蝎镇痛抗肿瘤缬精甘肽的两个氨基酸残基对镇痛活性的影响

目的研究蝎镇痛抗肿瘤缬精甘肽(analgesic-antitumor peptide,AGAP)中2个氨基酸残基对其镇痛活性的影响。方法利用已构建成功的2个突变体W38G和R58D,采用金属离子螯合亲和层析和阳离子交换层析方法对突变体蛋白进行纯化。采用小鼠醋酸扭体法测定两个突变体的镇痛活性。结果在大肠杆菌BL21(DE3)中实现了可溶性表达。采用金属离子螯合层析和阳离子交换层析方法获得了电泳纯样品。突变体W38G镇痛活性与未突变重组蛋白(recombinant analgesic-antitumor peptide from Buthus martensii Karschr,BmK AGAP)相同,突变体R58D镇痛活性降低。结论突变体与rBmK AGAP相比镇痛活性发生变化,说明所选取的突变位点对蝎镇痛抗肿瘤缬精甘肽的镇痛活性具有一定的调节作用。     

镇痛抗肿瘤缬精甘肽BmK AGAP的两对二硫键C16-C36和C22-C46对镇痛活性的影响

目的研究二硫键C16-C36和C22-C46对镇痛抗肿瘤缬精甘肽BmK AGAP镇痛活性的影响。方法使用本实验室已构建成功的2个二硫键双突变体(C16S,C36S)和(C22S,C46S),在大肠杆菌中诱导表达,通过金属离子螯合亲和层析和阳离子交换层析方法对突变体蛋白分离纯化,采用小鼠醋酸扭体法测定2个突变体的镇痛活性,圆二色谱分析BmK AGAP和2个二硫键突变体的二级结构变化差异,利用生物信息学的方法模建BmK AGAP的三维空间结构。结果 a.目的蛋白在大肠杆菌BL21(DE3)中实现了可溶性表达;b.采用金属离子螯合亲和层析和阳离子交换层析方法获得了电泳纯样品;c.2个双突变体(C16S,C36S)和(C22S,C46S)的镇痛活性与阳性对照组rBmKAGAP比较均下调,具有显著性差异。结论二硫键C16-C36和C22-C46在保持和调节BmK AGAP的镇痛生物活性方面具有重要作用。     

镇痛活性肽VRD突变体(S54A)的构建与表达以及对活性的影响

目的研究54位丝氨酸对镇痛活性肽(analgesic peptide,AGP)VRD生物活性的影响。方法利用一步聚合酶链式反应(polymerase chain reaction,PCR),用Ala替代镇痛活性肽VRD第54位的Ser,从而构建突变体S54A。采用本实验室构建的pSYPU作为表达载体在大肠杆菌BL21(DE3)中表达重组蛋白。通过金属离子螯合亲和柱色谱和阳离子交换柱色谱方法对重组蛋白进行分离纯化。小鼠扭体法测定重组蛋白的镇痛活性。结果重组蛋白主要以可溶性形式存在,通过两步色谱方法获得了电泳纯样品。突变体S54A的镇痛活性明显低于AGP-VRD(P<0.05)。结论镇痛活性肽VRD中54位丝氨酸与其镇痛活性密切相关。     

蝎镇痛抗菌活性肽BmK AS的融合表达及蛋白切割

目的构建镇痛抗菌活性肽BmK AS原核融合表达载体,实现可溶性表达,获得重组活性肽BmK AS。方法利用本实验室已构建成功的pET 28a-AS质粒,设计引物经过聚合酶链式反应,将目的基因克隆至pET 32a载体中,构建融合表达载体pET 32a-AS,优化诱导表达条件,实现BmK AS在大肠杆菌BL21(DE 3)中的融合可溶性表达,通过金属离子螯合亲和薄层色谱法对重组蛋白进行初步分离纯化,获得重组融合蛋白后采用凝血酶进行切割去除纯化标签,经SDS-PAGE检测切割效果。结果成功构建重组表达质粒,优化得到低温诱导促进蛋白可溶性表达的方法;采用金属离子螯合亲和薄层色谱法获得重组蛋白;电泳结果表明凝血酶可以切割融合蛋白。结论实现活性肽BmK AS在大肠杆菌中的融合可溶性表达,经凝血酶切割后获得重组镇痛抗菌活性肽BmK AS,为后续药理活性研究奠定基础。     

两种具有双功能活性蝎毒蛋白融合体的构建

目的首次报道利用东亚钳蝎镇痛抗肿瘤缬精甘肽-BmKAGAP产生两种不同的融合蛋白,目的是获得镇痛和抗肿瘤的双功能活性。方法融合蛋白是以嵌合毒素的形式进行构建,由三部分组成,包括促性腺激素释放激素(luteinizing hormone-releasing hormoneL,HRH),两种不同类型的柔性连接物,以及BmKAGAP。克隆编码两种融合蛋白的基因并在大肠杆菌中实现可溶性表达。通过金属离子螯合亲和层析和阳离子交换层析方法对融合蛋白分离纯化,采用小鼠醋酸扭体法和MTT法测定融合蛋白的镇痛活性与细胞抑制活性。结果融合蛋白在小鼠模型中体现出镇痛活性(rLHRH-Tag(His)AGAP>rTag(His)-LHRH-L8-AGAP>rBmKAGAP),同时对肝癌细胞株Hep3B具有细胞毒活性(rLHRH-Tag(His)-AGAP>rBmKAGAP>rTag(His)-LHRH-L8-AGAP)。结论得到优化的双功能融合蛋白候选者为嵌合毒素rLHRH-Tag(His)-AGAP。     

来源于豆豉的纳豆激酶基因在大肠杆菌中活性表达研究

目的构建大肠杆菌表达质粒pTYB10 2 ,实现纳豆激酶基因 (nattokinasegene)在大肠杆菌中高活性表达。方法以纳豆芽孢杆菌基因组DNA为模板 ,采用PCR方法分别扩增编码前肽、成熟肽的序列 (pro NK) ,构建大肠杆菌表达质粒pTYB10 2 ,转化大肠杆菌ER2 5 6 6。在IPTG诱导下 ,分别在 37℃ (2h)、30℃ (3h)和 15℃ (14h)培养。结果pTYB10 2能表达有活性的纳豆激酶。SDS PAGE表明 ,15℃表达杂蛋白质更少。结论证明具有 77个氨基酸序列的前肽 (pro序列 )对纳豆激酶的活性表达是必不可少的。     

重组人肽抗生素hPAB-β及其突变体的活性测定与筛选

目的 测定重组人肽抗生素hPAB—β及其突变体的抗菌活性，筛选理想的活性分子。方法 应用平板法和MIC测定法检测重组表达的hPAB—β及突变体hPAB—β38、hPAB—β34对8株实验室保存菌株及10株临床分离菌株的杀菌能力。并以化学合成的肽抗生素hPAB—β作对照；根据各目的分子抗菌能力的强弱，筛选理想的目标分子，并分析突变体结构的变化对其功能的影响。结果 重组hPAB—β及突变体hPAB—β38与化学合成的天然分子抗菌活性相当．对革兰氏阳性菌的MIC在125～250μg／ml之间，对革兰氏阴性菌的MIC在31～125μg／ml范围内；突变体hPAB—β34活性下降4倍左右；结构分析表明hPAB—β38与hPAB—β参与二级结构中α螺旋和β片层构成的氨基酸残基完全相同，而hPAB-β34则有显著不同，α螺旋少了2个残基，3个β片层中除β2与hPAB—β相同外，β1和β3的构成均多2个残基，推测hPAB—β34二级结构的改变是其活性降低的主要原因。结论 重组肽抗生素hPAB—β具有抗菌活性，删除3个端残基的突变体hPAB—β38活性不变，可作为hPAB—β走向临床应用的一个新侯选者。     

人β-防御素DEFB113的重组表达及活性鉴定

DEFB113为人β-防御素家族新成员,其生物学功能尚不清楚。为了探索一种可以较低成本和较高产量表达活性DEFB113多肽的方法,将编码DEFB113成熟肽的序列克隆到载体pTWIN1上,并转化至E.coliBL21(DE3)中进行表达。结果表明融合蛋白主要以可溶形式存在;重组载体中融合标签的几丁质结合域(CBD)和内含肽(intein)分别使亲和层析纯化和融合标签自剪切一步完成。最终得到DEFB113多肽的产率为6～7 mg/LLB。经活性鉴定,该重组DEFB113多肽对于S.aureus,E.coli K12D31和C.albicans均表现出较强的抑制作用;另外DEFB113无明显的溶血活性,是一种较理想的多肽抗生素。该研究为进一步研究DEFB113的生理功能以及其它人β-防御素结构与功能奠定了基础。     

Y111是维持血管内皮细胞生长抑制因子生物活性的关键氨基酸

目的血管内皮细胞生长抑制因子(Vascu lar endothe-lial cell growth inh ib itor,VEG I)是近年来发现的一类TNF家族新成员,具有抑制内皮细胞增殖与新生血管生成的作用。但目前其结构功能关系研究尚为空白,该文研究VEG I空间结构上重要残基的作用极其对生物活性的影响。方法采用定点突变技术突变了四个关键氨基酸E45R,G47A,Y111F,Y111T,构建了表达载体并在大肠肝菌中表达及纯化,采用人脐静脉内皮细胞增殖和鸡胚尿囊膜血管生成实验研究了四个突变体的活性。结果E45R突变体的活性显著下降,G47A突变体的活性略低于野生型VEG I,Y111F,Y111T突变体的活性比野生型VEGI下降10倍以上。结论VEGI的第111位氨基酸Y是发挥其功能的关键氨基酸,可能与受体形成直接结合,且氨基酸Y上的苯环与酚羟基均对其活性的发挥具重要作用;第45位氨基酸E对其活性的发挥也具重要作用,而第47氨基酸对其活性的发挥可能不具重要作用。     

石榴花挥发油化学成分的GC-MS分析及体外抗氧化活性测定

目的:分析石榴花挥发油的化学成分,并测定其体外抗氧化活性。方法:采用水蒸气蒸馏法提取石榴花中的挥发油,用GC-MS对其进行分析;以对金属离子螯合作用和ABTS自由基清除作用为指标评价石榴花挥发油的抗氧化活性。结果:从石榴花挥发油中分离鉴定23个化合物,占挥发油总量的88.49%;20.00%石榴花挥发油对金属离子的螯合作用和ABTS自由基的清除作用优于0.10 mg·mL-1的EDTA或VC。结论:石榴花挥发油中含有多种有应用价值的化学成分,具有显著的体外抗氧化活性。     

Roles of disulfide bridges in scorpion toxin BmK M1 analyzed by mutagenesis

Abstract: The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12–63, 16–36, 22–46 and 26–48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S‐78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na⁺ channels (rNa_v1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long‐chain scorpion toxins, loss of either bridge C22?C46 or C26?C48 is fatal for the general folding of the molecule. Bridge C16?C36 mainly contributes to the global stability of the folded scaffold, and bridge C12?C63 plays an essential role in the functional performance of scorpion toxins.     

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage,co-expressing with chaperones and fermenting in fed-batch mode

The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.     

Differential involvement of disulfide bridges on the folding of a scorpion toxin

Leiurotoxin I is a neurotoxin, blocker of Ca²⁺-activated apamin-sensitive K⁺ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys³-Cys²¹, Cys⁸-Cys²⁶ and Cys¹²-Cys²⁸. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were replaced by α-aminobutyrate residues to suppress one disulfide bridge, were analyzed by ¹H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys³-Cys²¹ and a loss of organized structure for another analog lacking disulfide bridge Cys¹²-Cys²⁸. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys¹²-Cys²⁸ is essential for the folding process. In contrast, the lack of disulfide bridge Cys³-Cys²¹ does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I.     

Proteomic analysis of serum-derived extracellular vesicles in ankylosing spondylitis patients

BackgroundAnkylosing spondylitis (AS) is a chronic inflammatory disease, whose pathogenesis is still unclear. Many studies show the proteins in extracellular vesicle (EVs) would change regularly in many diseases. The study aims to explore the proteins contents of serum-derived EVs in AS patients.MethodsEVs were separated by ExoQuick^TM kit. The protein profiles of AS patients and healthy subjects were analyzed by Label-free-liquid chromatography mass spectrometry (LC-MS/MS) technology. Enzyme-linked immunosorbent assay (ELISA) was used to verify the levels of the differently expressed proteins. Receiver operation characteristic (ROC) curves and bioinformatic analysis were conducted.ResultsSix hundred and ten serum-derived EVs proteins from AS patients were detected. Seventy-three diferentially expressed proteins were found in AS group, compared with healthy subjects. Of these, 31 proteins were up-regulated in AS group, while 42 proteins were down-regulated. ELISA result showed that EVs-derived serum amyloid A-1 (SAA1) was higher in AS group, which was consistent with the Label-free-LC-MS/MS data. ROC curves result revealed that the area under curve (AUC) value of EVs-derived SAA1 for AS was 0.768 (0.652–0.885). Bioinformatic analysis revealed that the differently expressed proteins in AS group were significantly involved in “complement and coagulation cascades”, “staphylococcus aureus infection”, “systemic lupus erythematosus” and “PI3K-Akt signaling pathway”.ConclusionsThe protein profiles of serum-derived EVs in AS patients and healthy subjects were different. EVs-derived SAA1 may be a potential biomarkes of AS. The function analysis indicated that the differentially expressed proteins may potentially participate in immune response.     

Covalent structures of BmK AS and BmK AS-1, two novel bioactive polypeptides purified from Chinese scorpion Buthus martensi Karsch.

Complete amino acid sequences of two novel bioactive polypeptides, each containing 66 amino acid residues, BmK AS and BmK AS-1 purified from the venom of Chinese scorpion Buthus martensi Karsch, have been determined by Edman sequencing and mass spectrometry on native proteins, reduced and S-carboxymethylated proteins and their peptides obtained after cleavage with proteolytic enzymes. Sequence analysis showed 86.4% structural identity between BmK AS and BmK AS-1 and also a high sequence similarity between BmK ASs and AaH IT4, a unique anti-insect toxin and a ligand of Na+ channels obtained from Sahara scorpion A. australis Hector, but poor sequence homology between BmK ASs and those of the known alpha-, beta-type and long-chain insect-selective type scorpion neurotoxins. The positions of four disulfide bridges in BmK AS-1 were established as Cys-12 and Cys-62, Cys-16 and Cys-37, Cys-23 and Cys-44, and Cys-27 and Cys-46, which are the same as those in alpha- and beta-scorpion neurotoxins. These results suggest that BmK ASs and AaH IT4 may form a new group sharing similar structural and functional properties in the family of scorpion neurotoxic polypeptides.     

Chemical constituents and analgesic activity of Aconitum kusnezoffii Reichb.

In the present study,we aimed to investigate the chemical constituents and analgesic activity of Aconitum kusnezoffii Reichb. The isolation and purification of components were achieved by a series of chromatography, including silica gel, Sephadex LH-20 and HPLC. By using spectroscopic analysis, their structures were identified. Using PDE-4A as analgesic target, moleculardocking was conducted between isolated compounds by using Schrodinger software. Neoline is a typical non-ester diterpene alkaloid. It was studied by using the mouse torsion body method and hot plate method. A total of 12 diterpene alkaloids were obtainedand identified as Mesaconitine(1), Bewutine (2), Bewudine (3), Songoramine (4), Songorine (5), Neoline (6), Talasamine (7), isotalatizidine (8), Hokbusine A (9), Mesaconine (10), 8-OEt-14-benzoylmesaconine (11), 8-Methoxy-14-benzoyl-beiwutinine (12).Compounds 9 and 12 were isolated from Aconitum kusnezoffii Reichb. for the first time. Twelve diterpenealkaloids could act on the analgesic target. Neoline is a typical non-ester diterpene alkaloid. It had significant analgesic effect. Diterpene alkaloids were the main components of Aconitum kusnezoffiiReichb., and they had good analgesic activity.     

Correlation between antimicrobial activity and chelating ability of 2-(N,N,N-trialkylammonio)alkyl hydrogen phosphates.

The chelating abilities of phosphobetaines [2-(N,N,N-trialkylammonio)alkyl hydrogen phosphates; 1] for divalent cations (e.g., calcium ion, zinc ion, copper ion, and cadmium ion) were investigated to elucidate the correlation between the antimicrobial activity and the chelating ability. The maximal chelating ability was obtained with the compound having a dimethylene bridge as an intercharge distance between the phosphate and quaternary ammonium group; in contrast, the minimum was obtained with the compound with a trimethylene bridge. These behaviors paralleled the antimicrobial activity of these compounds. The antimicrobial activities, in terms of log (1/MIC) (minimum inhibitory concentration, mumol/L) of homologues of 1 against Candida albicans and Propionibacterium acnes were found to correlate linearly with the chelating ability for the calcium ion. This linear relationship is discussed with respect to the structure of the phosphobetaine moiety in the molecule of 1.