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目的 克隆人细胞角蛋白CK9的cDNA,通过原核表达系统表达融合蛋白并进行纯化和鉴定。方法 从人角质形成细胞系（HaCaT）中提取RNA,以RT-PCR反转录cDNA,使用特异性引物PCR扩增出人CK9编码区全长并克隆到pET-28a载体上,再用IPTG诱导融合蛋白his-CK9的表达。表达的融合蛋白通过Ni 2＋亲和层析纯化后,进行SDS-PAGE和Western blot鉴定。结果 测序鉴定构建表达载体中CK9的cDNA序列正确;融合蛋白his-CK9可在大肠杆菌中诱导表达;纯化的融合蛋白his-CK9纯度较高;纯化的融合蛋白his-CK9可与商品化CK9抗体特异性结合。结论 成功构建原核表达载体pET-28a-CK9,并成功诱导及纯化融合蛋白his-CK9。

目的构建CHD4／Mi-2β基因的截短体原核表达重组质粒，在大肠杆菌中诱导表达并对GST融合蛋白进行纯化和初步鉴定。方法采用PCR方法扩增CHD4／Mi-2β的染色质调节区（CHD4-C）、解螺旋酶模序（CHD4-H）及DNA结合区（CHD4-D）基因片段，将目的基因片段插入原核表达载体pGEX-5T构建带GST标签的重组质粒，阳性克隆行DNA序列测定。IPTG诱导GST-CHD4-C、GST—CHD4D和GST—CHD4H原核表达，谷胱甘肽琼脂糖珠纯化融合蛋白，并对融合蛋白Westernblot鉴定。结果构建了3个CHD4／Mi-2β基因的截短体重组质粒；IPTG诱导显示，GST—CHD4-C、GST—CHD4-D和GST—CHD4-H融合蛋白主要以可溶性蛋白形式在细胞裂解液上清中表达，表达产物的相对分子质量分别为130、110、90ku。谷胱甘肽琼脂糖珠纯化融合蛋白，纯化产物的纯度最高可达94．5％。Westernblot证实各融合蛋白与抗GST单克隆抗体发生特异性结合反应，分子质量与估计值相符，提示为GST融合表达的CHD4／Mi-2β截短体蛋白。结论成功纯化了较高纯度的GST—CHD4-C、GST—CHD4-D和GST—CHD4H融合蛋白，为进一步研究CHD4蛋白在染色质重塑中的作用奠定了实验基础。

目的构建肾癌G250抗原肽与HBcAg重组基因的原核表达质粒pET28a（＋）／C—G250肽-C，并通过大肠杆菌进行原核表达并纯化，分析表达融合蛋白的免疫原性及抗原性。方法PCR扩增编码G250抗原中249～268氨基酸的基因片段，将G250肽插入去除主要免疫优势区的重组质粒pGEMEX／HBcAg中，获得重组质粒pGEMEX／C—G250肽-C，为更换酶切位点，以pGEMEX／C—G250肽-C为模板，PCR扩增C—G250肽一C基因片段，最终将目的基因亚克隆入原核表达质粒pET28a（＋）中，IPTG诱导融合蛋白表达，利用Ni2＋NTA亲和层析法纯化重组融合蛋白，SDSPAGE鉴定融合蛋白的表达，纯化后融合蛋白经腹腔注射免疫BALB／c小鼠，间接ELISA法检测小鼠血清中抗体的滴度。结果酶切及基因测序鉴定证实融合基因正确重组于表达质粒pET28a（＋）中，原核表达并纯化后，该分离纯化融合蛋白纯度达95％以上，其相对分子质量约为22．35。BALB／c小鼠经4次免疫后，所有小鼠血清中均可检测到抗体，免疫第6周其血清中抗体滴度可达到1：5．12×10。，抗G250肽抗体滴度达到1：6．4×10^4，抗HBcAg抗体滴度不超过1：4×10^3。结论成功构建肾癌G250抗原肽与HBcAg重组基因原核表达质粒pET28a（＋）／C—G250肽-c，在大肠杆菌中可高效表达，纯化后融合蛋白具有较强的免疫原性，能诱导小鼠产生高滴度和高特异性的抗体。

目的克隆Graves’病患者甲状腺组织T淋巴细胞的CTLA-4胞外段和TCRVβ8目的基因，重组为CTLA-4-TCRVβ8基因并表达出融合蛋白。方珐RT—PCR从Graves’病患者甲状腺组织中克隆T淋巴细胞的CTLA-4胞外段和TCRVβ8基因，依次与表达质粒进行连接，双酶切及测序鉴定获得正确的重组子；原核表达融合蛋白，SDS-PAGE及Western-Blotting检测蛋白表达情况。结果基因克隆扩增出CTLA-4和TCRVβ8目的基因片段，测序结果证实序列与已发表的一致。重组基因原核表达出与预期分子质量大小相符的目的蛋白。结论成功构建和表达了CT-LA-4-TCRVβ8基因，为探索Graves’病免疫耐受治疗提供了基因产品。

目的构建以增强型绿色荧光蛋白（EGFP）为报告基因的脑源性神经营养因子（BDNF）融合蛋白真核表达质粒，并对其表达和定位进行研究。方法以人血白细胞DNA为模板，利用一对删除终止密码子的引物进行PCR获得BDNF基因片段，并将其与pEGFP—N1质粒载体连接，构建BDNF-EGFP融合蛋白真核表达质粒。以脂质体Lipofectamine2000转染Hela细胞，提取总RNA，通过RT—PCR方法检测BDNF-EGFP在转录水平的表达，Western blot及荧光显微镜进一步观察融合蛋白的表达定位。结果酶切和PCR鉴定证实BDNF基因片段正确克隆入载体质粒中，转染Hela细胞后，RT—PCR证实BDNF-EGFP融合蛋白在转录水平有表达，荧光显微镜观察显示BDNF-EGFP融合蛋白主要分布于细胞质中，Western blot结果显示Hela细胞培养液中有BDNF-EGFP融合蛋白存在。结论成功构建了BDNF-EGFP融合蛋白真核细胞表达质粒，BDNF-EGFP融合蛋白能够在Hela细胞中表达，主要位于细胞质中并可以分泌到细胞外。

目的 获取人白介素24（IL-24）cDNA，构建原核表达载体以表达含有谷胱甘肽S转移酶（GST）的融合蛋白GST-IL-24。方法 以人外周血单核细胞（PBMC）总RNA为模板，RT-PCR扩增IL-24cDNA，克隆入原核表达载体pGEX-4T-2，测序结果显示在IL-24cDNA第223位G→A，370位T→C，从而导致其编码蛋白的氨基酸发生改变：A75T，H124Y，通过二次PCR将其纠正，重组载体pGEX-IL-24转化大肠杆菌BL21（DE3），异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，SDS-PAGE，Western blot检测显示有融合蛋白GST-IL-24表达。结果 通过RT-PCR及二次PCR得到人IL-24cDNA，构建其原核表达载体，经IPTG诱导在相对分子量约为50000处有融合蛋白表达。结论 IL-24的重组载体在大肠杆菌BL21（DE3）可以表达GST—IL-24融合蛋白。

目的 应用基因工程技术制备TGFβ1-(78-109)-NBc融合蛋白，作为TGFβ1疫苗，用于抗纤维化研究。方法合成TGFβ1-(78-109)编码区DP4A，连接于质粒载体，再在其下游连接NBc编码区，测序证实后，进行原核表达，表达产物以别色PJlC龙、比ISA等方法鉴定。趁果经DP4A序列分析证实融合基因序列完全正确，SDS-PAGE证实原核表达产物相对分子质量为25kD，ELISA检测证实TGFβ1-(78-109)和HBc的抗原表位均可正确暴露。结论 本研究成功构建了TGFβ1-(78-109)-NBc融合基因，进行了原核表达，并初步证实了表达产物的抗原性，为下一步进行TGFβ1疫苗抗纤维化的研究打下了基础。

Mitochondria form networks. Formation of mitochondrial networks is important for maintaining mitochondrial DNA integrity and interchanging mitochondrial material, whereas disruption of the mitochondrial network affects mitochondrial functions. According to the current view, mitochondrial networks are formed by fusion of individual mitochondria. Here, we report a new mechanism for formation of mitochondrial networks through KIF5B-mediated dynamic tubulation of mitochondria. We found that KIF5B pulls thin, highly dynamic tubules out of mitochondria. Fusion of these dynamic tubules, which is mediated by mitofusins, gives rise to the mitochondrial network. We fur- ther demonstrated that dynamic tubulation and fusion is sufficient for mitochondrial network formation, by recon- stituting mitochondrial networks in vitro using purified fusion-competent mitochondria, recombinant KIF5B, and polymerized microtubules. Interestingly, KIF5B only controls network formation in the peripheral zone of the cell, indicating that the mitochondrial network is divided into subzones, which may be constructed by different mecha- nisms. Our data not only uncover an essential mechanism for mitochondrial network formation, but also reveal that different oarts of the mitochondrial network are formed bv different mechanisms.

The AAA （ATPase-associated with various cellular activities） ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation （ERAD）. In membrane fusion, p97 is thought to function in analogy to the related ATPase NSF （N-ethylmaleimide- sensitive fusion protein）, which promotes membrane fusion by disassembling a SNARE complex. In ERAD, p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome. Here, we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 （EEA1） as a new p97 substrate. We demonstrate that a fraction of p97 is localized to the early endosome membrane, where it binds EEA1 via the N-terminal C2H2 zinc finger domain. Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes, which is associated with an altered trafficking pattern for an endocytic cargo. Mechanistically, we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane. We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.

G protein-coupled receptors （GPCRs） are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic tar- gets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression char- acteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between dif- ferent fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.