人突触相关蛋白抗原表位分析及多克隆抗体的制备

目的　泡沫化细胞差异表达基因FRG4经生物信息学分析 ,与人突触相关蛋白基因有很高同源性 ,经联机检索 ,有关突触相关蛋白的研究很少 ,与脂蛋白代谢、泡沫细胞形成和凋亡的关系没有报道。本文在对泡沫化相关基因FRG4分析的基础上 ,利用计算机软件进行抗原表位分析 ,制备特异的抗人突触相关蛋白的多克隆抗体 ,为进一步深入研究其表达和功能提供条件。方法与结果　①选取抗原多肽 :利用ExPASy软件和BioEdit软件对FRG4cDNA序列进行亲水性分析 ,预测其二级结构 ;使用Lasergene99软件预测其抗原决定族 ;根据亲水性、二级结构、偶联难易及实验难度选取抗原 13肽PKLVKEEVFWRNY。②多肽合成及交联 :采用美国ABI公司的多肽自动合成仪 ,以固相合成法合成 ,合成产物经高效液相色谱纯化检测 ,纯度达到 90 %以上 ;抗原多肽与血兰蛋白用BDB法交联 ,与不完全弗氏佐剂等体积混合包被。③动物免疫 :多点皮下注射抗原免疫新西兰兔 ,每两周强化免疫一次 ,第三次免疫后 10天取耳血测抗体效价 (ELISA法 )。④抗血清收获及检测 :第四次免疫后第十至十四天经颈动脉放血 ,分离抗血清 ,冷冻抽干 ,- 2 0℃保存。高效液相色谱检测显示抗体纯度达 82 .79% ,抗体滴度为 1∶16 0 0 0 ,免疫印迹方法检测在 4 0kDa处有一条带 ,与软件分析     

抗弓形虫水通道蛋白多肽抗体的制备及鉴定

目的 研制抗弓形虫水通道蛋白(TgAQP)的特异性多肽抗体,并应用于检测弓形虫ME49株中水通道蛋白的表达和亚细胞定位。方法 根据TgAQP氨基酸序列设计B细胞抗原多肽,并将合成的多肽与KLH偶联作为免疫原免疫新西兰大耳白兔制备多克隆抗体,通过ELISA、Western blot和免疫荧光的方法对所得抗体进行鉴定。结果 选定并合成抗原表位多肽,与KLH偶联作为免疫原制备多克隆抗体;经ELISA测定其多肽抗体效价达1∶40 000; Western blot表明制备的抗体能特异地识别天然弓形虫中29.9 kDa处的条带,与预测的TgAQP相对分子量大小相符;免疫荧光检测到抗血清识别的蛋白定位于弓形虫胞质中。结论 制备了抗弓形虫水通道蛋白多肽的多克隆抗体,为进一步研究弓形虫水通道蛋白的生物学功能和代谢特点奠定了基础。     

细粒棘球绦虫水通道蛋白9的多肽特征及免疫定位研究

目的 研制细粒棘球绦虫水通道蛋白9 (EgAQP9)的特异性多肽抗体,并用于检测其在棘球蚴囊壁、生发细胞及原头蚴中的组织分布情况。 方法 根据EgAQP9特异氨基酸序列设计合成B细胞抗原多肽,再用合成的多肽偶联KLH作为免疫原注射免疫新西兰兔以制备多克隆抗体。酶联免疫吸附试验(ELISA)方法检测多肽抗体滴度,蛋白免疫印迹法(Western blot)测定多肽抗体免疫活性,免疫荧光实验分析EgAQP9的亚细胞定位,免疫组化实验分析EgAQP9组织定位。 结果 免疫新西兰兔成功制备出多肽抗体;间接ELISA检测其多肽抗体效价高达1∶256 000;Western blot结果显示多肽抗体能够特异识别细粒棘球绦虫中36 kDa处的条带,与EgAQP9预测的相对分子量大小相符;免疫荧光实验结果显示EgAQP9分布于棘球蚴生发细胞的胞质、胞膜中;免疫组化实验显示该蛋白主要分布在原头蚴表面及棘球蚴囊泡生发层。结论 本研究以制备的EgAQP9兔源多克隆抗体定位了AQP9在棘球蚴生发细胞、原头蚴及棘球蚴囊壁中的分布状况。     

应用合成多肽技术制备肺癌转移相关蛋白1多克隆抗体的研究

目的应用人工合成多肽技术制备肺癌转移相关蛋白1(Lung Cancer Metastasis-Related Protein1,LCMR1)的多克隆抗体。方法运用生物信息学技术和人工合成多肽方法制备LCMR1特异性多肽,鉴定纯度后与牛血清白蛋白、匙孔血蓝蛋白进行偶联制备多肽抗原,然后免疫新西兰雄性白兔,获得抗血清,经亲和层析法纯化后,用ELISA法鉴定抗体效价,用Western Blot鉴定特异性。结果成功获得了LCMR1的多克隆抗体,ELISA检测抗体效价大于1:100 000,Western Blot免疫印迹结果显示其具有抗原特异性识别。结论应用免疫原性肽抗体技术成功制备了LCMR1的多克隆抗体,为今后深入了解LCMR1基因的生物学功能提供了有用的研究工具。     

血管紧张素Ⅱ相关新基因BC097361融合蛋白的表达、纯化及多克隆抗体的制备

目的 构建血管紧张素Ⅱ相关新基因BC097361的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗BC097361蛋白多克隆抗体并进行鉴定.方法 应用RT-PCR技术,以LX02细胞总RNA为模板,扩增BC097361目的 基因片段,构建原核表达载体pET-32a(+)-BC097361.转化大肠埃希菌BL21(DE3),异丙基-β-半乳糖苷诱导并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析,Western blot分析证实融合蛋白表达的特异性.大量表达后利用Ni+亲和柱对表达蛋白进行纯化及柱上复性.纯化蛋白免疫新西兰兔,获得抗BC097361蛋白的多克隆抗体.以纯化的BC097361蛋白为抗原,分别以免疫前后的新西兰兔血清作为第一抗体,利用Western blot和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测.结果 扩增获得BC097361基因片段,成功表达了BC097361相关蛋白,经十二烷基硫酸钠一聚丙烯酰胺凝胶电泳和Western blot分析得到证实.成功获得融合蛋白及兔抗BC097361多克隆抗体.酶联免疫吸附法检测证实多克隆抗体效价>1:320 000,Western blot、免疫组织化学检测证明多克隆抗体的特异性良好.结论 利用大肠埃希菌BL21(DE3)能够成功表达BC097361蛋白,获得高特异性、高效价兔抗BC097361蛋白的多克隆抗体,为今后研究BC097361蛋白的生物学特性奠定了基础.     

不同牛PrP合成肽段的抗原性研究

目的研究PrP多肽的抗原性以及不同偶联方法对多肽免疫原性的影响。方法选择4段牛PrP多肽(BoP1、BoP2、BoP3、BoP4)为半抗原,KLH为载体,MBS、戊二醛两种偶联方法进行偶联,免疫BALB/c小鼠,间接ELISA方法检测小鼠血清中抗体效价,并对两种偶联方法进行比较。结果两组动物均出现死亡,戊二醛偶联多肽免疫小鼠出现不同程度的腹水,MBS偶联多肽免疫小鼠未见其它异常反应;戊二醛偶联的4种多肽产生的抗体效价无显著性差异(P>0.05),MBS偶联的KLH-BoP1、KLH-BoP2、KLH-BoP4产生抗体效价无显著性差异(P>0.05),MBS偶联的KLH-BoP3抗体效价低于其它3种多肽(P<0.05);对同一种多肽而言,不同偶联方法KLH-BoP1、KLH-BoP2、KLH-BoP4所产生抗体效价无显著性差异(P>0.05),戊二醛偶联KLH-BoP3产生的抗体效价明显高于MBS偶联KLH-BoP3产生的抗体效价(P<0.05)。结论PrP多肽可作为半抗原刺激小鼠强烈的免疫反应,不同偶联方法,不同肽段抗原性不同,为抗PrP多克隆及单克隆抗体的制备奠定了基础。     

斑马鱼抗podocin抗体的制备、鉴定及在足细胞损伤模型中的应用

目的:利用合成的podocin多肽制备针对斑马鱼podocin的多克隆抗体,鉴定其特异性,并应用于斑马鱼足细胞损伤研究中。方法:(1)设计、合成斑马鱼podocin抗原多肽,将合成后的多肽与钥孔血蓝蛋白(KLH)偶联,免疫新西兰大白兔制备抗血清,protein A亲和纯化得到抗podocin多克隆抗体;(2)ELISA和免疫组化检测抗体效价及组织特异性;(3)构建足细胞特异性表达硝基还原酶(NTR)的转基因斑马鱼Tg(pod:Gal4;UAS:NTR-m Cherry),NTR/MTZ(甲硝唑)系统特异性地诱导足细胞损伤;用podocin反义寡核苷酸阻断正常斑马鱼胚胎podocin mRNA的翻译过程,利用抗podocin抗体对上述两种疾病模型进行免疫荧光染色以验证合成的podocin抗体能否应用于斑马鱼足细胞研究中。结果:(1)化学合成的多肽纯度为92.6%,达到免疫用抗原标准;(2)ELISA和免疫组化染色结果表明抗体效价分别达1∶5.12×105和1∶106;(3)免疫荧光染色表明无论在斑马鱼前肾还是中肾,podocin均特异性地表达于足细胞,且沿着毛细血管袢呈连续、线性分布,表明抗体具有良好的组织特异性;(4)MTZ诱导足细胞损伤24h后见podocin呈粗糙的颗粒状分布,48h后阳性表达面积占整个肾小球面积比值明显下降;(5)显微注射podocin反义寡核苷酸的斑马鱼胚胎出现心包水肿、体轴弯曲的表型,第3天时几乎无Podocin表达,随后逐渐恢复至正常,第5天时表达与正常组无异。结论:所制备的斑马鱼抗podocin抗体效价高、组织特异性强,能够反应足细胞损伤情况,是斑马鱼足细胞研究的有力工具。     

兔抗阴道毛滴虫重组蛋白AP65多克隆抗体的制备、纯化及鉴定

目的制备、纯化并鉴定兔抗阴道毛滴虫重组蛋白AP65的多克隆抗体。方法日本大耳兔多次免疫重组蛋白AP65．间接ELISA法检测抗体效价，用饱和硫酸铵法和nprotein Asepharose4FF柱纯化多克隆抗体，Western blot检测免疫反应性。结果第3次加强免疫后，间接ELISA法检测血清效价达到1：25600，用硫酸铵法纯化多克隆抗体纯度为56％，而后采用亲合层析柱nprotein Asepharose 4FF再次纯化。纯度达到80％。Western blot鉴定兔抗重组蛋白AP65多克隆抗体能识别重组蛋白AP65。结论成功制备了高效价、高纯度的抗阴道毛滴虫重组蛋白AP65多克隆抗体。     

抗淋病奈瑟菌外膜蛋白Ⅰ多克隆抗体的制备及其ELISA双抗夹心法的建立

目的制备抗淋病奈瑟菌外膜蛋白Ⅰ多克隆抗体,建立检测外膜蛋白Ⅰ的双抗夹心法.方法以淋病奈瑟菌及其外膜蛋白Ⅰ作为抗原,分别免疫健康雄性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白Ⅰ多克隆抗体,并采用ELISA法检测抗体效价.结果与讨论经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白Ⅰ呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白Ⅰ可获满意结果.     

抗淋病奈瑟菌外膜蛋白I多克隆抗体的制备及其ELISA双抗夹心法的建立

目的 制备抗淋病奈瑟菌外膜蛋白I多克隆抗体,建立检测外膜蛋白I的双抗夹心法。方法 以淋病奈瑟菌及其外膜蛋白I作为抗原,分别免疫健康性家兔及雌性BALB/C小鼠,制备兔抗淋病奈瑟菌和鼠抗外膜蛋白I多克隆抗体,并采用ELISA法检测抗体效价。结果与讨论 经ELISA证明兔抗淋病奈瑟菌多抗可与淋病奈瑟菌外膜蛋白I呈高效价反应,以此建立的ELISA双抗夹心法检测外膜蛋白I可获满意效果。     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

N-acetyl-L-cysteine combined with mesalamine in the treatment of ulcerative colitis： Randomized,placebo-controlled pilot study

AIM： To evaluate the effectiveness and safety of oral N-acetyl-L-cysteine （NAC） co-administration with mesalamine in ulcerative colitis （UC） patients.
METHODS： Thirty seven patients with mild to moderate UC were randomized to receive a four-wk course of oral mesalamine （2.4 g/d） plus N-acetyl-L-cysteine （0.8 g/d） （group A） or mesalamine plus placebo （group B）. Patients were monitored using the Modified Truelove-Witts Severity Index （MTWSI）. The primary endpoint was clinical remission （MTWSI ≤ 2） at 4 wk. Secondary endpoints were clinical response （defined as a reduction from baseline in the MTWSI of ≥ 2 points） and drug safety. The serum TNF-α, interleukin-6, interleukin-8 and MCP-1 were evaluated at baseline and at 4 wk of treatment. RESULTS： Analysis per-protocol criteria showed clinical remission rates of 63% and 50% after 4 wk treatment with mesalamine plus N-acetyl-L-cysteine （group A） and mesalamine plus placebo （group B） respectively （OR = 1.71; 95% CI： 0.46 to 6.36; P = 0.19; NNT = 7.7）. Analysis of variance （ANOVA） of data indicated a significant reduction of MTWSI in group A （P = 0.046） with respect to basal condition without significant changes in the group B （P = 0.735） during treatment. Clinical responses were 66% （group A） vs 44% （group B） after 4 wk of treatment （OR = 2.5; 95% CI： 0.64 to 9.65; P = 0.11; NNT = 4.5）. Clinical improvement in group A correlated with a decrease of IL-8 and MCP-1. Rates of adverse events did not differ significantly between both groups.
CONCLUSION： In group A （oral NAC combined with mesalamine） contrarily to group B （mesalamine alone）, the clinical improvement correlates with a decrease of chemokines such as MCP-1 and IL-8. NAC addition not produced any side effects.     

Delorme's transrectal excision for internal rectal prolapse

Surgical therapy of functional outlet obstruction in patients with internal rectal intussusception may include abdominal, perineal, or transrectal procedures. Because abdominal procedures often result in significant physiologic impact but unrelieved constipation, the authors have elected Delorme's transrectal excision for management of these patients. Since a short-term placebo effect attends many therapies, this report describes results of transrectal excision only after a threeyear postoperative period. Delorme's transrectal excision of internal intussusception accomplished sustained symptomatic relief in over 70 percent of otherwise refractory constipated patients. The association of internal intussusception with other abnormalities underscores the importance of defining both anatomic and functional components when selecting patients whose constipation may require surgical therapy. Critical technical elements, surgical pitfalls, and potential complications of the procedure are discussed.Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, Toronto, Canada, June 11 to 16, 1989.     

The oral glucose tolerance test: A comparison of the time points on the basis of limit values,normal dispersion,and reproducibility

Summary Time points in the glucose tolerance test (GTT) are compared on the basis of limit values, dispersion within a reference population, and reproducibility. We suggest using the distance between a limit value and the median reference value as a measure of the magnitude of abnormality. The distance between 140 mg/100 ml and the median fasting plasma glucose value is chosen as a standard distance and limits for other points in the GTT are calculated to equal this standard distance of abnormality. We suggest that the probability of correctly interpreting an inividual result is directly related to the reproducibility of the test and inversely related to the percentage of the total range of values which is dispersed among the normal population. The ratio of reproducibility to percentage normal dispersion is proposed as an index of the probability of correctly interpreting an individual result. According to this index, the probability of correct interpretation varies in order: fasting plasma glucose concentration>3-h>2-h>0.5-h>1-h plasma glucose concentration.