hGIF), is attractive due to its distinct neuronal growth inhibitory activity, which is not shown by other human MT isoforms. It has been reported that the neuronal growth inhibitory activity arises from the N-terminal β-domain rather than its C-terminal α-domain. However, previous bioassay results have shown that the single β-domain is less effective at inhibiting the neuron growth than that in intact hMT3 on a molar basis, which suggests that the α-domain is indispensable to the neuronal growth inhibitory activity of hMT3. In order to confirm this assumption, we constructed two domain-hybrid mutants, the β(MT3)–β(MT3) mutant and the β(MT3)–α(MT1) mutant, and investigated their structural and metal binding properties by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, etc. The results showed that stability of the Cd₃S₉ cluster of the β(MT3)–β(MT3) mutant decreased significantly while the Cd₃S₉ cluster of the β(MT3)–α(MT1) mutant had a similar stability and solvent accessibility to that of hMT3. Interestingly, the bioassay results showed that the neuronal growth inhibitory activity of the β(MT3)–β(MT3) mutant decreased significantly, while the β(MT3)–α(MT1) mutant showed similar inhibitory activity to hMT3. Based on these results, we conclude that the α-domain is indispensable and plays an important role in modulating the stability of the metal cluster in the β-domain by domain–domain interactions, thus influencing the bioactivity of hMT3. Z.-C. Ding and Q. Zheng contributed equally to this work.     

小鼠金属硫蛋白 Ⅰ cDNA编码序列的克隆及其在昆虫细胞中的表达

将质粒ｐＢＸ－ＭＴ上的小鼠ＭＴ－ⅠｃＤＮＡ片段切下作为模板,通过ＰＣＲ方法删除该片段的非编码序列,将编码序列克隆到质粒ｐＢＳ－ＳＫ中,经ＤＮＡ序列测定后证明其克隆序列正确．再将ＭＴ－ⅠｃＤＮＡ编码序列插入到转移载体ｐＢａｃＰＡＫ８的ＢａｍＨⅠ和ＥｃｏＲⅠ位点之间,通过磷酸钙／ＤＮＡ共转染方法将其导入昆虫细胞Ｓｆ９中,以Ｗｅｓｔｅｒｎｂｌｏｔ和ＤｏｔＥＩＡ方法对表达产物进行了检测,表达量为１ｍｇ／Ｌ     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.     

牛疱疹病毒Ⅰ型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活作用

由含有ＢＨＢＶ－１（ＢｏｖｉｎｅＨｅｒｐｅｓＶｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＢＩＣＰＯ（ＢＨＶ－１ＩｎｆｅｃｔｅｄＣｅｌｌＰｒｏｔｅｉｎＯ）的ＤＮＡ序列至表达载体ｐＳＶＫ３,构建质粒ｐＳＶ２．９。将该质粒与ｐＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶＬＴＲ启动子控制下的荧光素酶基因的表达。根据ｐＳＶ２．９与含有ＢＩＶＬＴＲ不同区段缺失的质粒ｐＤ－３１９－Ｌｕｃ、ｐＤ－１１５－Ｌｕｃ、ｐＤ－５２－Ｌｕｃ共转染小牛肺细胞的实验结果,推测ＢＩＶＬＴＲ－３１９位上游区的ＤＮＡ序列影响ＢＩＣＰＯ基因产物对ＢＩＶＬＴＲ表达的激活作用。     

Activated v-myc and v-ras oncogenes do not transform normal human lymphocytes.

Activated v-myc (pSV v-myc) and v-Ha-ras (GT10) oncogenes were introduced into normal human lymphocytes, NIH 3T3 fibroblasts, B-lymphoblastoid cells, and human epithelial cells, using a reconstituted Sendai virus envelope-mediated gene transfer technique. Efficient transfer of the plasmid in each cell type was demonstrable within 1.5 h of transfection by Southern blotting of extrachromosomal DNA extracts, which unexpectedly revealed that v-myc plasmid DNA was unstable in normal lymphocytes but not in the other cell types. The v-myc plasmid was stabilized when cotransfected into lymphocytes together with v-Ha-ras. The transfected v-Ha-ras plasmid was stable in all the cell types tested. v-myc plasmid expression was clearly detectable by 5 h in all cell types except human lymphocytes. Lymphocytes expressed v-myc when transfected together with v-Ha-ras. Transfected ras oncogene was efficiently expressed in all the cell types tested. Expression of the transfected genes increased at 24 and 48 h after transfection. Even though plasmid stability and expression were achieved in myc-ras-cotransfected lymphocytes, no effects on cellular DNA synthesis or immortalization were observed, in contrast to efficient transformation of NIH 3T3 fibroblasts by the same procedure. Our data suggest that efficient expression of transfected myc and ras oncogenes in normal quiescent human lymphocytes is not sufficient for the induction of cell growth and immortalization.