Implementation of a single quad MS detector in routine QC analysis of peptide drugs

A newly developed single quad mass spectrometry （MS） detector was coupled to a ultra-high perfor- mance liquid chromatography （UPLC） system and implemented in the routine quality control （QC） and impurity analysis of four therapeutic peptides, namely bleomycin sulfate, tyrothricin, vancomycin HCl and bacitracin, which were selected given their multi-component drug nature and their closely struc- turally related impurity profiles. The QC and impurity profiling results obtained using the ultra-high performance liquid chromatography ultraviolet/mass spectrometry （UPLC-UV/MS） detection system were analyzed against the results obtained using traditional high performance liquid chromatography- ultraviolet detection （HPLC-UV） methods derived from pharmacopoeial methods. In general, the used stationary phases of sub-2 μm particle （UPLC） technology resulted in lower limits of detection and higher resolution separations, which resulted in more detected impurities and shorter overall run times con- trasting the traditional HPLC columns. Moreover, online coupling with a single quad MS detector allowed direct peak identification of the main compounds as well as small impurities, hereby increasing the information content without the need of reference standards.

Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.

Type-2 Diabetes Mellitus and Glucagon-Like Peptide-1 Receptor toward Predicting Possible Association

Aim: This study aimed to investigate the effect of non-synonymous SNPs (nsSNPs) of the Glucagon-like peptide-1 Receptor (GLP-1R) gene in protein function and structure using different computational software. Introduction: The GLP1R gene provides the necessary instruction for the synthesis of the insulin hormones which is needed for glucose catabolism. Polymorphisms in this gene are associated with diabetes. The protein is an important drug target for the treatment of type-2 diabetes and stroke. Material and Methods: Different nsSNPs and protein-related sequences were obtained from NCBI and ExPASY database. Gene associations and interactions were predicted using GeneMANIA software. Deleterious and damaging effects of nsSNPs were analyzed using SIFT, Provean, and Polyphen-2. The association of the nsSNPs with the disease was predicted using SNPs & GO software. Protein stability was investigated using I-Mutant and MUpro software. The structural and functional impact of point mutations was predicted using Project Hope software. Project Hope analyzes the mutations according to their size, charge, hydrophobicity, and conservancy. Results: The GLP1R gene was found to have an association with 20 other different genes. Among the most important ones is the GCG (glucagon) gene which is also a trans membrane protein. Overall 7229 variants were seen, and the missense variants or nsSNPs (146) were selected for further analysis. The total number of nsSNPs obtained in this study was 146. After being subjected to SIFT software (27 Deleterious and 119 Tolerated) were predicted. Analysis with Provean showed that (20 deleterious and 7 neutral). Analysis using Polyphen-2 revealed 17 probably damaging, 2 possibly damaging and 1 benign nsSNPs. Using two additional software SNPs & GO and PHD-SNPs showed that 14 and 17 nsSNPs had a disease effect, respectively. Project Hope software predicts the effect of the 14 nsSNPs on the protein function due to differences in charge, size, hydrophobicity, and conservancy between the wild and mutant types. Conclusion: In this study, the 14 nsSNPs which were highly affected the protein function. This protein is providing the necessary instruction for the synthesis of the insulin hormones which is needed for glucose catabolism. Polymorphisms in this gene are associated with diabetes and also affect the treatment of diabetic patients due to the fact that the protein acts as an important drug target.

Construction and expression of bone morphogenetic protein single chain Fv

objective: To construct the bone morphogenetic protein (BMP) single chain Fv (scFv) and obtain its expression. Methods: Using a synthesized peptide linker containing 15 amino acids, the N end of the heavy chain gene fragment of a strain of murine anti-BMP McAb was connected with the C end of its light chain gene fragment with subcloning. Then the recombinant BMPscFv was cloned into pGEX-4T-1 plasmid and in duced to express in E. coli JM 109. Results: The full length of the recombinant BMPscFv gene was 705 bp and its fusion protein was about 52 kD. Conclusion: Subcloning is a rapid, simple and reliable method for construction of scFv.

Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery

Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide （Aβ, anti-Aβ single-chain Fv, scFv） via adeno-associated virus （AAV） inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5＇ end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer＇s disease. Key Words： Alzheimer＇s disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration

火麻仁黄嘌呤氧化酶抑制肽的酶解制备工艺研究

为寻找开发火麻仁蛋白,提高火麻应用价值的最佳条件,以火麻蛋白为原料,通过酶解法制备火麻仁黄嘌呤氧化酶抑制肽,采用正交试验法优化中性蛋白酶酶解火麻蛋白制备多肽的最佳工艺条件。以中性蛋白酶用量、酶解时间、酶解温度进行单因素试验,通过正交试验分析获得制备火麻仁黄嘌呤氧化酶抑制肽的最佳酶解条件为pH值6.5,料液比1∶5,酶用量6000 U/g,酶解温度50℃,酶解时间5 h。在此条件下进行的验证试验中,多肽抑制率为78.98%,表明该工艺稳定、可行,具有较高的应用价值。

棉籽肽制备工艺的初步研究

本文以水解度和DPPH·清除能力作为评价指标,采用单因素试验和响应面法优化棉籽肽制备工艺。结果表明,最佳工艺条件为酶解温度53.5℃,液料比18,酶解时间4 h。对最佳工艺进行验证,棉籽蛋白的水解度为22.7%,质量浓度为1.23 mg/mL的棉籽肽DPPH·清除能力为85.12%。

应用噬菌体展示随机12肽库筛选诺如病毒抗原模拟表位

目的:利用Ph.D.-12噬菌体展示肽库筛选诺如病毒(NoV)的抗原模拟表位。方法:包被与GⅡ.4、GⅡ.6、GⅡ.17型NoV具有高特异性及中和能力较强的单链可变片段抗体(scFv),用Ph.D.-12噬菌体展示肽库进行3轮生物淘选。ELISA鉴定淘选所得噬菌体与scFv的结合活性及其与NoV P蛋白的竞争作用;阳性克隆测序后进行生物信息学分析,合成多肽鉴定其抗原性。结果:发现1段与GⅡ.6 VP1区同源性较高的氨基酸序列“MG-D-W”,综合分析提示其可能为GⅡ.6 NoV的抗原模拟表位,且合成的包含“MG-D-W”的多肽可竞争抑制P蛋白与人类组织血型抗原(HBGAs)受体的结合。结论:“MG-DW”是与NoV单链抗体高亲和力的肽段,可能模拟了GⅡ.6 NoV与scFv结合的抗原表位。

靶向程序性细胞死亡蛋白配体-1多肽类放射性核素标记分子探针研究进展

程序性细胞死亡蛋白配体-1(PD-L1)是一个重要的免疫检查点分子,在调节机体免疫反应方面发挥重要作用。临床研究表明,肿瘤组织中PD-L1的表达水平与免疫检查点抑制剂疗效密切相关。由于肿瘤的时空异质性,临床上常用的免疫组织化学方法不能全面准确地反映患者体内PD-L1的表达水平,而核医学分子影像技术可在分子水平上无创、实时、动态、可视化监测机体内PD-L1的表达水平。本文主要针对靶向PD-L1多肽类放射性分子探针的研究进行综述,为寻找新型免疫成像的多肽类分子探针及免疫治疗适宜患者的筛选和疗效评估等提供指导。

MTHFR基因A1298C(rs1801131)位点多态性与阿尔茨海默病患者脑脊液生物标志物、认知水平及脑结构变化的关系

目的探讨亚甲基四氢叶酸还原酶(MTHFR)基因A1298C(rs1801131)位点多态性与阿尔茨海默病(Alzheimer’s disease,AD)患者脑脊液β-淀粉样蛋白1-42亚型(Aβ_(1-42))水平、认知水平及脑结构变化的关系。方法选取AD神经影像学倡议计划(ADNI)数据库中符合纳入标准的1271例研究对象,采用多元线性回归模型分析MTHFR基因A1298C(rs1801131)位点多态性与AD患者脑脊液Aβ_(1-42)水平、认知水平及脑结构变化的关系。采用中介分析模型分析脑脊液Aβ_(1-42)水平在MTHFR基因A1298C(rs1801131)位点多态性与认知水平关联中的中介作用。结果多元线性回归模型分析显示,A1298C-C等位基因与AD患者脑脊液Aβ_(1-42)水平、海马体积及认知水平呈负相关(β=-0.121~-0.084,t=-3.308~-1.953,P<0.05),与认知状态、脑室体积呈正相关(β=0.108、0.126,t=3.749、3.653,P<0.05)。中介分析模型显示,脑脊液Aβ_(1-42)水平在A1298C-C等位基因与记忆功能(ADNI_MEM)评分的关系中介导比率为26.0%,脑脊液Aβ_(1-42)水平在A1298C-C等位基因与简易精神状态检查表评分的关系中介导比率为19.9%。结论MTHFR基因A1298C(rs1801131)位点多态性影响AD患者脑脊液生物标志物、认知水平及海马体积和脑室体积,A1298C-C等位基因可能通过降低脑脊液Aβ_(1-42)水平促进AD的发生发展。MTHFR基因靶向修饰可能成为未来高AD遗传风险人群的重要干预靶点。

基于Aerolysin纳米孔道的单个β-淀粉样多肽N-端片段分析

淀粉样蛋白(β-amyloid,Aβ)引起的淀粉样蛋白异常沉积被认为是诱发阿尔茨海默症的因素之一.与人类不同,啮齿动物较少出现这类特征性病变.与人类Aβ相比,啮齿动物Aβ的第5,10和13位氨基酸由Arg,Tyr和His分别变为Gly,Phe和Arg.本文采用分子动力学模拟和Aerolysin纳米孔道单分子分析技术对人类Aβ_(1—15)和啮齿动物Aβ_(1—15)的结构差异进行研究.实验结果表明,与人类Aβ_(1—15)相比,啮齿动物Aβ_(1—15)穿过纳米孔道时具有更低的阻断频率和能垒,证明了Aerolysin纳米孔道可以辨别具有细小结构差异的Aβ多肽分子.本文以硫酸盐K_(2)SO_(4)作为糖胺聚糖(Glycosaminoglycans,GAGs)的简化模型,对Aβ_(1—15)与硫酸根离子的相互作用进行研究.统计分析显示,两种多肽均能与硫酸根离子结合,降低它们被纳米孔道捕获的频率,人类Aβ_(1—15)的捕获频率降低25%,啮齿动物Aβ_(1—15)的捕获频率降低59%.然而,加入硫酸根离子后,两种多肽阻断时间的变化存在明显差异,与未加入硫酸根时相比,人类Aβ_(1—15)的阻断时间延长了14%,啮齿动物Aβ_(1—15)的阻断时间则缩短了7%.由实验结果推测,两种多肽不同的序列和构象导致它们与硫酸根离子结合的方式和强度不同,对过孔行为产生了不同影响.本文研究结果对于筛选用于阿尔茨海默症治疗的小分子抑制类药物具有参考价值.

酶联免疫法测定甘精胰岛素中单链前体残留研究

目的 建立酶联免疫法检测甘精胰岛素中单链前体残留,为甘精胰岛素药物质量控制和安全性评价提供新方法。方法采用双抗夹心酶联免疫法,以甘精胰岛素原为标准品,建立单链前体残留检测方法,验证方法的重复性、准确度、范围和精密度,并应用该方法检测9批甘精胰岛素原料药。结果在0.156~10.00 ng·mL^(-1)浓度范围内,对甘精胰岛素原标准品进行四参数曲线拟合,曲线相关系数R~2大于0.99,方法准确度较高、重复性良好。重复检测3批甘精胰岛素原料药,平均加标回收率在104%~113%范围内,RSD%小于10%,方法精密度良好。9批样品中单链前体残留量均小于1 ng·mg^(-1)。结论建立的酶联免疫法可用于甘精胰岛素及其他重组人胰岛素类似物中单链前体残留量的质量控制和安全性评价。

Self-assembling bacterial pores as components of nanobiosensors for the detection of single peptide molecules

Nano-sized bacterial pores were inserted into a lipid membrane as a nanobiosensor for the detection of single peptide molecules. Due to the intrinsic properties of single-channel conductance, the transit of individual molecules through the pore can be studied. The analysis of both the blockage current and duration is able to provide specific structural information and allows the detection of specific peptides in bulk mixtures.

北极甜虾废弃物多肽钼螯合物的抗氧化性研究

本研究通过单因素实验,探索影响北极甜虾多肽钼螯合物抗氧化性的因素。实验表明,利用北极甜虾虾壳为原料,可以制备多肽钼螯合物。

膨腹海马酶解物制备工艺优化与抗氧化活性

以膨腹海马(Hippocampus abdominalis)为原料,1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为指标,优化海马酶解物提取工艺。根据DPPH自由基清除率,从碱性蛋白酶、风味蛋白酶、胰酶、木瓜蛋白酶、胰蛋白酶、胃蛋白酶中筛选出最优碱性蛋白酶进行工艺优化,在单因素试验基础上,通过Box-Behnken中心复合设计数学模型,采用响应曲面对酶解时间、pH值、酶底物比例(enzyme/substrate,E/S)进行考察,筛选出最佳的酶解工艺,并进行体外抗氧化能力的验证。结果表明,膨腹海马最佳酶解工艺为料液比1∶20、pH 11、E/S 7.581%、酶解温度50℃、酶解时间3 h,在此条件下DPPH自由基清除率能达64.62%。因此,响应曲面优化膨腹海马酶解物的提取工艺能够在较短时间内酶解得到抗氧化活性较高的活性肽。

TMEM106B基因多态性与阿尔茨海默病发病的关系及其机制

目的探讨TMEM106B参与阿尔茨海默病(Alzheimer’s disease,AD)发病的可能机制。方法从阿尔茨海默病神经影像学计划(ADNI)研究队列纳入接受TMEM106B rs1990622基因分型和AD脑脊液(CSF)核心蛋白、神经影像学评估的受试者308名,根据CSFβ淀粉样蛋白42(Aβ_(1-42))水平将其分为AD病理组和非AD病理组。收集患者的年龄、性别、受教育年限、基因型、AD CSF核心蛋白[包括Aβ_(1-42)、总tau蛋白(T-tau)和磷酸化tau蛋白(P-tau)]水平及神经影像学指标[包括海马、内嗅皮质及颞叶内侧MRI结果及氟脱氧葡萄糖正电子发射计算机断层扫描(FDG-PET)标准摄入值(SUV)]。采用卡方检验或t检验比较两组受试者上述指标,并采用多因素线性模型分析TMEM106B rs1990622位点多态性与AD关键表型的关联性。结果两两比较结果显示,AD病理组及非AD病理组受试者的Aβ_(1-42)、T-tau、P-tau水平及载脂蛋白E?4携带状态、FDG-PET SUV、各脑区(海马、内侧颞叶和内嗅皮质)体积比较差异有显著性(t=-6.137~50.792,P<0.05)。多因素线性模型分析结果显示,AD病理组女性受试者的TMEM106B rs1990622位点多态性与CSF Aβ_(1-42)水平存在显著关联(β=58.637,t=2.664,P<0.001)。结论TMEM106B rs1990622位点多态性与CSF Aβ_(1-42)水平存在关联,TMEM106B可能是通过影响CSF Aβ_(1-42)代谢参与AD的疾病过程。

抗弓形虫靶向抗菌肽的构建及其效应的初步评价

目的构建弓形虫主要表面抗原1单链抗体S1与爪蟾抗菌肽(magainin)的融合基因,在大肠杆菌中诱导表达,观察纯化的靶向抗菌蛋白抗弓形虫感染的效果。方法根据弓形虫主要表面抗原1单链抗体S1的编码序列设计引物从噬菌粒S1/pIT-2中扩增到S1基因;以其为模板,加入接头抗菌肽序列,采用重叠PCR法扩增得到S1基因与爪蟾抗菌肽的融合基因(S1M),将其克隆到原核表达载体pET-32c中,构建成S1与爪蟾抗菌肽基因的重组表达质粒S1M/pET-32c;测序验证后转化E.coliBL21,以IPTG诱导表达,用Ni2+螯合柱亲和纯化融合蛋白S1M,SDS-PAGE检测融合基因的表达和纯化的融合蛋白S1M;分别采用体内和体外试验观察纯化的靶向抗菌蛋白抗弓形虫感染效果。结果测序结果表明,成功地将弓形虫主要表面抗原1单链抗体S1与爪蟾抗菌肽的融合基因构建到原核表达载体pET-32c中;SDS-PAGE显示IPTG诱导表达的融合蛋白S1M大小约为43kDa,与预期的大小相一致,以包涵体的形式存在;通过变性条件下Ni2+亲和柱纯化获得S1M重组融合蛋白;体内和体外试验证实,经靶向抗菌蛋白处理过的弓形虫对小鼠的致病能力下降,应用靶向抗菌蛋白的弓形虫感染小鼠的存活时间与对照组相比有明显的提高。结论以抗弓形虫速殖子人源单链抗体作为靶向分子,以爪蟾抗菌肽作为效应分子,构建成功的靶向抗菌蛋白通过体内、外试验证实,具有一定的抗弓形虫感染的作用,虽然还不能完全杀灭弓形虫,但是在弓形虫生物治疗药物的研制方面进行了有益的探索,为弓形虫病的生物治疗提供了新的思路。

双酶法水解对大豆寡肽苦味的影响

大豆寡肽具有多种生理功能,但大豆分离蛋白在酶水解过程中生成的苦味限制了大豆寡肽在食品中的应用.文中利用单一酶Promatex和双酶Promatex/Flavourzyme对大豆分离蛋白进行水解,在相同的水解度和肽链长度下,比较水解方法对大豆寡肽溶液苦味的影响.结果表明,采用双酶Promatex/Flavourzyme分步水解大豆分离蛋白生产大豆寡肽,不但水解液的苦味值大大降低,而且生产周期明显缩短.

抗菌肽CM4组分对K562癌细胞染色质DNA断裂作用的SCGE研究

单细胞凝胶电泳法（ｓｉｎｇｅｃｅｌｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ，ＳＣＧＥ）是一种快速，敏感的检测单个哺乳动物细胞ＤＮＡ断裂的技术，也叫彗星实验（ｃｏｍｅｔａｓｓａｙ）．此实验首次通过ＳＣＧＥ法观察抗菌肽ＣＭ４组分对人髓样白血病Ｋ５６２细胞和正常人白细胞核染色质ＤＮＡ的影响，从而进一步研究抗菌肽抗癌作用的机制．荧光显微镜观察显示经抗菌肽ＣＭ４组分处理过的Ｋ５６２癌细胞核染色质ＤＮＡ出现断裂，形成一个亮的荧光头部和彗星似的尾部，而经同样处理的正常人白细胞和未经抗菌肽处理的Ｋ５６２癌细胞核染色质ＤＮＡ未出现断裂，核完整，呈圆形．经彗星尾长分析，前者ＤＮＡ损伤率平均为７３６２％，统计学处理Ｐ＜０００１，具高度显著性差异．这表明，抗菌肽ＣＭ４对Ｋ５６２癌细胞核染色质ＤＮＡ有明显的断裂作用，而对正常人白细胞则没有断裂作用．

ANP/NPRA基因多态性与心肌梗死后心力衰竭的相关性研究

目的 探讨心肌梗死后患者心钠素（ANP）T2238C及利尿钠肽A型受体（NPRA）G1023C基因多态性与心肌梗死后严重心力衰竭（CHF）发生的关系.方法 记录研究人群的临床特征,测定血浆ANP、脑钠肽（BNP）水平;并采用聚合酶链反应和限制型片断长度多态性（PCR-RFLP）的方法,分析研究人群的ANP T2238C与NPRA G1023C基因多态性.结果 TT ANP 基因型在严重心力衰竭组中分布显著高于对照组（P＜0.05）,经多因素logistic回归分析校正混杂变量后仍显示差异有统计学意义（P＝0.033,OR：1.96,95%CI：1.05~3.27）.对照组中,CC NPRA G1023C基因型患者血浆BNP水平显著低于GG基因型患者.结论 ANP T2238C基因多态性显著影响心肌梗死后患者的心力衰竭病程及其预后,NPRA G1023C基因多态性可能通过调控BNP水平作用于心力衰竭的神经体液过程.