酸性条件下糖对抗HpaAIgY的保护作用

目的探讨酸性条件下5种糖对抗HpaA蛋黄抗体(IgY)的保护作用,为寻找提高IgY耐酸性的保护剂,制备口服防治幽门螺杆菌(H.pylori)感染的IgY制剂提供理论依据。方法建立检测抗HpaAIgY的间接ELISA法,用不同pH值的模拟胃液分别配制IgY液(IgY终浓度为1.0mg/ml),以pH1.0、2.0和3.0的模拟胃液分别配制含20%菊糖、山梨醇、葡萄糖、蔗糖、葡聚糖的IgY液(IgY终浓度为1.0mg/ml),用pH2.0的模拟胃液分别配制含不同浓度的菊糖、山梨醇的IgY液(IgY终浓度为1.0mg/ml)。上述IgY液均于37℃孵育2h,采用间接ELISA法测定IgY的抗体效价。结果 IgY的活性随着模拟胃液pH值的降低而明显下降;在酸性环境下,5种糖对IgY均有一定的保护作用,以山梨醇和菊糖的保护作用较强;在pH2.0时,40%山梨醇和30%菊糖对IgY的保护作用最强,分别能保存80.3%和73.4%的IgY活性。结论在酸性条件下,糖对IgY均有一定的保护作用,菊糖和山梨醇可作为特异性IgY的保护剂。     

抗流感病毒鸡卵黄免疫球蛋白IgY的制备和性质

用流感病毒免疫产蛋鸡,获得的免疫卵黄经两步盐析和一步凝胶过滤分离得到的IgY经SDS-PAGE分析其纯度在95%以上,纯化的IgY经胃蛋白酶(pH=4 0,37℃)酶解后,得到的片段抗体为Fab。采用竞争抑制ELISA法对IgY和Fab的免疫反应活性进行了对比,表明IgY经胃酶切解后保留了约70%的反应活性。     

抗轮状病毒IgY和Fab'的分离与纯化

抗轮状病毒IgY可用两步盐析结合凝胶过滤从蛋黄中分离出来,用SDS—PAGE检测其纯度可达到95％以上。纯的IgY经胃蛋白酶分解得到的抗体片断（Fab’）,经SDS-PAGE和MALDI质谱法测定,其纯度达到99％以上。结果表明,所设计的分离抗轮状病毒IsY和Fab’的方法简单、有效。经ELISA法检测,Fab’的活性仍保持在IgY原始活性的70％以上。     

IgY抗体对幽门螺旋菌菌体抗原细胞毒活性的中和作用

以超声震荡及高速离心法裂解空泡毒素基因阳性 (VacA+ )HP临床分离株 ;采用Hela细胞体外培养法分析此HP菌体蛋白的细胞毒活性及其免疫产卵母鸡制备的IgY抗体的保护作用。HP菌体抗原制备物经SDS PAGE显示至少有十余个蛋白区带 ,主要区带位于约 940 0 0和 6 70 0 0。以此HP菌体抗原包被的ELISA显示 ,球部溃疡患者血清IgG抗体滴度明显增高。体外培养中HP菌体蛋白制备物对Hela细胞具有明显的细胞毒活性 ,LD50 小于 12 μg/ml。以此抗原免疫产卵母鸡 ,提取的IgY抗体浓度依赖地阻断HP菌体蛋白的Hela细胞毒活性 ;提示局部应用抗体制剂做被动免疫 ,有可能成为治疗HP感染性疾病的有效方法     

缓释定向释放型IgY微胶囊的制备及其效果评价

目的制备缓释定向释放型IgY微胶囊,并评价其效果。方法采用喷雾干燥法,以明胶、海藻酸钠为微胶囊壁材,制备抗仔猪病原性腹泻IgY微胶囊。以IgY的活性、包封率、体外释放性能及稳定性为评价指标,通过单因素分析和L_9(3_4)正交试验,分别从成囊材料的配比对微胶囊性能的影响、制备微胶囊成囊乳化液的稳定性影响因素以及喷雾干燥条件3方面对IgY微胶囊制备条件进行优化,并评价微胶囊的IgY活性、包封率、在人工胃液(simulated gastric fluid,SGF)和人工肠液(simulated intestinal fluid,SIF)中的缓释效果及稳定性。结果筛选出的制备微胶囊的最佳工艺条件为:明胶浓度4%、海藻酸钠浓度0.75%,按IgY与明胶-海藻酸钠质量比为3∶4加入IgY,乳化后按进风温度170℃,进料速度10 ml/min,干燥时间10 s,出口温度70℃进行喷雾干燥。在此工艺条件下制备的IgY微胶囊包封率为77.4%,活性保持在85%以上;在SGF中2 h释放率为8.6%,在SIF中4 h释放率为81.2%;90℃加热30 s仍可保持50%以上的活性,常温(25~30℃)放置10个月活性仍保持在90%以上,4℃保存10个月活性仅下降5%。结论本工艺制备的明胶-海藻酸钠IgY微胶囊具有生物活性高,稳定性强的特点,且对特异性IgY具有缓释和靶向作用。     

IgY的冻融法脱脂条件研究

目的寻求IgY的最佳脱脂条件。方法用人IgG免疫产蛋母鸡,待蛋黄中抗体效价达1∶64以上时,选用不同的稀释度、酸碱度、冻融次数、离心力等条件分离IgY。测定除脂率、效价、蛋白含量,以确定最佳脱脂条件。结果稀释度为1∶7,pH为5·2,冻融1~2次,离心力大于或等于3000g,离心20min以上,其除脂率可超过98%,IgY的特异性抗体效价未降低,蛋白含量平均为8·033mg/ml。结论用冻融法分离IgY能有效去除蛋黄液中的脂肪,此工艺便于批量生产低脂IgY产品。     

幽门螺杆菌重组VacA蛋黄抗体的制备

目的研制高效价、高特异性的抗细胞空泡毒素VacA的鸡蛋黄抗体(IgY),为研制幽门螺杆菌(H.pylori)治疗性抗体奠定基础。方法用纯化的重组细胞空泡毒素抗原免疫22周龄洛曼产蛋母鸡,取免后所产鸡蛋,将蛋黄稀释、离心,收集上清,加硫酸铵沉淀。以ELISA间接法检测抗体效价,Bradford法检测蛋白含量,SDS-PAGE法检测相对分子质量及纯度。结果母鸡初次免疫后76d,抗体效价达到1∶6400,最高可达1∶12800。蛋黄抗体经纯化浓缩后,用SDS-PAGE分析,出现2条蛋白带,纯度可达80%以上,其含量为25·66mg/ml。结论已成功制备了抗重组细胞空泡毒素的特异性蛋黄抗体。     

抗甲型肝炎病毒卵黄免疫球蛋白的制备及应用

目的制备抗甲型肝炎病毒(hepatitis A virus,HAV)卵黄免疫球蛋白(immunoglobulin of yolk,IgY),并建立双抗体夹心ELISA法,用于测定甲肝疫苗中的HAV抗原含量。方法用纯化的HAV免疫健康产蛋母鸡,制备抗HAV IgY,采用多步骤分离纯化IgY,紫外分光光度法测定纯化IgY的蛋白含量,还原型SDS-PAGE分析IgY的相对分子质量及纯度,间接ELISA法检测IgY的效价、特异性及其热稳定性和酸碱稳定性。以纯化的IgY作为包被抗体,HRP标记的抗HAV单克隆抗体作为二抗,建立双抗体夹心ELISA法,对疫苗生产过程中的HAV抗原含量进行测定,并与市售ELISA试剂盒的检测结果进行比较。结果亲和层析纯化后的抗HAV IgY浓度为3.67 mg/ml;重链和轻链的相对分子质量分别为66 000和27 000,纯度为96.78%;效价最高为1∶16 000;纯化的抗HAV IgY只与HAV呈阳性反应,与脊髓灰质炎病毒和肠道病毒71(enterovirus 71,EV71)均不反应;纯化的抗HAV IgY在25⁷0℃条件下处理15 min及经pH 3.070℃条件下处理15 min及经pH 3.0¹0.0的Tris-HCL缓冲液处理2 h,其抗体效价基本无影响。建立的双抗体夹心ELISA法HAV浓度在15.6210.0的Tris-HCL缓冲液处理2 h,其抗体效价基本无影响。建立的双抗体夹心ELISA法HAV浓度在15.62² 000.00 ng/ml范围内,HAV浓度的对数值与A450值之间呈线性相关,R2=0.935,最低检测量为7.81 ng/ml,与市售试剂盒检测结果具有良好的相关性,相关系数为0.952 7。结论制备的抗HAV IgY浓度、纯度、效价均较高,特异性较强,稳定性良好,建立的双抗体夹心ELISA法可用于检测甲肝疫苗生产过程中HAV的抗原含量。     

抗金黄色葡萄球菌IgY的功能性试验

目的　观察抗金黄色葡萄球菌IgY的生物学效应及稳定性。方法　将金黄色葡萄球菌稀释至适宜浓度 ,用微量凝集法测定IgY抗体的生物学效应及稳定性。结果　抗体IgY(10mg ml)溶液能明显抑制金黄色葡萄球菌生长 ,且能使感染性病灶症状消失 ,治愈病灶局部未检出葡萄球菌。抗体IgY在 2 5℃、37℃条件下活性稳定 ;巴氏消毒 6 4℃条件下 15min活性下降 4 0 % ,15min以后迅速下降 ;70℃作用 15min活性下降 70 % ;在pH4 0～ 7 9环境中活性改变不显著。结论　抗金黄色葡萄球菌IgY具有很好的生物学效应和高度稳定性。     

抗氯霉素卵黄抗体的制备及其分离纯化

将合成的氯霉素(CAP)与牛血清白蛋白(BSA)的偶联物(CAP-BSA)作为免疫抗原注射到母鸡体内得到氯霉素卵黄抗体(IgY). 比较了不同的提取方法,用优化的辛酸-硫酸铵沉淀法从卵黄液中初步提取卵黄抗体. 选用3,3￠-二氨丙基亚胺(DADPA)作为亲和凝胶与配基氯霉素之间的"间隔臂",合成了分离纯化抗氯霉素特异性IgY的亲和层析柱. 将IgY粗品经过亲和层析柱,得到的特异性IgY的抗体活性提高了10倍,收率约为3.3%.     

胃蛋白酶对抗狂犬病毒IgY活性的影响

目的 观察抗狂犬病毒IgY经胃蛋白酶酶解24h后，抗体活性的变化。方法 高效价抗狂犬病毒IgY粗提后，经胃蛋白酶酶解，纯化，用ELISA及小鼠中和实验测其抗体活性。结果 获得单一IgY解片段并能够中和狂犬病毒。结论IeY经胃蛋白酶酶解24h仍保持其中和抗体的活性，为制备口服免疫制剂奠定基础。     

福氏志贺菌IgY的抑菌作用

目的探讨口服福氏志贺菌IgY在动物体内的抑菌作用。方法以福氏志贺菌制备免疫原,免疫母鸡,收集鸡蛋,提取IgY。以不同剂量的IgY口服免疫6日龄乳鼠,并分别以不同剂量的福氏志贺菌于不同时间攻击,观察体内抑菌作用。结果攻菌前口服特异性IgY的乳鼠,其直肠内分离菌的百分率明显低于攻击后口服IgY的乳鼠。抑菌作用随着口服IgY剂量的减少和攻菌量的增加而降低。口服大于3mg/只的特异性IgY,可使肠内特异菌减少50%以上。口服4mg/只可抵御10亿菌的攻击。结论福氏志贺菌特异性IgY可有效抑制该菌在乳鼠肠内的繁殖。     

幽门螺杆菌VacA-HpaA融合蛋白的表达及其免疫学特性

目的构建H.pylori细胞空泡毒素VacA与黏附素HpaA融合基因的原核表达载体,诱导其表达融合蛋白,并检测表达产物的抗原性与免疫原性。方法用PCR从pQE30-VacA质粒扩增出VacA基因,克隆至pTrc99A-HpaA载体中,与HpaA基因融合后,插入原核表达载体pQE30中,再将pQE30-VacA-HpaA转化入大肠杆菌DH5α,诱导表达并提纯融合蛋白,SDS-PAGE检测融合蛋白的表达,Bradford法检测融合蛋白含量,Western blot鉴定特异性。将融合蛋白免疫家兔,得到多克隆抗血清,用双向免疫扩散和ELISA检测免疫原性。结果SDS-PAGE显示融合蛋白相对分子质量约为65000,表达量在35%以上,主要以包涵体形式表达,蛋白含量为0·72mg/ml,具有良好的VacA和HpaA抗原性与免疫原性。结论VacA-HpaA融合蛋白已成功表达,且具有良好的免疫原性,为进一步研究制备H.pylori疫苗创造了条件。     

Pigment removal from canola oil using chlorophyllase

Frost-damaged or prematurely harvested canola seed (rapeseed) may yield oil with a high chlorophyll content (50–60 μg/ml). Enzymatic hydrolysis of chlorophyll, added to buffer/surfactant, buffer/acetone or buffer/acetone/canola oil, to produce water-soluble chlorophyllide (green pigment) was studied using a crude chlorophyllase preparation (acetone-dried chloroplasts) from 15 to 20-day-old sugar beet seedlings. In buffer/surfactant, the optimum pH for enzyme activity was temperature dependent. At 30 C and 0.24% Triton X-100 (or 30% acetone), chlorophyllase showed maximum activity toward a crude chlorophyll preparation over the range of pH 8–10. At 60 C, the activity was more than twofold higher, with a sharp maximum at ∼pH 8. Mg²⁺ enhanced the activity with an optimal concentration of 50 mM. At pH 7.5, 50 C and in the presence of only 6% acetone, the enzyme showed high affinity for chlorophyll (Km=15μM or 13.5 μg/ml), suggesting that the natural chlorophyll concentrations found in green canola oils might facilitate high enzymatic efficiencies. The crude enzyme was stable in buffer/acetone at pH 7.5 and 50 C for at least two hr. With acetone concentrations as low as 6%, maximum enzyme activities in buffer and buffer/canola oil required intensive mixing (homogenization) of the various substrate, enzyme and liquid phases. In general, the rate and extent of chlorophyll hydrolysis were greater in buffer than in buffer/oil. In both reaction systems, chlorophyll hydrolysis slowed down with time due to accumulation of phytol, which proved to be a competitive inhibitor (K_i=11 μM or 3.3 μg/ml). The other hydrolysis product, chlorophyllide, did not affect enzymatic activity. Crude canola oil used in the reconstitution of green oil did not support enzymatic chlorophyll hydrolysis without prior degumming and desoaping. The optimum buffer/oil ratio of the reaction mixtures was above 2/1 (v/v).     

Adsorption behaviors of avian immunoglobulins and purification of immunoglobulin Y from chicken serum with mixed-mode resins

The importance of immunoglobulin Y(IgY) as a specific antibody equivalent to mammalian immunoglobulin G(IgG) is well recognized. However, production of highly purified IgY is still difficult due to the lack of specific purification methods. In this study, adsorption behaviors of Ig Y on four mixed-mode resins with functional ligands of 4-mercatoethyl-pyridine(MEP), 2-mercapto-1-methyli-midazole(MMI), 5-aminobenzi-midazole(ABI) and tryptophan-5-aminobenzi-midazole(W-ABI) were evaluated. The results showed that high adsorption ratio were found at p H 6.0–7.0 with little adsorption under acidic conditions. The resin with ABI ligand was then used to separate IgY from immunized chicken serum. An efficient process with Ig Y purity of 95% and recovery of 90% was developed after optimization of loading and elution p H and injection volume. The biological activity of the purified Ig Y was fully maintained. These results indicated that mixed-mode chromatography with specially-designed ligands has great potential for the separation of Ig Y from crude feedstock.     

氨基树脂固定胃蛋白酶的方法及性质研究

采用氨基树脂作为载体,戊二醛作为交联剂,对胃蛋白酶的固定化进行了研究,并对固定化条件和固定化胃蛋白酶的部分酶学性质进行了分析。确定固定条件为：戊二醛浓度为5%,载体处理温度为室温（25℃）,处理时间为5h,m（胃蛋白酶）：m（氨基树脂）为1：25,pH为3.0,固定时间为12h。此条件下固定化的胃蛋白酶活力为30U/g,酶的活力回收率为60%。与非固定化相比最适水解温度由50℃升高到60℃,最适pH值由2.0升高到4.0,游离酶米氏常数3.08g/L,固定化酶米氏常数1.2g/L,固定化胃蛋白酶的储存半衰期约为25天。对珠蛋白的操作半衰期为9天。     

HPLC measurement of testicular long chain acyl-CoA synthetases with different substrate specificities

Acyl-CoA synthetase activity with various long chain fatty acid substrates was measured in microsomes from rat testes, isolated spermatids and testes of hypophysectomized adult rats, using reversed-phase high performance liquid chromatography (HPLC). The spectrophotometric HPLC method produced results comparable to those of parallel radiometric assays and was highly specific for acyl-CoA products. At optimal pH and cofactor concentrations, specific activity from whole testis was similar for 18∶1, 20∶4 and 22∶5 but somewhat lower for 16∶0 over the substrate range 0.01–3.2 mM. Activity from spermatids or from testes of hypophysectomized rats was much lower with 22∶5 than with 18∶1 or 20∶4, whereas activities with 18∶1 and 20∶4 were similar at all substrate concentrations. All substrates exhibited Michaelis-Menten type saturation kinetics and linear Lineweaver-Burke plots at lower substrate concentrations but inhibited activity at higher concentrations. Apparent values of K_M for 16∶0, 18∶1 and 20∶4 were more than twice that of 22∶5, whereas both observed and calculated maximum velocities were similar for the four fatty acids. Differences in pseudokinetic parameters and differential expression of the testicular acyl-CoA synthetase activities with different fatty acids suggest the presence of multiple enzymes, at least one of which may be hormonally regulated.