蛋白激酶PKR的克隆表达及与其相互作用的蛋白质的亲和纯化

目的:克隆并表达双链RNA调节的蛋白激酶(PKR)基因,以纯化分离与PKR相互作用的蛋白质。方法:设计特异性引物,PCR扩增分别得到FLAG-PKR-HA和HA-PKR-FLAG基因片段,克隆至表达载体pSG5中。将重组质粒通过脂质体法转染PKR稳定沉默(PKRkd)的HeLa细胞,Western blot检测PKR及标签表达情况;最后,利用HA、FLAG串联亲和纯化系统(TAP)纯化分离与PKR相互作用的蛋白,Western blot技术及银染SDS-PAGE验证PKR蛋白复合物的纯化和分离情况。结果:成功构建了PKR融合蛋白表达载体及FLAG、HA亲和纯化系统,SDS-PAGE和银染结果显示TAP系统分离得到了PKR蛋白带和两条可能与PKR相互作用的蛋白质片段。结论:成功纯化和分离了与PKR相互作用的蛋白质,为进一步研究奠定了基础。     

一种新的研究蛋白相互作用的技术——串联亲和纯化

蛋白质组学的发展及分析技术的进步，客观上要求建立新的生物纯化策略。串联亲和纯化(tandem affinity purification,TAP)是一项新的纯化蛋白复合物的技术，它与质谱技术结合使用，已成为当前蛋白质组学研究的重要工具。本文从该技术的每一层面人手，对此方法的研究进展进行概述。     

串联亲和纯化技术:一种极具潜力的分离、鉴定蛋白复合体的工具

大多数细胞的生物学功能都是由蛋白复合体而非单一蛋白来完成的,所以识别和分析蛋白复合体的组分是研究蛋白功能所必需的。串联亲和纯化技术(TAP)能在生理条件下有效地分离、鉴定蛋白复合体,而无需知道复合体的组成或功能,目前已成功地应用于酵母及其他生物体中的蛋白复合体的成分分析。与质谱技术联合应用可以识别与目的蛋白相互作用的蛋白,进而揭示蛋白质相互作用网络及其功能。     

病毒对宿主PKR信号转导途径抑制作用的研究进展

干扰素(interferon,IFN)诱导的双链RNA激活的蛋白激酶(double-stranded RNA-activated protein kinase,PKR)的激活是细胞抵御病毒感染的重要机制之一,活化的PKR可磷酸化真核起始因子2(eukaryotic initiation factor2,elF2)的α亚基,导致病毒蛋白合成停止,因此,PKR信号转导途径异常与病毒持续感染密切相关.@@1 PKR的结构与功能@@人类PKR基因位于染色体2p21-22,属于丝/苏氨酸蛋白激酶亚家族的成员,为一种双链RNA (double-stranded RNA,dsRNA)结合蛋白,相对分子质量为68kDa,含551个氨基酸.PKR由氨基末端的调节结构域和羧基末端的催化结构域构成,后者含有11个保守的激酶亚结构域,其中位于296位的赖氨酸在磷酸化反应时结合ATP.     

应用Profinity eXact标签在真核表达系统中纯化EGFP蛋白

目的研究Profinity eXact标签在真核蛋白表达系统中的纯化效果。方法通过PCR扩增Profinity eXact标签,构建中间载体pUC18ETagEGFP,构建Profinity eXact标签绿色荧光蛋白真核表达载体FUETagEGFP,采用DNA-磷酸钙方法转染293FT细胞,收集过量表达Profinity eXact标签绿色荧光蛋白的细胞,随后提取蛋白进行纯化,采用荧光检测EGFP蛋白结合、纯化效率,最后利用SDS-PAGE检测纯化蛋白的纯度。结果经PCR扩增、酶切和测序,Profinity eXact标签、中间载体pUC18ETagEGFP、Profinity eXact标签真核表达载体构建正确,经荧光显微镜镜下观察Profinity eXact标签真核表达载体转染293FT细胞效果良好,经荧光检测确定EGFP蛋白结合、纯化效率,经SDS-PAGE发现Profinity eXact标签在真核蛋白系统中纯化效果良好。结论 Profinity eXact标签在真核蛋白表达系统中能正常工作,并有较好的纯化效果。     

抗天花粉蛋白单抗用于天花粉蛋白免疫毒素的亲和纯化

天花粉蛋白（ＴＣＳ）是一种单链致核糖体失活的蛋白，用细胞融合技术筛选到一株分泌抗该蛋白单抗的杂交瘤细胞株（Ｔ１Ａ９），并将单抗Ｔ１Ａ９制成免疫亲和凝胶，用此亲和凝胶纯化以ＴＣＳ和抗人黑色素瘤单抗Ｎｇ７６构建的免疫毒素，能有效地分离掉样品中的游离抗体，提高免疫毒素的毒效。体外细胞毒性实验表明，亲和纯化的免疫毒素对黑色素瘤细胞（Ｍ２１）的杀伤作用比分子筛（ＳｅｐｈａｃｒｙｌＳ－２００）层析后的样品提高了６．３倍。它对Ｍ２１细胞的半致死浓度（ＩＣ＿（５０））为１．０×１０￣（－１０）ｍｏｌ／Ｌ。比ＴＣＳ和Ｎｇ７６的混合物的毒性（ＩＣ＿（５０）＝２．５×１０￣（－７）ｍｏｌ／Ｌ）提高了２５００倍。ＴＣＳ是制备免疫毒素的一种有效和常用的“弹头”，用此法纯化这类免疫毒素将会有一定的普遍性。     

亲和标签在重组蛋白纯化领域的研究进展

亲和纯化标签在重组蛋白纯化领域中发挥着重要作用,经过标准化流程即可得到高产量高纯度的靶蛋白。近年来,许多不同种类的纯化标签陆续被设计出来,其中包括短肽标签、表位标签、折叠蛋白结构域标签、非层析法标签,以及最近研究较多的复合标签。除了通过经典的蛋白酶去除靶蛋白所携带标签的方法之外,自我裂解去除标签的方法因其简便廉价等优点近年来逐渐受到重视。本文主要对重组蛋白纯化标签的研究进展进行综述。     

人CaMKKβ蛋白的原核表达、纯化及活性测定

目的对原核体系表达的CaMKKβ蛋白体外活性进行探索,为针对CaMKKβ、AMPK药物研发提供科研基础。方法克隆人CaMKKβ基因,建立原核表达载体pET28a-CaMKKβ,将其转化入E.coli Rosetta(DE3)感受态细胞内,在16℃、0.02 mmol/L IPTG条件下诱导重组表达CaMKKβ蛋白,Ni-NTA纯化6His-CaMKKβ蛋白,并利用Glo?Max Assay方法检测其活性。结果通过基因测序表明pET28a-CaMKKβ质粒构建成功;重组人CaMKKβ蛋白可溶性表达量较高,Ni-NTA纯化6His-CaMKKβ蛋白纯度可达80%以上,活性检测结果表明,大肠杆菌体系内表达的人CaMKKβ蛋白在CaM/Ca2+存在与否情况下,酶活基本一致。结论成功构建原核表达载体pET28a-CaMKKβ,实现重组人CaMKKβ在原核体系内可溶性表达,活性测定表明原核体系内表达的CaMKKβ自主酶活性较强,受CaM/Ca2+影响较小。     

miR-92a对心肌缺血再灌注小鼠损伤PKR/NLRP3的影响

目的 研究miR-92a对心肌缺血再灌注小鼠损伤双链RNA依赖性蛋白激酶/炎性小体(PKR/NLRP3)的影响.方法 60只C57BU6小鼠随机分为对照组、模型组、阴性模型组及inhibitor模型组.对照组行假手术处理,模型组为结扎冠脉降支的IR模型小鼠,阴性模型组及inhibitor模型组小鼠分别通过静脉注射空载质...     

自身抗原Ro60kD重组融合蛋白的纯化和鉴定

将表达Ro60kD GST融合蛋白的重组体导入大肠杆菌JM109 中表达重组抗原, 经GST亲和层析柱纯化后用标准抗Ro血清鉴定, 经免疫印迹法证实, 纯化重组蛋白具有Ro 抗原性, 该抗原可用于临床检测及研究抗原表位与疾病的相关性     

The carboxy-terminal, M3 motifs of PACT and TRBP have opposite effects on PKR activity

PKR is an interferon(IFN)-induced, serine-threonine protein kinase, which plays a crucial role in IFN's antiviral and antiproliferative actions. The three known activators of PKR are dsRNA, heparin, and PACT. PACT activates PKR by direct protein-protein interaction in response to cellular stress. The human TAR (trans-activating region)-binding protein (TRBP), which is very homologous to PACT, also interacts with PKR, leading to an inhibition of PKR activity. Since these two highly homologous proteins have opposite effects on PKR, we examined if they interact with PKR differently by assaying their interaction with various point mutants of PKR. Our results indicate that TRBP and PACT interact with PKR through the same residues, and no differences were identified in these two interactions. Domain swap experiments between PACT and TRBP indicated that the inhibitory effects of TRBP on PKR activity are mediated through its carboxy-terminal residues, which contain TRBP's third dsRNA-binding motif.     

Protein kinase PKR catalytic activity is required for the PKR-dependent activation of mitogen-activated protein kinases and amplification of interferon beta induction following virus infection

The protein kinase regulated by RNA (PKR) enhances both activation of mitogen-activated protein kinases and the induction of interferon beta (IFN-β) by measles virus defective in C-protein expression (C^ko). Here we used complementation of human cell lines stably deficient in PKR (PKR^kd) to probe the basis of these PKR-mediated responses. We found that PKR^kd HeLa and amnion U cell lines were defective for virus-mediated activation of IFN induction signaling components compared to PKR-sufficient control cells. Complementation of PKR^kd cells with wildtype PKR, but not with PKR mutants defective in either catalytic activity or dsRNA-binding activity, restored JNK, p38 and ATF-2 phosphorylation and enhanced IFN-β induction following infection. By contrast to mammalian PKR, the Z-DNA binding domain-containing fish homologue of PKR, PKZ, lacked the capacity to enhance C^ko virus-mediated IFN-β induction. Furthermore, inhibition of virus growth was observed with C^ko-infected PKR^kd cells complemented with PKR but not with PKZ.     

人酪氨酸激酶6原核表达载体的构建、表达、纯化与鉴定

目的构建人络氨酸激酶6(PTK6)与His融合蛋白重组表达载体,在大肠杆菌中诱导表达,并对重组蛋白进行纯化。方法通过PCR在编码PTK6的cDNA序列两端添加EcoR I和BamH I酶切位点,双酶切后将编码PTK6的cDNA序列亚克隆至原核表达载体PHUE构建重组蛋白表达载体PHUE/PTK。重组载体经鉴定后转化大肠杆菌表达菌株BL21(DE3)感受态细胞。IPTG诱导重组蛋白表达。收集菌体裂解后,用尿素洗涤和溶解包涵体。溶解上清经Ni-NTA亲和层析柱纯化,并用SDS-PAGE和Western blotting检测纯化产物。结果成功构建人PTK6重组蛋白原核表达载体,重组蛋白以包涵体形式表达,其相对分子质量为55 000,与预期一致。亲和层析纯化产物的SDS-PAGE和Western blotting鉴定表明获得纯度大于80%的目的重组蛋白。结论人PTK6 His融合蛋白的纯化为多克隆及单克隆抗体的制备以及结构和功能的研究奠定了基础。     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

应用蛋白质L检测和纯化培养上清液中的单链抗体

目的 探索一种检测和纯化单链抗体的新方法。方法 使用不同的酶标记物，观察ELISA法检测培养上清液中单链抗体的相对灵敏度。含单链抗体的培养上清液经超滤、313g／L饱和硫酸铵沉淀及蛋白质L琼脂糖亲和层析纯化单链抗体。结果 HRP标记蛋白质L可检测到1：16孔的阳性结果，而HRP标记蛋白质A仅检测到1：2孔的阳性结果。用9E10单抗检测培养上清液中的单链抗体时，除加入未经稀释培养上清液孔略有显色外，其他加入稀释后的培养上清液孔均不显色。蛋白质L亲和层析纯化的单链抗体经SDS-PAGE及ELISA鉴定，所得到的单链抗体纯度高、活性保持良好。结论 蛋白质L是一种能灵敏检测和有效纯化单链抗体的结合物，本实验建立的方法是一种有效、简便实用的方法。     

结核分枝杆菌rPstS1-HspX融合蛋白的表达、纯化及其免疫反应性分析

目的 构建结核分枝杆菌rPstS1-hspX (rph)融合基因及其原核表达载体pET-23b(+)-rPstS1-hspX[pET-23b(+)-rph],表达、纯化rPstS1-HspX (rPH)融合蛋白,并分析其免疫反应性.方法 采用基因拼接技术将PstS1和HspX编码基因通过多肽接头(GSGSG)的DNA序列进行连接,构建融合基因rph.将融合基因定向克隆入原核表达载体pET-23b(+),构建重组原核表达质粒pET-23b(+)-rph.将重组质粒转化大肠杆菌E.coli BL21 (DE3) pLysE感受态细胞,IPTG诱导融合蛋白表达.SDS-PAGE和Western印迹法鉴定其表达情况.用镍离子鳌合亲和层析柱纯化融合蛋白,Western印迹法初步评价融合蛋白的免疫反应性.结果 融合基因rph及其原核表达载体pET-23b (+)-rph构建成功.融合蛋白rPH主要以可溶性非包涵体形式表达,相对分子质量为51 000,表达量约占菌体总蛋白的23％.经亲和层析后得到了纯度达92％的融合蛋白.Western印迹证实融合蛋白能与结核病阳性血清发生特异性免疫反应.结论 成功构建了原核表达载体pET-23b(+)-rph,获得了rPH融合蛋白,为rPH融合蛋白在结核病诊断中的应用提供了依据.     

c-Jun N-terminal kinase and p38 mitogen-activated protein kinase mediate double-strand RNA-induced inducible nitric oxide synthase expression in microglial cells

Double-stranded RNA (dsRNA) has been implicated as a potential immune stimulant in activating microglia, which can cause chronic neurodegeneration. In this study, we examined the involvement of different types of mitogen-activated protein kinases (MAPKs) in the induction of inducible nitric oxide synthase (iNOS) by dsRNA in microglial cells. Nitric oxide production was increased after exposure of microglia to 50 μg/mL dsRNA. Levels of dsRNA-induced nitrite production in a line of immortalized murine microglia (BV2) and in primary cultures of murine microglia were decreased by inhibition of JNK or p38 MAPK, but were increased by inhibition of extracellular signal-regulated kinase. Similar results were shown in the levels of dsRNA-induced iNOS gene expression in BV2 cells. Phosphorylation levels of p38 MAPK were increased, depending on p38 MAPK inhibitor concentrations, while activation levels of MAPKAPK2, a known p38 substrate, were inhibited. Thus, it is likely that SB203580 inhibited the kinase activity of p38 MAPK, resulting in the loss of a feedback inhibition regulatory loop of p38 MAPK in BV2 cells. These findings suggest that dsRNA stimulated iNOS expression via MAPK signaling pathways, including JNK and p38 MAPK.