重组链激酶及其三种突变体的活性研究

利用PCR技术从Streptococucspyogenes的基因组DNA中扩增了链激酶的编码基因ska,并进行了序列分析 ,利用基因删除及定点位变技术获得了删除了C-末端 42个氨基酸残基编码区的突变链激酶基因skaΔC42 ,第 5 9位Lys残基突变为Glu的突变链激酶基因skaK5 9E以及删除C-末端 42个氨基酸且第 5 9位Lys残基突变为Glu的突变链激酶基因skaΔC42K5 9E ,将ska及其三种突变体分别克隆到表达载体pET 1 5b上 ,构建分别表达野生型链激酶 (SK)、C-末端缺失 42个氨基酸残基的突变体 (SKΔC42 )、第 5 9位Lys残基突变为Glu的突变体 (SKK5 9E)及C-末端缺失 42个氨基酸且第 5 9位Lys残基突变为Glu突变体 (SKΔC42K5 9E)的表达载体pSK ,pSKΔC42 ,pSK K5 9E ,pSKΔC42K5 9E ,分别转化E .coliBL2 1 (DE3) ,IPTG诱导后在大肠杆菌中实现了高效表达 ,经亲和层析、离子交换层析及分子筛层析 ,获得了rSK、rSKΔC42、rSKK5 9E及rSKΔC42K5 9E ,活性分析表明rSK与其三种突变体具有相同的比活性。     

TBK1相关激酶活性缺失突变体和泛素样结构域突变体的构建、表达及生物活性鉴定

目的:构建TANK结合激酶1(TBK1)相关激酶活性缺失突变体和泛素样结构域突变体真核表达载体,检测该基因相关突变体在293细胞中的表达,并利用萤光素酶报告基因实验检测其生物活性。方法:根据文献报道的突变序列及QuickChange Site-Directed Mutagenesis实验设计手册,设计合成2条针对TBK1相关激酶活性缺失突变体和泛素样结构域突变体的引物,以实验室之前构建的TBK1野生型真核表达载体为模板,构建TBK1激酶活性缺失突变体和泛素样结构域突变体真核表达载体,分别命名为pcDNA3-Flag-TBK1(KD)、pcDNA3-Flag-TBK1(ΔULD)。以LipofactAMINE2000转染试剂转染至293细胞中进行瞬时表达,利用萤光素酶实验检测2种TBK1突变体诱导β干扰素(IFN-β)转录的情况。结果:测序结果表明,TBK1相关激酶活性缺失突变体和泛素样结构域缺失突变体真核表达载体构建成功,Western印迹检测表明其在293细胞中获得有效表达;用萤光素酶报告基因实验检测,与野生型TBK1相比,其相关激酶活性缺失突变体和泛素样结构域缺失突变体诱导IFN-β转录激活的作用明显降低。结论:真核表达的TBK1相关激酶活性缺失突变体和泛素样结构域突变体具有相应的生物学活性,为研究其功能奠定了基础。     

hHO-1结构预测及突变体的构建、表达、纯化和活性测

人血红素加氧酶-1(human heme oxygenase-1,hHO-1)是血红素代谢的限速酶,直接调节体内胆红素水平.利用软件Spdbv程序对hHO-1进行结构模拟预测,以Ala取代第25位His残基,hHO-1活性部位的结构有较明显的改变,但据模拟推测突变后hHO-1与血红素仍然具有结合性.依据模拟结果,构建野生型和突变体表达载体pBHO-1和pBHO-1(M),并分别转化大肠杆菌DH5α,IPTG诱导表达目的蛋白.表达产物经30%-60%(NH4)2SO4盐析后纯度提高3.6倍,再经两次Q-Sepharose Fast Flow阴离子交换树脂分离则表达产物的纯度提高30倍.酶活性测定显示,突变体hHO-1(△hHO-1)较野生型hHO-1(whHO-1)活性下降了91.21%.本研究显示hHO-1的第25位His在酶与底物血红素氧化反应中起着重要作用,为有效调控酶活性发挥其生物学作用提供依据.     

NDM-1的同源建模及其模型活性位点分析

近期研究发现的超级病原菌耐药的原因,是其含有一种特殊的金属内酰胺酶—NDM-1。采用计算生物学技术,通过分子建模、使NDM-1与其它金属β-内酰胺酶类相比对,探索NDM-1对于β-内酰胺类抗生素具有广谱的水解能力的分子机理,为相关新药的开发提供理论依据。将NDM-1序列用BLAST进行同源性搜索,挑选一些相似性较高的序列进行多序列比对的同源建模,对所得的模型进行评估。根据已知的VIM-2晶体结构,使计算的NDM-1模型活性位点与已知VIM-2金属酶结构进行比较分析;结果发现NDM-1在锌离子结合位点的几个关键的氨基酸残基,与VIM-2金属酶较为相似。同时活性位点附近的氨基酸残基的立体折叠结构也与VIM-2存在相似性。NDM-1水解谱的广泛性,可能在于活性位点附近一些氨基酸残基的差异,后者可通过改变空间结构,从而增加了NDM-1的水解活性。     

天花粉蛋白R122G突变体的构建及活性研究

天花粉蛋白Ｒ１２２Ｇ突变体的构建及活性研究袁惠东１柯一保孔梅柯欣永夏其昌１张祖传１聂慧玲＊（中国科学院上海细胞生物学研究所,上海２０００３１;１中国科学院上海生物化学研究所,上海２０００３１）关键词天花粉蛋白;突变体;ＲＮＡＮ－糖苷酶天花粉蛋白（ｔｒ...     

睫状神经营养因子突变体蛋白的活性研究

为了进一步研究我室应用计算机分子模拟设计并表达纯化的睫状神经营养因子突变体蛋白的生物学活性,分别采用鸡胚背根神经节无血清培养法、TF-1细胞增殖法、正常小鼠减重法对其活性进行研究。结果是突变体蛋白能促进鸡胚背根神经节的生长;促进TF-1细胞增殖,MTT测定法表明突变体蛋白与国际参考品相比,比活不低于2.0×106U/mg;使正常小鼠的体重减轻,摄食量减少,脂肪指数下降,并且体重的减轻与突变体蛋白的给药剂量呈现良好的剂量依赖关系,其ED50为：150.986?g/kg/d。以上实验表明CNTF突变体蛋白具有促神经生长、促TF-1细胞增殖和减重的生物学活性。从而为其进一步的应用和开发提供了线索。     

原纤维蛋白1 C末端的系统发育和活性位点预测生物信息学分析

人前原纤维蛋白1 (profibrillin-1, FBN1)经过弗林蛋白酶(furin)酶切后得到成熟的原纤维蛋白-1和C末端多肽白脂素(asprosin),白脂素是新发现的参与调解糖代谢的热点激素。本研究采用整合生物信息学方法,对FBN1 C末端的系统发育和白脂素的功能位点进行分析,探究白脂素在糖代谢相关疾病发生中的分子机制。从UniProt数据库中搜索物种FBN1序列信息并进行比对;通过blastp方法检索脊椎动物FBN1 C末端氨基酸,构建进化树和进化轨迹,进行系统发育生物信息学分析;运用I-TASSER构建白脂素三级结构预测模型;运用COFACTOR和COACH法进行白脂素的配体活性位点分析。本研究发现：脊椎动物FBN1 C末端具有Furin酶识别位点(R-X-K/X-R)的高保守性,进化树结果显示FBN1 C末端蛋白有26个高保守位点,白脂素活性位点预测为“4RL”和“MAN”。本研究首次揭示原纤维蛋白1 C末端蛋白可能在脊椎动物中广泛存在,分析了白脂素的功能和结构的关系,为其在糖代谢性疾病中的功能机制研究和治疗提供新的思路。     

利用优化的反向PCR策略高效构建多位点突变体

蛋白质的突变体是研究其结构和功能的基础,文中旨在建立一种高效、快捷的多位点突变体构建方法。当要突变4个及以上相邻的氨基酸残基时,设计两长两短(长引物Ⅰ/Ⅰ、短引物Ⅱ/Ⅱ) 4条引物:长引物包含突变位点,且突变碱基数≤20 bp,短引物不包含突变位点;两条引物的GC含量≤80%、退火温度之差≤40℃,分别以Ⅰ/Ⅱ和Ⅰ/Ⅱ两对引物和模板进行两组反向PCR扩增。扩增后各体系均可得到含有突变位点的非甲基化线性质粒,且以Ⅰ/Ⅱ和Ⅰ/Ⅱ为引物扩增得到的两组线性质粒的断开位点分布在突变位点两侧。用DpnⅠ酶切回收后等摩尔比混合的PCR产物除去甲基化模板,再进行一轮变性和退火处理,两组线性质粒在95℃变性后互相以来自对方的单链DNA为模板退火形成开环质粒,转化大肠杆菌感受态细胞即可得到包含突变位点的转化子。结果表明,该方法可同时突变4–11个连续氨基酸残基(8–20 bp,将大幅简化多位点突变体的构建,从而进一步提高蛋白质结构和功能研究的效率。     

旋毛虫基因重组抗原的纯化

根据旋毛虫基因重组抗原蛋白的特点,筛选出裂解处理工程菌菌体的方法,并探索了用ＳｅｐｈａｃｒｙｌＳ－３００（ＨＲ）柱层析纯化重组蛋白的程序。用本法处理和纯化后,重组蛋白的纯度可达９０％以上。所纯化的三种重组抗原蛋白均能与猪旋毛虫病血清发生特异性反应而不与正常猪血清和猪囊虫病血清反应,其中以重组蛋白ＲＰ３４的特异性最强,ＲＰ３７较弱,ＲＰ４６介于两者之间。研究结果表明,本纯化方法易于操作、设备简单和特异性蛋白回收率高,是处理和纯化旋毛虫基因重组抗原的最佳程序。     

HIV-1整合酶四聚体结构模拟及其活性位点分析

HIV-1复制需要HIV-1整合酶将其环状DNA整合进宿主DNA中,这其中包括2个重要反应,即“3′-加工”和“链转移”,两者均由HIV-1整合酶催化完成.阻断其中的任一反应,都能达到抑制HIV-1复制的目的.因此,了解HIV-1整合酶的完整结构和聚合状态,对深入探讨其作用机理及设计新型抑制剂具有重要的指导作用.然而,迄今为止仅有HIV-1整合酶单独结构域的晶体结构可供参考,而其全酶晶体结构尚未获得解析.本研究利用分子模拟技术,通过蛋白质 蛋白质/DNA分子对接、动力学模拟等方法,构建了全长整合酶四聚体的结构模型、HIV-1 DNA与整合酶复合物的结构模型,进一步从理论上证实HIV-1整合酶是以四聚体形态发挥催化作用,明确“3′-加工”和“链转移”在HIV-1整合酶上的催化位点.同时,通过与作用机理相似的细菌转座子Tn5转座酶等的结构比对,推测HIV-1整合酶的核心结构域中应有第2个Mg^２＋存在,其位置螯合于Asp64与Glu152之间.在HIV-1整合酶结构研究的基础上,有望进一步设计出新的抗艾滋病药物.     

Changes in the inhibitory responses to electrical field stimulation of intestinal smooth muscle from Trichinella spiralis infected rats

Functional motor changes and morphological alterations have been associated with intestinal inflammation. The aim of this work was to study functional motor changes in inflamed and non-inflamed intestinal segments of Trichinella spiralis infected rats. Thickness of muscle layers and cell infiltration during infection were also evaluated. Segments of rat jejunum and ileum were placed in organ bath and relaxations of the longitudinal muscle in response to electrical field stimulation (EFS) were recorded. During the post-infection (PI) period EFS-induced relaxations in ileum were decreased. Maximal decreases in relaxation were found on day 14-23 PI for ileum, whereas non significant changes were observed in jejunal samples throughout the experimental period. The sensitivity of the EFS-induced relaxations to the NO synthase inhibitor Nω-nitro-l-arginine (L-NNA) and to the soluble guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) was decreased on day 14 PI for jejunum, whereas in the ileum it lasted from day 14-23 PI. The sensitivity of EFS-induced relaxations to apamin (a small conductance calcium activated potassium channel blocker) disappeared between day 6-23 PI for both jejunum and ileum. In contrast, the sensitivity of the EFS-induced relaxations to the K⁺ channel blockers tetraethylamonium (TEA) and tetrapenthylammonium (TPEA) chloride was similar for healthy tissue and for tissue obtained form infected animals. Distribution and density of NADPH-diaphorase positive neurons was similar in tissue obtained form healthy and infected animals. In conclusion, intestinal inflammation induces functional and structural changes in both worm-free and worm-positive intestinal segments. Increased muscle thickness was similar for both inflamed and noninflamed segments but the most prominent functional changes i.e. a long-lasting decrease of EFS-induced relaxation was found in non-inflamed ileal segments.     

旋毛虫Ts87抗原的免疫学特性及保护性的初步研究

应用Western btot和ELISA方法对纯化的重组蛋白PET—28a( )／Ts87进行免疫学特性鉴定及保护性研究。Western blot和ELISA结果显示,Ts87抗原可被人工感染旋毛虫的兔血清、病猪血清、病人血清及抗Ts87的兔血清所识别。Ts87抗原免疫BABL/c小鼠,较对照组减虫率为29％,说明Ts87抗原可作为旋毛虫免疫诊断和疫苗的候选抗原。     

Proteomic analysis of mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis

Infection with the intestinal nematode Trichinella spiralis induces profound, but stereotypic pathological changes to the epithelium, which are common to many nematode infections. This study describes changes in jejunal epithelial protein expression that reflect these stereotypic responses. Adult male BALB/c mice were infected with T. spiralis, and groups (n = 4) examined on day 14/15 (time of worm rejection) were compared with uninfected controls (n = 4). Jejunal epithelium was harvested and extracted for two-dimensional gel electrophoresis. Tryptic peptide mass fingerprinting was used to create a reference map consisting of a total of 52 landmark spots. Of these, 16 were observed to change in intensity during infection. The changes observed at day 14/15 were of relevance to such mechanisms as lipid utilization and transport (increase in triacylglycerol lipase, and reduction in intestinal fatty acid binding protein) and innate immunity (appearance of intelectin-2). As a result, candidate molecules have been identified for further focused studies on their role in the host response to intestinal nematode infection.     

旋毛虫感染小鼠肠道菌群变化的研究

目的通过观察黑龙江株旋毛虫感染小鼠肠道分泌物中分泌型免疫球蛋白A、肠道菌群的变化,探讨感染小鼠肠道菌群的变化。方法分别于小鼠感染黑龙江株旋毛虫后7、14、21、28和35d,观察模型组及对照组小鼠肠道分泌物中的分泌型免疫球蛋白A、肠道双歧杆菌、乳酸杆菌、肠杆菌、肠球菌的菌群变化。sIgA采用放射免疫法检测。结果模型组sIgA分泌水平在感染后14d达高峰,随后缓慢下降但始终保持高水平（P〈0．01）。模型组肠道双歧杆菌的数量在感染后7d略低于对照组（P〈0．05）,第14天降至最低水平,随后逐渐升高,至感染后35d恢复正常水平。乳酸杆菌的数量在感染后7d略低于对照组,第14天降至最低水平,随后逐渐增加（P〈0．05）。肠杆菌的数量在感染后7d略高于对照组,感染后14d明显高于对照组,随后始终保持下降趋势（P〈0．05）。肠球菌在感染后7d略高于对照组（P〈0．05）,在14d明显高于对照组,随后缓慢下降,至感染后35d恢复正常水平。结论旋毛虫感染小鼠sIga的分泌在肠道免疫中发挥重要作用,同时也影响肠道菌群;肠道菌群的变化可能与旋毛虫感染小鼠免疫系统中sIgA的分泌有关。     

Protective effects of recombinant 53-kDa protein of Trichinella spiralis on acute lung injury in mice via alleviating lung pyroptosis by promoting M2 macrophage polarization

Trichinella spiralis represents an effective treatment for autoimmune and inflammatory diseases. The effects of recombinant T. spiralis (TS) 53-kDa protein (rTsP53) on acute lung injury (ALI) remain unclear. Here, mice were divided randomly into a control group, LPS group, and rTsP53 + LPS group. ALI was induced in BALB/c mice by LPS (10 mg/kg) injected via the tail vein. rTsP53 (200 µl; 0.4 μg/μl) was injected subcutaneously three times at an interval of 5 d before inducing ALI in the rTsP53+LPS group. Lung pathological score, the ratio and markers of classic activated macrophages (M1) and alternatively activated macrophages (M2), cytokine profiles in alveolar lavage fluid, and pyroptosis protein expression in lung tissue were investigated. RTsP53 decreased lung pathological score. Furthermore, rTsP53 suppressed inflammation by increasing IL-4, IL-10, and IL-13. There was an increase in alveolar M2 macrophage numbers, with an increase in CD206 and arginase-1-positive cells and a decrease in alveolar M1 markers such as CD197 and iNOS. In addition, the polarization of M2 macrophages induced by rTsP53 treatment could alleviate ALI by suppressing lung pyroptosis. RTsP53 was identified as a potential agent for treating LPS-induced ALI via alleviating lung pyroptosis by promoting M2 macrophage polarization.     

Trichinella spiralis: histamine secretion induced by TSL-1 antigens from unsensitized mast cells

Mast cells' hyperplasia and activation are prominent features in Trichinella spiralis infection. Recently, it was shown that TSL-1 antigens from T. spiralis muscle larvae induce IL-4 and TNF release by unsensitized, normal mast cells (MC) involving an Ig-independent mechanism. In this study, we characterized histamine secretion induced by TSL-1 antigens from normal, unsensitized rat peritoneal MC. Maximum histamine secretion (30+/-5.3% SEM, n=13) was achieved with 30 ng/mL TSL-1 antigens. However, TSL-1 did not induce an increase in beta-hexosaminidase release or NADPH oxidase activity by MC. Interestingly, histamine secretion by TSL-1 was completed at 10s, and was inhibited by both Bordetella pertussis toxin and neuraminidase V, characteristics similar to those involved in substance P-induced histamine secretion. However, in contrast to substance P, TSL-1 induced histamine secretion in the absence of detectable changes in intracellular Ca(2+). We are investigating the molecular pathways involved in MC activation by TSL-1.     

Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle

Trichinella spiralis is an intracellular nematode parasite of mammalian skeletal muscle. Infection of the muscle cell leads to the formation of a host-parasite complex that results in profound alterations to the host cell and a re-alignment of muscle-specific gene expression. The role of parasite excretory-secretory (ES) proteins in mediating these effects is currently unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, a global proteomics approach was used to analyse the ES proteins from T. spiralis muscle larvae. Following 2-DE of ES proteins,MALDI-TOF-MS and LC-MS/MS were used to identify the peptide spots. Specific Trichinella EST databases were assembled and used to analyse the data. Despite the current absence of a Trichinella genome-sequencing project, 43 out of 52 protein spots analysed were identified and included the major secreted glycoproteins. Other novel proteins were identified from matches with sequences in the T. spiralis database. Our results demonstrate the value of proteomics as a tool for the identification of Trichinella ES proteins and in the study of the molecular mechanism underpinning the formation of the host-parasite complex during Trichinella infections.     

Exploring the drug resistance mechanism of active site,non-active site mutations and their cooperative effects in CRF01_AE HIV-1 protease: molecular dynamics simulations and free energy calculations

Human immunodeficiency virus type 1 protease is essential for virus replication and maturation and has been considered as one of the important drug target for the antiretroviral treatment of HIV infection. The majority of HIV infections are caused due to non-B subtypes in developing countries. Subtype AE is spreading rapidly and infecting huge population worldwide. Understanding the interdependence of active and non-active site mutations in conferring drug resistance is crucial for the development effective inhibitors in subtype AE protease. In this work, we have investigated the mechanism of resistance against indinavir (IDV) due to therapy selected active site mutation V82F, non-active site mutations PF82V and their cooperative effects PV82F in subtype AE-protease using molecular dynamics simulations and binding free energy calculations. The simulations suggested all the three complexes lead to decrease in binding affinity of IDV, whereas the PF82V complex resulted in an enhanced binding affinity compared to V82F and PV82F complexes. Large positional deviation of IDV was observed in V82F complex. The preservation of hydrogen bonds of IDV with active site Asp25/Asp25′ and flap residue Ile50/50′ via a water molecule is crucial for effective binding. Owing to the close contact of 80s loop with Ile50′ and Asp25, the alteration between residues Thr80 and Val82, further induces conformational change thereby resulting in loss of interactions between IDV and the residues in the active site cavity, leading to drug resistance. Our present study shed light on the effect of active, non-active site mutations and their cooperative effects in AE protease.

Communicated by Ramaswamy H. Sarma     

Effect of myrrh and thyme on Trichinella spiralis enteral and parenteral phases with inducible nitric oxide expression in mice

Trichinellosis is a serious disease with no satisfactory treatment. We aimed to assess the effect of myrrh (Commiphora molmol) and, for the first time, thyme (Thymus vulgaris L.) against enteral and encysted (parenteral) phases of Trichinella spiralis in mice compared with albendazole, and detect their effect on inducible nitric oxide synthase (iNOS) expression. Oral administration of 500 mg/kg of myrrh and thyme led to adult reduction (90.9%, 79.4%), while 1,000 mg/kg led to larvae reduction (79.6%, 71.3%), respectively. Administration of 50 mg/kg of albendazole resulted in adult and larvae reduction (94.2%, 90.9%). Positive immunostaining of inflammatory cells infiltrating intestinal mucosa and submucosa of all treated groups was detected. Myrrh-treated mice showed the highest iNOS expression followed by albendazole, then thyme. On the other hand, both myrrh and thyme-treated groups showed stronger iNOS expression of inflammatory cells infiltrating and surrounding encapsulated T. spiralis larvae than albendazole treated group. In conclusion, myrrh and thyme extracts are highly effective against both phases of T. spiralis and showed strong iNOS expressions, especially myrrh which could be a promising alternative drug. This experiment provides a basis for further exploration of this plant by isolation and retesting the active principles of both extracts against different stages of T. spiralis.     

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).