抗重组人巨细胞病毒单克隆抗体的制备与鉴定

为了获得抗人巨细胞病毒(HCMV)单克隆抗体(McAb).采用重组HCMV(rhCMV)gp52蛋白片段免疫Balb/c小鼠,将免疫鼠脾细胞与Sp2/0细胞融合,经ELISA法筛选阳性克隆及测定效价,用免疫印迹法进行特异性鉴定.最终获得4株(3E9,5A6,4G12,2H2)能稳定分泌高效价抗rhCMV McAb的杂交瘤细胞.其培养上清的ELISA效价分别为12048,11024,11024,1512;腹水效价分别为1×10-7,1×10-7,1×10-6,2×10-6.它们分别识别gp52蛋白上的2个不同的抗原表位,2H2识别gp52蛋白上的一个表位,3e9,5A6和4G12识别另一个表位.该单抗可作为快速诊断CMV感染的特异性抗体.     

相思子毒素单克隆抗体的制备纯化及鉴定

目的制备相思子毒素单克隆抗体并鉴定其特性。方法以甲醛处理的相思子毒素毒蛋白为抗原免疫BALB/c小鼠;取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,经间接ELISA法筛选、融合细胞有限稀释法克隆、克隆化杂交瘤细胞株的亚类鉴定等方法筛选出单克隆抗体杂交瘤细胞株,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行单抗的纯化,并对单克隆抗体的特异性进行鉴定。结果获得了4株可稳定分泌单克隆抗体的杂交瘤细胞2D3、4E6、1C8和1E5,诱生的腹水效价分别为1∶1×107、1∶1×106、1∶1×105、1∶1×106,亚类鉴定表明2D3为IgG1,其余3株均为IgG2b;特异性鉴定显示它们与多种毒素均无交叉反应,经过亲和层析,获得了纯化的单抗。结论获得了特异性的相思子毒素单克隆抗体,为建立相思子毒素的检测及纯化方法奠定了基础,其中4E6的效价最高,可作为检测相思子毒素的核心试剂。     

抗delta-like-1单克隆抗体的制备与鉴定

目的 制备抗delta-like-1的单克隆抗体并鉴定其特异性.方法 用delta-like-1多肽-BSA蛋白复合物作为免疫原免疫Balb/c小鼠,取其脾细胞与小鼠骨髓瘤细胞(SP2/0)融合,经多次筛选及克隆化,建立可以稳定分泌抗delta-like-1单抗的杂交瘤细胞株.用ELISA、Western blot、细胞免疫组化对此抗体特性进行鉴定.结果 筛选到两株能稳定分泌抗delta-like-1单抗的细胞株3H11C11A9E3和2D5D11F8F1,亚类鉴定两株均为IgG1,轻链为kappa链;ELISA法测定两株细胞腹水抗体效价分别为1∶4.096×105和1∶1.024×105,抗体亲和常数分别为3.98×107、3.28 × 107L·mol-1;Western blot显示此单抗能特异性识别神经胶质瘤细胞株U251中的天然delta-like-1蛋白;细胞免疫组化进一步显示delta-like-1分布于细胞核中.结论 成功制备了抗delta-like-1单克隆抗体,可为研究delta-like-1与脑胶质瘤等神经系统疾病的关系奠定基础.     

SARS冠状病毒核衣壳蛋白单克隆抗体的制备及鉴定

目的:SARS 冠状病毒(SARS-CoV)核衣壳蛋白 N(nucleocapsid protein)单克隆抗体(monoclonal antibody,McAb)的制备及鉴定。方法:用基因重组的 SARS-CoV N 蛋白免疫 BALB/c 小鼠制备 McAb,并采用间接 ELISA、免疫印迹法进行筛选和鉴定。结果:筛选出2株抗 SARS-CoV N 蛋白 McAb 杂交瘤细胞株,间接 ELISA 证实这组单克隆抗体仅与 SARS-CoV 产生特异性反应,而与其他病原体无交叉反应,IgG 亚类鉴定1株为 IgG1,另1株为 IgG2b。2株抗体亲和常数分别为4.14×10~(-9)和3.19×10~(-9)。结论:获得特异性针对 SARS-CoV 的单克隆抗体,为 SARS-CoV 早期诊断试剂的研制奠定了基础。     

接头蛋白c-Crk 单克隆抗体的制备和鉴定

目的制备接头蛋白c-Crk单克隆抗体。方法以c-Crk为抗原,免疫Balb/c小鼠,用杂交瘤技术建立稳定分泌抗c-CrkmcAb的杂交瘤细胞,用ELISA,western-blot等鉴定其生物活性。结果获得两株能稳定分泌c-CrkmcAb的杂交瘤细胞（3C7、3G11）,亚类均为IgG1;腹水效价为1∶105;western-blot、免疫组织化学分析和ELISA检测证实两株mcAb均与c-Crk有较高的特异性。结论本实验成功制备了两株抗c-Crk特异性的mcAb,可用于c-Crk免疫检测方法的建立。     

抗HBsAg单链抗体特异性单克隆抗体的制备及应用

目的获得抗HBsAg单链抗体(HBScFv)的单克隆抗体,并初步研究其在抗HBsAg类小分子抗体及其融合蛋白质的纯化、活性鉴定方面的应用。方法利用杂交瘤技术,以大肠杆菌表达的重组抗HBScFv为抗原,免疫Balb/c小鼠,再将小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合。通过ELISA法、Western-blot鉴定特异性单克隆抗体,通过免疫双扩散法鉴定单抗亚型,并将筛选到的高效价单抗用于抗HBsAg Fab抗体等重组蛋白质的亲和色谱及ELISA鉴定。结果建立了9株能够稳定分泌抗HBScFv特异性单克隆抗体的细胞株,其细胞上清效价为1∶160~1∶12 800,腹水效价为1∶50 000~1∶400 000;单克隆抗体的亚类分析结果显示有6株为IgG1,其余3株为IgG2a。初步应用结果表明制备的单克隆抗体14F7可用于抗HBsAg Fab抗体等重组蛋白质的亲和色谱及活性鉴定。结论成功建立了能够稳定分泌HBScFv特异性单克隆抗体的细胞株。     

抗哇巴因单克隆抗体的制备及初步鉴定

目的制备抗哇巴因单克隆抗体。方法以半抗原哇巴因与蛋白质载体卵清蛋白(OVA)的耦联物为抗原,免疫BALB/C小鼠,取脾细胞与SP2/0细胞融合,获得两株分泌抗哇巴因的单克隆抗体的细胞株(OUA1,OUA2)。结果两株细胞染色体数目均为100条左右,证实为杂交瘤细胞;所分泌抗体为小鼠IgG1亚类;小鼠腹水效价测定分别为5×106,8×106;和地高辛交叉反应率分别为1.342%和2.323%;ELISA相加试验表明,两株单克隆抗体可能针对同一抗原决定簇。结论成功制备出小分子物质哇巴因的单克隆抗体。     

新型冠状病毒抗原表位融合蛋白的设计制备及免疫特性研究

新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus 2,SARS-CoV-2)感染人体后可引起发热、咳嗽等呼吸道症状,重症者可发生新型冠状病毒肺炎(Coronavirus Disease 2019,COVID-19)、严重急性呼吸综合征、甚至死亡。目前针对新型冠状病毒的感染无特效治疗药物,主要依靠疫苗接种来阻断其传播。在新型冠状病毒的结构蛋白中,刺突蛋白(Spike, S)和核衣壳蛋白(Nucleocapsid, N)是其主要的抗原蛋白,也是制备新型冠状病毒疫苗和研发抗体检测试剂的重要候选蛋白。使用原核表达系统表达重组新型冠状病毒抗原表位融合蛋白,并对其免疫原性进行验证。本研究通过分析筛选出新型冠状病毒结构蛋白S1、N及E蛋白的抗原表位,将筛选出的含强抗原表位的片段串联成融合蛋白,利用基因工程技术构建了表达融合蛋白的工程菌,纯化复性后获得了可溶性的融合蛋白。不同剂量的融合蛋白配伍不同的佐剂后进行小鼠免疫实验,通过检测抗血清的效价及相关细胞因子的水平,评价融合蛋白诱导的体液免疫和细胞免疫效果。设计的融合蛋白以包涵体的形式表达,经透析复...     

整合素阻断剂AP25单克隆抗体的制备与鉴定

目的 制备整合素阻断剂AP25的单克隆抗体,鉴定其效价和特异性.方法 从抗原免疫BALB/c小鼠分离免疫脾细胞,通过杂交瘤技术将免疫脾细胞与小鼠骨髓瘤细胞SP2/0融合,筛选杂交瘤细胞株并进行扩大培养.采用小鼠体内诱生法制备腹水,进行纯化后得到抗AP25单克隆抗体,用ELISA和Western blot鉴定其效价和特异性.结果 ELISA检测抗AP25单克隆抗体效价可达1∶2×106,Western blot鉴定抗体特异性良好.结论 获得了能够高表达抗AP25单克隆抗体的杂交瘤细胞株;腹水制备抗体,纯化后得到效价高、特异性良好的AP25单克隆抗体.     

人心肌肌钙蛋白Ⅰ单克隆抗体的制备与鉴定

为了建立抗人心肌肌钙蛋白Ⅰ(CTnI)单克隆抗体(McAb)的杂交瘤细胞株。以重组表达并纯化后的CTnI免疫BalB/C小鼠,采用传统单克隆抗体技术筛选能稳定分泌抗CTnI McAb的杂交瘤细胞株。结果:建立了3株稳定分泌抗CTnI McAb的杂交瘤细胞株2E3,3D4和3E6,腹水效价分别为4×10-5,1×10-6,1×10-5,亲和常数分别为4.8×108,5.2×109,3.6×108,最低检测浓度分别为20,40,20μg/L,其中2E3和3D4可以识别CTnI的不同表位。     

Affective and Anxiety Disorders and Alcohol and Drug Dependence:

Depression and anxiety frequently coexist in patients with substance use disorders. This clinically-oriented article examiens the relationship between these conditions and emphasizes data showing that substances of abuse can cause signs and symptoms of both depression and anxiety. These substance-related syndromes appear to have a different course and prognosis than uncomplicated, independent anxiety and major depressive disorders, and clinicians should consider the role of alcohol and other drugs in all patients presenting with these complaints. The authors will also outline an approach for diagnosing and managing patients with the combination of a substance use and depressive or anxiety disorder.     

Synthesis and anti-nociceptive and anti-inflammatory effects of gaultherin and its analogs

The synthesis of gaultherin (1) and its analogs was carried out to provide 11 glycosides under phase-transfer catalytic conditions. The activities of all synthesized compounds were evaluated by nitric oxide production inhibitory assay in vitro. Methyl 2-O-(4-O-β-d-galactopyranosyl)-β-d-glucopyranosylbenzoate (5f) showed significantly anti-nociceptive and anti-inflammatory effects by the evaluation in vivo. Structure–activity relationships within these compounds were discussed.     

Modelling and simulation of variability and uncertainty in toxicokinetics and pharmacokinetics

Two important methodological issues within the framework of the variability and uncertainty analysis of toxicokinetic and pharmacokinetic systems are discussed: (i) modelling and simulation of the existing physiologic variability in a population; and (ii) modelling and simulation of variability and uncertainty when there is insufficient or not well defined (e.g. small sample, semiquantitative, qualitative and vague) information available. Physiologically based pharmacokinetic models are especially suited for separating and characterising the physiologic variability from the overall variability and uncertainty in the system. Monte Carlo sampling should draw from multivariate distributions, which reflect all levels of existing dependencies in the intact organism. The population characteristics should be taken into account. A fuzzy simulation approach is proposed to model variability and uncertainty when there is semiquantitative, qualitative and vague information about the model parameters and their statistical distributions cannot be defined reliably.     

成骨细胞的功能与损伤和骨质疏松的防治

骨质疏松是一种全身性骨骼疾病,导致骨折风险增加。成人的骨量通过破骨细胞的骨吸收和成骨细胞的骨形成作用来维持动态平衡,治疗骨质疏松症的理想策略是抑制破骨细胞的骨吸收和/或增强成骨细胞的骨形成功能。目前针对保护成骨细胞及增强其功能的骨质疏松疗法相对较少。因此,本文针对成骨细胞相关功能蛋白、各种细胞损伤机制（内质网应激、氧化应激、机械过载、微小RNA和长链非编码RNA的影响等）及骨质疏松的治疗与预防作一综述,以期为针对增强成骨细胞功能的骨质疏松治疗策略提供新思路。     

Self-stimulation and amphetamine: Tolerance to d and l isomers and cross tolerance to cocaine and methylphenidate

The effects of the d and l isomers of amphetamine on self-stimulation responding were tested following acute and chronic administration. Tolerance and post-drug depression of responding occurred in tests with both isomers, indicating no role for p-hydroxynorephedrine (PHN) which is one of the metabolites of d-amphetamine. In the second experiment, d-amphetamine, methylphenidate and cocaine all produced quantitatively and qualitatively similar effects on self-stimulation responding following acute administration. Following chronic administration of d-amphetamine, animals showed tolerance to all three drugs, indicating cross-tolerance among them. These data are consistent with an hypothesis that tolerance and post-drug depression following chronic amphetamine treatment are the result of decreases in postsynaptic receptor sensitivity, which would lead to a decreased effectiveness of all three drugs, regardless of their pre-synaptic mechanisms.     

益生菌与肿瘤防治

益生菌广泛存在于自然界中,通过维持宿主体内菌群平衡、影响肠屏障功能和调节免疫应答等作用,提高宿主健康水平,被公认为"肠道健康卫士".一些益生菌可以增强机体的免疫功能,抑制致癌物质,影响肿瘤细胞的基因表达,对肿瘤具有拮抗作用.大量研究表明,益生菌在未来的肿瘤防治中有很好的应用和发展前景.     

Acamprosate and naltrexone treatment effects on ethanol and sucrose seeking and intake in ethanol-dependent and nondependent rats

Rationale  Two pharmacotherapies are approved for treating alcohol craving (acamprosate and naltrexone), but both have shown mixed findings in animals and humans. Objectives  The present experiments utilized a “reinforcer blocking” approach (i.e., rats were able to consume ethanol during treatment) to better understand the efficacy of these treatments for ethanol seeking and drinking using ethanol-dependent and nondependent rats. Materials and methods  In “nondependent” experiments, drugs (acamprosate 50, 100, and 200 mg/kg; naltrexone 0.1, 0.3, and 1.0 mg/kg) were administered over 3-week periods prior to operant sessions with a low response requirement to gain access to reinforcers for 20 min. For “dependent” experiments, rats were made dependent in vapor/inhalation chambers. Results  Acamprosate and naltrexone had similar effects on intake in nondependent and dependent rats; neither drug was selective for ethanol over sucrose drinking. In nondependent animals, naltrexone was more efficacious at more doses than acamprosate, and acamprosate’s effects were limited to a dose that also had adverse effects on body weight. Both pharmacotherapies showed more selectivity when examining reinforcer seeking. In nondependent rats, acamprosate and naltrexone had response-attenuating effects in ethanol, but not sucrose, groups. In dependent animals, acamprosate had selective effects limited to a decrease in sucrose seeking. Naltrexone, however, selectively decreased ethanol-seeking in nondependent rats. Conclusions  The naltrexone-induced decreases in seeking suggested a change in incentive motivation which was selective for ethanol in nondependent rats. The “nondependent” paradigm may model early stages of “problem drinking” in humans, and the findings suggest that naltrexone could be a good intervention for this level of alcohol abuse and relapse prevention.     

Antiinfective and encrustation-inhibiting materials--myth and facts

Catheters, urethral and ureteral stents and other urological implants are frequently affected by encrustration and infection due to their permanent contact with urine. Indwelling urinary catheters provide a haven for microorganisms and thus require extensive monitoring. Several surface modification techniques have been proposed to improve the performance of devices including the immobilization of biomolecules, the incorporation of hydrophilic grafts to reduce protein adsorption, the creation of hydrophobic surfaces, the creation of microdomains to regulate cellular and protein adhesion, new polymers and antimicrobial coatings. Physico-chemical explanation to elucidate the mechanism of such encrustation or infection inhibiting materials is still not available. Our series of experiments showed a marked decrease of silver-activity in biological fluids which corresponds with the controversial clinical results obtained with silver coated urinary catheters. Rifampicin/minocycline coated catheters had very low activity against Gram-negative rods, enterococci and Candida spp., the main causing organisms of urinary catheter infection. Surface engineered materials and antimicrobial drug delivery systems will be the next generation of sophisticated urinary catheters and stents, if both efficacy as well as efficiency has been proved clinically.     

Alprazolam and lorazepam single and multiple-dose effects on psychomotor skills and sleep

Summary The effects of alprazolam 0.5 mg and lorazepam 2 mg on cognitive and psychomotor skills were assessed in twelve normal volunteer subjects in a randomised, double-blind, crossover design. Single and multiple dose effects were monitored using a battery of tests comprising critical flicker fusion threshold (CFFT), choice reaction time (CRT), simulated car tracking, and subjective ratings of perceived sedation (LARS) and of sleep behaviour (LSEQ). Compared with placebo baseline scores, treatment with lorazepam 2 mg (both single and multiple doses) resulted in a widespread impairment of CRT, tracking accuracy, and CFFT. Single doses of alprazolam 0.5 mg reduced CFFT with respect to the placebo baseline. Single and multiple dose treatment with both drugs resulted in subjective reports of sedation, a reduction of sleep onset latency, and improved sleep quality. Only lorazepam 2 mg significantly disrupted the integrity of behaviour on waking from sleep. These results suggest important pharmacodynamic differences between the two drugs in the doses used.