新型HIV-1 gp41融合抑制剂CP32M的构效关系研究

目的研究新型融合多肽CP32M中VEWNEMT序列的长短、残基的极性及其他部位残基的极性对其抑制gp41融合活性的影响。方法在多肽CP32M的基础上,通过对前7个氨基酸序列进行截取、部分替换、全部替换及改变其他位置氨基酸残基的极性设计合成系列多肽,测定多肽抑制HIV-1融合活性。应用圆二色谱、分子排阻色谱测定多肽与N36的结合功能。结果截取、替换氨基酸后的肽抑制gp41融合的活性都不同程度降低,与N36结合形成六束螺旋的能力减弱。在CP32M中部及C端i与i+4位置引入Lys增加Glu-Lys离子对作用后,肽活性降低。用C34的C端氨基酸序列NEKDLLE及可与gp120结合的序列RINNIPWSEAM置换QIWNNMT后,多肽仍有很高活性。结论片段VEWNEMT是关键片段,其中VE及MT是关键功能氨基酸。其他片段替代该片段后仍有较强活性,也与N36形成螺旋结构,提示该片段含有共性结合区。     

HIV-1gp41NHR序列靶点专一性的荧光共振能转移测定

目的确定并利用荧光共振能转移法(FRET)对以HIV-1 gp41 N端七联重复序列(NHR)为靶点的融合抑制剂进行筛选和作用机制研究。方法 FRET采用金属络合物多肽技术设计针对不同结合位点、结合强度可调、涵盖全部NHR序列的靶点和探针,对HIV融合抑制剂进行高通量筛选。由于HIV在进行膜融合时其gp41的N端NHR和C端CHR可形成稳定的六螺旋结构,因此,利用圆二色谱仪对FRET所使用的靶点/探针对的结合强度进行验证,确定对应的靶点/探针对可形成稳定的六螺旋结构;同时,借助细胞活性测试测定抑制剂的活性,验证FRET是否可用于筛选以HIV-1 gp41 NHR为靶点的抑制剂。结果与结论 FRET中使用的靶点/探针均可形成螺旋度较高的六螺旋结构,其中Fe(Env2.0)3/CP2及Fe(Env5.0)3/CP5形成的α螺旋度分别高达89.6%和84.7%。FRET所使用的靶点/探针对专一性强、结合作用强,可用于进行HIV-1融合抑制剂的筛选和机制研究。     

尖吻蝮蛇去整合素基因克隆与功能分析

目的：获得尖吻蝮蛇去整合素基因，并分析重组蛋白生物学活性。方法：提取尖吻蝮蛇毒腺组织总RNA．经RT—PCR获得去整合素基因片段。将该去整合素基因片段重组入pMAL-p2x栽体中诱导表达，以直链淀粉树脂柱亲和纯化，并检测融合蛋白的抑制血小板聚集活性。结果：谊片段编码的氨基酸序列中含有去整合素保守的RGD三肽序列及12个Cys残基，与GenBank中其他蛇毒去整合素氨基酸序列最高同源性为86％，重组融合蛋白具有抑制ADP诱导的血小板聚集活性。结论：发现了一个新的去整合素基因，谊基因编码蛋白具有抑制血小板聚集的功能。     

抗心肌缺血损伤活性多肽的分离纯化与鉴定

目的：分离纯化心肌肽混合溶液中具有心肌损伤保护活性的单一有效成分。方法：采用体外活性测定追踪、反相色谱分离结合生物质谱检测技术对心肌肽溶液中的活性成分进行纯化及结构鉴定。结果：从心肌肽混合溶液中分离出一活性多肽并确定了其氨基酸序列为KGAW SNVLRGMGGAF;人工合成上述肽段,体外多柔比星（阿霉素）损伤心肌细胞活性测定结果表明,此多肽能明显抑制阿霉素对心肌细胞的损伤。结论：得到一种新的具有心肌细胞保护活性的多肽。     

活性肽及其在运动中的应用

1　前言近年来 ,功能活性肽的研究已成为国际生命科学研究的热点。 2 0世纪 90年代开始 ,寡肽及其相关营养品的开发研究备受重视。所谓的肽 (Ｐｅｐｔｉｄｅ)即是蛋白质的结构片段 ,是氨基酸的有机合成物 ,也是蛋白质的水解产物 (ＰｒｏｔｅｉｎＨｙｄｒｏｌｙｓａｔｅ)。两个氨基酸通过肽键 (-ＣＯ -ＮＨ - )连接成二肽 ,三个氨基酸通过肽键连接成三肽 ,而多个氨基酸通过肽键连接成的肽叫多肽。一般把 2～ 10个氨基酸残基组成的肽叫寡肽 (Ｏｌｉｇｏｐｅｐｔｉｄｅ ,ＯＰ)。因为肽的分子相对于蛋白质分子小得多 ,但生物活性非常高 ,少量的…     

肝癌特异性结合肽的筛选及鉴定

目的探讨利用噬菌体随机肽库筛选肝癌细胞特异性结合多肽或肝癌细胞优势结合多肽的可行性。方法①利用噬菌体随机十二肽库，对人肝癌细胞系HepG2和人正常肝细胞系L02进行4轮差异筛选、富集，获得与肝癌细胞亲和的噬菌体克隆。②用酶联免疫吸附测定(ELISA)法测定其与肝癌细胞、正常肝细胞的亲和力，进一步筛选出高度亲和肝癌细胞的特异性阳性噬菌体克隆。比较其与其他肿瘤细胞的亲和力。③提取阳性噬菌体DNA并进行DNA序列测定，获得与肝癌细胞亲和的多肽序列。④用免疫荧光共聚焦成像进一步确定展示阳性多肽序列的噬菌体与肝癌细胞的结合部位。⑤根据筛选的多肽序列合成十六肽WH16，利用竞争抑制实验鉴定其与肝癌细胞的结合。结果筛选及ELISA结果示得到与肝癌细胞特异性结合并内化入肝癌细胞的噬菌体克隆，测序结果示56．67％的被检噬菌体展示相同的多肽序列FLLEPHLMDTSM，推测FLEP是肝癌细胞特异性结合肽的基序。免疫荧光共聚焦成像进一步确定展示序列FLLEPHLMDTSM的噬菌体与肝癌细胞特异性结合并内化入细胞内。竞争抑制实验示WH16能有效抑制展示序列FLLEPHLMDTSM的噬菌体克隆与肝癌细胞的结合。结论利用噬菌体随机肽库获得能与肝癌细胞特异性结合的多肽序列FLLEPHLMDTSM，据此合成的十六肽WH16与肝癌细胞有亲和力。     

人禽流感HSN1病毒HA1蛋白抗原表位及主要免疫功能区特征的研究

目的 确定人禽流感H5N1病毒HA1蛋白的单克隆抗体(McAb)抗识别表位及主要免疫功能区.方法 将16条人禽流感H5N1病毒HA1基因重叠片段克隆入真核表达载体pDisplay,构建表达HA1蛋白的重叠肽段重组质粒,转染HeLa细胞后制备细胞抗原片.利用灭活的H5N1病毒免疫BALB/C小鼠,制备病毒特异单克隆抗体(单抗)和多克隆抗体(多抗),采用间接免疫荧光法分析单抗、多抗与HA1肽段的反应性.确定HA1蛋白的单抗识别表位及主要免疫功能区.结果 成功克隆、表达了16条不同长度的H5N1肽段,并制备、获得30株分泌抗H5N1型禽流感病毒McAb的杂交瘤细胞株,其中18株为抗HA的单抗,18株中有6株单抗具有中和病毒活性.利用表达的H5N1肽段对单抗识别表位进行分析,初步确定3B5、3D1、3132、M6、M3等18株McAb识别表位区域分别位于氨基酸残基aa133-143、aa155-165、aa166-196、aa166-176、aa34-66及aa296-328之间,其中3B5、3D1、3B2、M6等McAb识别表位为序列依赖型表位,M3和M7等具有中和活性的McAb识别表位为构象性中和表位.结论 利用制备的H5N1病毒特异性McAb和表达的病毒HA1蛋白重叠肽抗原,初步确定了18株抗HA McAb的识别表位和HA1蛋白的主要免疫功能区,为进一步研究开发新型疫苗提供了重要理论依据.     

新的M家族芋螺毒素的克隆及进化分析

目的从中国南海芋螺中寻找新的M家族芋螺毒素。方法应用3′RACE及巢式PCR进行基因克隆,将得到的目的基因连接入pGM-T载体、转化并测序,用进化分析软件对得到的新序列进行进化分析。结果从6种芋螺毒腺管中获得8个新的M家族芋螺毒素前体肽序列,其成熟肽含有15~21个氨基酸残基。结论这些新的M族毒素与已报道的M家族毒素序列同源性较低,是Mini-M家族的新成员。     

抗HIV融合多肽CP32M的质量分析

目的建立一种适用于分析抗HIV融合多肽CP32M含量及有关物质的方法,以及测定其水分和三氟乙酸（TFA）含量的方法。方法采用高效液相色谱法（HPLC）测定CP32M原料药中CP32M的含量。色谱条件：Zor-bax XDB-C18色谱柱,含0.1%TFA的水为流动相A,含0.1%TFA的乙腈为流动相B,梯度洗脱,流速1 ml/min,检测波长214 nm,柱温25℃。采用卡尔菲休库仑滴定法测定水分含量,采用高效液相色谱法测定样品中TFA含量。结果 3批CP32M原料药中,CP32M含量分别为（98.72±0.32）%,（99.48±0.52）%和（99.51±0.31）%（按无水物计算）,总有关物质含量为1.07%～1.18%,样品中水分含量为2.99%～3.14%,TFA含量为3.89%～4.23%。结论该方法快速简便,结果准确可靠,可用于CP32M的质量分析。     

B型利钠肽及N末端B型利钠肽原在严重创伤中的变化及其意义

B型利钠肽(Btype natriuretic peptide,BNP)主要由心肌细胞在心室舒张末期容量或压力负荷增高时分泌.通过增加肾小球滤过率、抑制肾素-血管紧张素-醛固酮轴、抑制交感神经活性、扩张血管等一系列调节,BNP能降低心脏过高的前负荷及后负荷[1].BNP的前体为B型利钠肽原(pro-B-type natriuretic peptide,proBNP),proBNP经加工分裂成两个片段,其中C端一个片段为BNP,N端片段无活性,称为N末端B型利钠肽原(N-terminal pro -B - type na-triuretic peptide,NT-proBNP).二者同源且等比例产生,具有相同的检测意义,都可以反映心肌细胞受到的容量负荷或压力负荷的大小[2].     

Characterization of 2-hydroxyadenine DNA glycosylase activity of Escherichia coli MutY protein

Purpose : 2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants. 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells. Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro. On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood. Materials and methods : Gel shift assays were used to assess the binding activity of E. coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides. Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined. Results : The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides. MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs. M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides. Conclusions : E. coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA. The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides.     

Characterization of 2-hydroxyadenine DNA glycosylase activity of Escherichia coli MutY protein

PURPOSE: 2-Hydroxyadenine (2-ohA) is an oxidation product of adenine generated in DNA by ionizing radiation and various chemical oxidants. 2-ohA has mutational potential comparable to that of 8-oxoguanine in bacteria and mammalian cells. Recent studies have shown that 2-ohA is removed from DNA by a human MutY homolog, MYH protein, in vitro. On the other hand, the repair mechanisms for 2-ohA in Escherichia coli are not yet understood. MATERIALS AND METHODS: Gel shift assays were used to assess the binding activity of E. coli full-length MutY protein and its N-terminal (residues 1-226) domain (M25) to 2-ohA/G-, 2-ohA/A-, 2-ohA/C- and 2-ohA/T-containing 24-mer oligonucleotides. Furthermore, whether these proteins specifically cleave 2-ohA-containing duplex oligonucleotides was examined. RESULTS: The purified MutY and M25 proteins had similar binding affinities to 2-ohA/G-, 2-ohA/A- and 2-ohA/C-containing oligonucleotides. MutY protein removed 2-ohA preferentially from 2-ohA/G mispairs. M25 protein showed the reduced catalytic activity for 2-ohA/G-containing oligonucleotides. CONCLUSIONS: E. coli MutY protein has a DNA glycosylase activity that removes 2-ohA from 2-ohA/G mispairs in DNA. The C-terminal domain is required for the removal of 2-ohA from DNA, but is not crucial for binding to 2-ohA-containing oligonucleotides.     

两种非核苷类逆转录酶抑制剂与已上市药物联合应用的体外抗HIV活性研究

目的：评价两种非核苷类逆转录酶抑制剂（JB25、JB26）与3种已上市药物齐多夫定（AZT）、依非韦仑（EFV）、沙奎那韦（SQV）联合应用的体外抗HIV活性。方法：将10个浓度的JB25、JB26分别与AZT、EFV、SQV的7个浓度组成各种浓度的组合,加入384孔细胞培养板,与HIV-1ⅢB急性感染的MT-2细胞共培养3d,最后利用TZMbl细胞报告基因检测HIV-1的表达,共重复3次。利用MacSynergyⅡ软件对数据进行分析,得到药物相互作用的情况。结果：JB25与AZT、EFV、SQV的平均协同/拮抗容量分别是244.45/-5.05,119.58/-65.93,145.83/-0.32（nmol/L）2%;JB26与AZT、EFV、SQV的平均协同/拮抗容量分别是398.90/0,103.62/-0.49,138.473/-0.27（nmol/L）2%。结论：两种非核苷类逆转录酶抑制剂与3种已上市的药物在体外联合应用,具有协同抗HIV作用,MacSynergyⅡ软件可以全面评价两种药物的联合作用情况。     

细胞电穿孔联合低浓度顺铂作用卵巢癌SKOV3细胞体外生长实验研究

目的 探讨细胞电穿孔联合低浓度顺铂对卵巢癌SKOV3细胞株体外生长作用的影响.方法 选用电场600 V/cm,脉冲个数5个,电容11μF作用于SKOV3细胞株.根据作用不同分为电穿孔＋药物（E＋M）组;电穿孔（E）组,药物（M）组和空白（C）组.每3 d一次观察处理后的生长细胞共24 d.描绘细胞生长曲线,观察各组细胞生长变化.结果 不同方式处理后细胞生长情况表明电穿孔＋药物组对SKOV3细胞的体外生长有良好抑制作用,差异显著（P〈0.01）;单纯电穿孔组对肿瘤细胞的抑制大于单纯药物组,低于电穿孔＋药物组;单纯药物组的低浓度顺铂对卵巢癌SKOV3细胞仅有微弱生长抑制.结论 以电场600 V/cm,脉冲个数5个,电容11μF作为细胞电穿孔参数并同时联合低浓度顺铂作用卵巢癌SKOV3细胞株时,有明显加强非细胞抑制剂量顺铂对体外SKOV3细胞生长的抑制作用.     

Homology between ABH-carrier alpha2-seminoglycoprotein and Mac-2 binding protein

alpha2-seminoglycoprotein (alpha2-SGP), purified from human seminal plasma, is a carrier of glycoprotein for the ABO blood grouping. The alpha2-SGP exists in the secretions of the seminal vesicle and various glands. However, the function of alpha2-SGP is, as yet, unknown. In this study, we determined that two internal amino acid sequences of 8 and 12 residues of alpha2-SGP were Ala-Val-Asp-Thr-Trp-Ser-Trp-Gly and Thr-Leu-Gln-Ala-Leu-Glu-Phe-His-Thr-Val-Pro-Phe. These sequences were completely coincident with the domain 3 of human Mac-2 binding protein (M2BP), which was identified as a tumor-associated antigen. In addition, we also confirmed an alpha2-SGP binding activity to galectin-3 that was one of a ligand for M2BP, and the immunological cross-reactivity between alpha2-SGP and M2BP. These findings strongly suggested that alpha2-SGP was identical with M2BP.     

Evaluation of [18F]fluoroxanomeline {5-{4-[(6-[18F]fluorohexyl)oxy]-1,2,5-thiadiazol-3-yl}-1-methyl-1,2,3,6-tetrahydropyridine} in muscarinic knockout mice

INTRODUCTION: We set out to develop a muscarinic M1-selective agonist (based on the structure of the functionally M1-selective xanomeline) that could be radiolabeled with fluorine-18 for use as an imaging agent for positron emission tomography. METHODS: The radiochemical synthesis was achieved, employing the arts of organic and radiochemical syntheses. Binding selectivity studies employed biodistribution studies, using autoradiography and/or tissue dissection, in wild-type or muscarinic receptor knockout mice. RESULTS: [(18)F]Fluoroxanomeline shows rather uniform uptake in all mouse brain regions and high specific binding, with a brain-to-blood ratio of 32 at 60 min postinjection. In addition, the specific binding is demonstrated by a 58% to 75% decrease in brain uptake upon coinjection with 5 nmol of unlabeled fluoroxanomeline or xanomeline. Brain uptake studies with [(3)H]xanomeline in muscarinic knockout mice show decreased uptake in M1 (17-34%) and M2 (2-20%) knockout mice compared with control. However, statistical significance was observed in only a few regions. Comparison of [(18)F]fluoroxanomeline in knockout mice showed no difference in M1 or M4 knockout mice but a general decrease in M2 (2-24%) knockout mice. The decrease of [(18)F]fluoroxanomeline uptake in M2 knockout mice reached statistical significance in brain stem, cerebellum, frontal cortex, hippocampus, inferior colliculus and superior colliculus. CONCLUSION: Although xanomeline displays highly selective M1 agonist activity in functional assays, little selectivity for muscarinic subtype binding was observed for xanomeline or its fluorine-containing analogue, fluoroxanomeline. This emphasizes the lack of correlation between functional selectivity and binding selectivity.     

新型光敏剂4SB的光谱特性及与血清蛋白相互作用的研究

目的研究新型光敏剂17,4-位氨基磺酸竹红菌乙素衍生物（17-4-amino-1-butane-sulfonic acid-hypocrellin B,4SB）的光谱特性及其与人血清白蛋白（human serum albumin,HSA）的相互作用规律,为4SB临床和基础研究提供实验依据。方法使用分光光度计和荧光光谱仪测量4SB在PBS和HSA溶液中的吸收光谱和荧光光谱,并与临床使用的光敏剂血卟啉衍生物（hematoporphyrin derivative,HpD）作对比,研究HSA对4SB光谱的影响。将4SB溶于HSA溶液后持续震荡,不同的时间点测定4SB吸收光谱和荧光光谱,观察4SB与HSA作用的时间规律。观察HSA对4SB吸收和荧光光谱的影响：将浓度为0、1.25、2.5、5.0、10和20μM/ml的HSA加入5μM/ml的4SB中,测定吸收光谱和荧光光谱。HSA与4SB相互的荧光淬灭：将不同浓度的4SB加入HSA中,测定4SB的荧光光谱;将不同浓度的4SB加入HSA中,测定HSA的荧光光谱。结果 4SB在600 nm以上的波段较HpD的吸收有很大提高,在人血清溶液体系中此峰红移且增强了2.3倍。4SB与HSA的结合时间是30 min。HSA与4SB是1对1的特异性结合,且位点在色氨酸残基附近。结论 4SB在红光波段吸收好,与白蛋白结合后增强了对光的吸收,与白蛋白1∶1结合,是一种有开发前景的光敏剂。     

CXCR4 HIV-1抑制剂的设计合成及活性研究

目的设计、合成一系列新型四氢喹啉-苄基或苯并咪唑多胺类化合物,作为潜在的靶向CXCR4辅助受体的新型抑制剂,并测定其抗HIV-1活性。方法组合HIV-1辅助受体CXCR4抑制剂三氮构效团与CCR5部分药效团,设计并合成一系列新化合物并经1H-NMR、MS表征,用HIV-1ⅢB病毒测定化合物抑制活性。结果与结论设计合成的10个目标化合物未见文献报道。活性测试结果显示,四氢喹啉-苯并咪唑多胺类化合物具有较好的抗HIV活性（IC50〈1μmol/L）,四氢喹啉-苄基多胺类化合物活性较差（IC50〉8μmol/L）。     

Preparation and preliminary evaluation of technetium-99m-labeled fragment E1 for thrombus imaging.

Fragment E1 labeled with 123I has been previously shown to permit imaging of thrombi in patients within as little as 20 min after injection. Because of the relatively rapid localization and blood disappearance of this protein, 99mTc would be the most clinically acceptable radionuclide for labeling Fragment E1. In this study, human fragment E1 was derivatized with a hydrazino nicotinate function to permit radiolabeling with reduced technetium. The modification reaction was carried out while the fragment E1 was protected in a complex, so that the modification occurred in nonfunctional regions of the fragment E1 molecule. After radiolabeling with 99mTc, the modified fragment E1 retained its functional activity, as judged by its binding to fragment DD in vitro. The ability of 99mTc-fragment E1 to produce images of venous thrombi was demonstrated in animal models. Images were focally positive within 20 min to 1 hr after injection. Thrombus-to-blood ratios exceeded those from 125I-fibrinogen in the same animals. This method of labeling appears to provide an alternative radiolabel to 123I without compromising the function of fragment E1.