特异性鸡卵黄免疫球蛋白（IgY）抗SARS-CoV作用的研究

制备了抗SARS CoVIgY ,探讨了该IgY中和SARS CoV的作用。灭活的SARS CoV免疫产蛋母鸡 ,水稀释法提取IgY。建立ELISA检测IgY结合SARS CoV的效价 ,采用病毒中和试验测定IgY的抗SARS CoV的作用。灭活SARS CoV免疫的母鸡可产生高效价的抗SARS CoVIgY ,该IgY中和SARS CoV的中和效价为 1∶5 12。因此 ,抗SARS CoVIgY可用于预防SARS CoV的感染及阻止SARS病毒的传播。     

抗蝰蛇毒IgY灌胃对蝰蛇伤小鼠保护作用的研究

目的 探讨抗蝰蛇蛇毒鸡卵黄抗体(immunoglobulin yolk,IgY)经腹腔注射或灌胃对蝰蛇伤小鼠保护作用,为其口服制剂的应用奠定理论基础.方法 用蝰蛇原毒单一抗原免疫母鸡,收集第12天以后的鸡蛋,用水稀释法提取抗蝰蛇毒IgY;间接法ELISA检测IgY的效价和灌胃小鼠体内IgY的吸收时间;以胃排空率实验确定蛇伤前灌胃IgY的最佳时间,以腹腔注射和不同浓度IgY灌胃实验分别检验IgY对蝰蛇伤小鼠的保护作用.结果 初次免疫后第12天开始收集抗体,水稀释法提取的IgY效价为1∶6400;不同浓度IgY灌胃后,2.5～3.5 h胃排空率均达61.8%以上,血浆中抗体效价亦相应达高峰;腹腔注射和IgY灌胃实验对蛇伤小鼠的保护作用明显,小鼠的存活时间与阴性IgY组小鼠相比差异显著(P＜0.05);同等效应下,灌胃IgY的有效剂量大约为腹腔注射的10～15倍.结论 抗蝰蛇蛇毒IgY经腹腔注射和灌胃实验均能有效保护蝰蛇伤小鼠.     

鸡卵黄抗眼镜王蛇毒抗体IgY的理化特性

目的 研制抗眼镜王蛇毒鸡卵黄抗体并研究该抗体的理化特性。方法 拟用减毒后的眼镜王蛇毒免疫母鸡后,从卵黄中制备抗眼镜王蛇毒抗体IgY。采用间接ELISA法对此抗体的理化特性进行研究。结果 ELISA显示免疫后12天开始出现特异性抗体,且抗体滴度逐渐升高,约在免疫后60天左右,最高可达1：100000,此最高滴度可维持30天,以后滴度逐渐降低,至免疫后100～110天,滴度仍可维持在最高滴度的一半(1：50000)。此抗体在10～65℃温度范围内活性保持稳定;在pH为4～12范围内,抗体活性稳定;此抗体经胰蛋白酶处理1h内,活性下降不明显,但1h以后活性迅速下降。结论从卵黄中制备抗眼镜王蛇毒抗体IgY,是可行的,稳定的。     

用斑点酶联免疫吸附试验检测轮状病毒抗原

我们用国产试剂和材料建立了一种以硝酸纤维素膜(NCM)作为固相载体的斑点酶联免疫吸附试验(Dot-ELISA),用于检测粪标本中的轮状病毒. 首先用铁笔将NCM划成5×5mm的小方格,然后将免抗人轮状病毒IgG打点至方格中心,室温凉干后,置封闭液中室温作用1小时,取出用PBST洗涤3～5次,每次5分钟,然后将膜直接浸入待检粪样、阳性抗原对照和阴性抗原对照中.置37℃ 30～60分钟,洗涤。随后将上述诊断膜浸入酶标兔抗人轮状病毒IgG中,室温45～60分钟,洗涤。用底物DAB显色数分钟,至阳性对照样本出现明显褐色斑点时,用自来水终止反应。根据斑点颜色深浅,肉眼即可判断阳性或阴性结果。     

纳豆激酶液态发酵条件的研究

考察纳豆杆菌在7.5 L发酵罐中分批发酵产纳豆激酶的条件,纳豆激酶酶活采用四肽底物测定。结果表明:纳豆杆菌生长和产酶的适宜条件不一致。发酵过程中发酵罐搅拌转速控制为500 r/min不变,0～12 h时控制pH为8.0、温度为37℃;12～36 h调整pH为7.0、温度为30℃;16 h时补加外源C源,连续发酵36 h。该过程中发酵液中比酶活最高达到3 232 U/mL,与摇瓶发酵相比,比酶活提高了58%。     

重组人干扰素α1 b滴眼液稳定性的研究

重组人干扰素α 1b滴眼液放置不同温度和不同时间后,定期测定其效价、无菌试验、pH值和外观,在4℃条件下,24个月内pH、无菌试验、外观和0月一致,没有明显变化;生物学活性24个月没有降低;室温（18～25℃）放置12个月活性明显降低,pH、无菌试验、外观和0月一致;37℃放置6个月活性基本丧失,pH、无菌试验、外观和0月一致.重组人干扰素α1b滴眼液在4℃条件下生物活性及其他理化性质稳定性较好.     

一株产木聚糖酶菌株的分离、鉴定及其酶学特性研究

以木聚糖为唯一碳源,采用平板水解圈筛选和摇瓶发酵相结合的方法,从土壤中分离、筛选到一株产木聚糖酶的细菌xy-7,根据其形态和生理、生化特性,并结合16S rDNA序列分析,初步鉴定为坎皮纳斯类芽孢杆菌(Paenibacillus campinasensis)。经测定,xy-7所产木聚糖酶的最适作用温度为60℃,最适作用pH为7.0。该酶热稳定性较好,60℃时保温2h酶活保持为原来的73%。此外,该酶的pH作用范围较广,pH 9.0时酶活仍能保持68%,属于耐碱性木聚糖酶。这些性质表明,该酶在制浆造纸等行业具有较好的应用前景。     

鸡血清与卵黄中抗中华眼镜蛇毒IgY动态变化研究

目的探索特异性IgY的产生和变化规律。方法用眼镜蛇毒原毒免疫产蛋母鸡,ELISA定期检测卵黄中的抗体效价变化,小鼠体外中和实验检测其生物活性。第1次免疫40周后,眼镜蛇毒攻击已免疫母鸡,检测攻击前后鸡血清中抗体效价变化情况,未经眼镜蛇毒免疫的母鸡作阴性对照。结果经免疫后第7天蛋黄中即可检测到抗体,经多次加强免疫,40周时蛋黄中还能保持高效价的抗体,通过分离纯化,此抗体可保护实验小鼠免受4 LD50眼镜蛇毒的攻击;同时,鸡血清中也保留着较高效价的抗体,可中和4 LD50以上的眼镜蛇毒。结论用眼镜蛇毒免疫鸡,经多次加强免疫,卵黄和鸡血清中可持久保持高效价的特异性抗体,初步检测此抗体可中和4 LD50的蛇毒。     

沙市婴幼儿腹泻中检出3株C群轮状病毒

1987～1988年在沙市165份婴幼儿急性腹泻标本中,用PAGE法检出轮状病毒37株(22.4％),其中3株为少见的轮状病毒,此种病毒经电镜观察,具有典型轮状病毒的形态结构,ELISA证实该病毒不具有A群和B群轮状病毒的群特异性抗原。RNA电泳分析表明,其基因组由11个双链RNA片段组成,电泳图型特殊,呈4:3:2:2的排列模式。上述试验表明,该病毒为世界上罕见的C群轮状病毒。免疫电镜证实,该病毒能被病人恢复期血清所凝集,提示该病毒是腹泻病儿的致病因子。     

抗胱抑素C鸡卵黄IgY抗体的制备及鉴定

目的:制备高效价、高特异性的抗人胱抑素 C 鸡卵黄 IgY 抗体,并对其基本特性进行分析和鉴定.方法:以人胱抑素 C 为抗免疫产蛋的罗曼鸡,采用水稀释-盐析法提取及纯化 IgY 抗体,采用蛋白质定量、SDS-PAGE、West?ern 印迹和 ELISA 法对 IgY 抗体进行分析和鉴定.结果:免疫后14 d 即可从鸡冠血中检测出抗胱抑素 C 的特异性抗体,抗体效价在28 d 达最高峰(1∶32000),并可维持2个月以上;收集高效价时的免疫鸡蛋,制备鸡卵黄抗体 IgY;还性 SDS-PAGE 显示抗体 IgY 为相对分子质量分别为65×103和21×103的2条带,抗体纯度可达92%,得率为每个鸡蛋36.5 mg,抗体检出敏感度为15.63 ng/mL;Western 印迹证明该抗体具有高度特异性.结论:制备了抗胱抑素 C 的高效价、高特异性 IgY 抗体.     

抗生殖器疱疹病毒卵黄免疫球蛋白(IgY)的研究

检测了鸡卵黄中抗生殖器疱疹病毒(HSV-2)抗体的产量、纯度、来源及稳定性。采用生殖器疱疹病毒(HSV-2)作为抗原免疫广州黄村鸡。通过改良水稀释法提取卵黄中的IgY。双紫外光波长测定抗体含量,SDS-PAGE电泳检测抗体纯度。Western blot免疫印迹法测定该抗体来源。ELISA检测IgY对温度、酸碱度的稳定性。结果,蛋黄液中抗体质量浓度13.6g.L-1,抗体纯度达96.2%。免疫印迹证明IgY与鸡血清中的IgG具有相同的分子量和抗原性。IgY具有良好的热稳定性,对酸碱具有一定的耐受力。WD水稀释法能得到高产量、高纯度的特异性IgY,而且有良好的生物学活性。     

Productivity and Some Properties of Egg Yolk Antibody (IgY) against Human Rotavirus Compared with Rabbit IgG

Productivity and some properties of anti-Human Rotavirus (HRV) hen egg yolk antibody (IgY) were compared with those of anti-HRV rabbit serum antibody (IgG). The hens immunized with HRV (Wa strain, serotype 1 and Mo strain, serotype 3) were found to continuously to lay eggs without any change in the egg laying rate and the yolk of the eggs laid over a year showed a high level of neutralization titer against HRV. The production of anti-HRV IgY by a hen (one year) was at least 15 times (anti-Wa) and 120 times (anti-Mo) more effective than those by an immunized rabbit in the neutralization titer of the antibodies.

The stability of anti-HRV IgY at temperature above 70°C and low pH 2–3 was less than that of anti-HRV rabbit IgG. The temperature corresponding to the maximum of denaturation endotherm (T_max) of IgY was 73.9°C while that of rabbit IgG was 77.0°C in the analysis by differential scanning calorimetry. This discrepancy in heat and acidic pH stability found between the two antibodies as discussed with regard to their protein structures.     

抗甲型流感鸡卵黄抗体的制备及效价测定

目的:研究抗甲型流感卵黄抗体的制备与纯化,并探讨其效价随免疫时间的变化关系。方法:用灭活甲型流感病毒复合抗原免疫蛋鸡,用PEG6000对卵黄抗体进行分离提取,SDS-PAGE法对其进行分子量测定,考马斯亮蓝法对其含量和纯度进行测定,用微量凝集法检测蛋鸡血清抗体和卵黄抗体的效价。结果:提取得到的卵黄抗体重链分子量为66 kDa、轻链分子量分26 kDa,每毫升卵黄液可得到纯度为95.80%的卵黄抗体9.98mg,回收率93.01%;高效价持续时间90 d以上;免疫蛋鸡血清和卵黄中3种特异性抗体的消长规律基本相似,但抗体水平之间存在明显的差异。结论:采用灭活甲型流感病毒复合抗原免疫蛋鸡可制备高效价、高纯度抗甲型流感卵黄抗体,为卵黄抗体在甲型流感防治中的应用研究奠定了基础。     

兔抗麻雀IgY酶标抗体的制备

目的制备辣根过氧化物酶（HRP）标记的兔抗麻雀IgY抗体,为禽类血清学检测体系的建立提供技术储备。方法硫酸铵盐析法粗提麻雀血清IgY,进一步在SDS-PAGE上分离后,切下带有目的条带的凝胶作为免疫原,免疫实验兔制备抗血清,Protein-A柱亲和纯化兔抗IgY血清IgG,,使用改良过碘酸钠法制备酶结合物。ELISA检测酶标抗体的工作浓度,western blotting检测酶标抗体的特异性。结果硫酸铵盐析法粗提IgY,可去除部分杂蛋白,SDS-PAGE上分离后切下带有目的条带的凝胶,可以得到足够纯度的抗原,将带有IgY的凝胶作为抗原免疫后获得的抗血清经Protein-A纯化后,二抗在SDS-PAGE上鉴定,纯度达到99%以上。改良的过碘酸钠法标记获得的抗体浓度为1.008 mg/mL,ELISA检测酶标抗体效价为1∶1000。Western blotting鉴定抗体具有特异性。结论获得了优质可靠的兔抗麻雀IgY酶标抗体。     

人NANOGP8蛋白的原核表达及多克隆抗体的制备

为获得抗人 NANOGP8基因的多克隆抗体,采用PCR法扩增了NANOGP8基因,经序列测定正确后,连接到pET-28a质粒上,将获得的重组质粒pET-28a-NANOGP8转化到大肠杆菌BL21（DE3）中,在16℃和37℃下分别进行诱导表达,经SDS-PAGE检测发现融合蛋白以包涵体形式存在于37℃诱导表达的沉淀中,大小约为45kDa。对包涵体进行洗涤、溶解处理,经Chelating Sepharose Fast Flow亲和层析柱进行纯化。对纯化得到的蛋白进行SDS-PAGE及Western blot免疫印记杂交检测,得到了单一条带。用获得的高纯度融合蛋白免疫新西兰兔,得到兔来源的抗人NANOGP8血清。对抗血清进行蛋白免疫印记杂交反应和酶联免疫吸附反应检测,结果显示成功制备了高滴度和特异性的兔抗人NANOGP8多克隆抗体,与市售抗人Nanog多克隆抗体相比,杂交更迅速,效果更明显。为进一步研究NANOGP8基因在肿瘤发生、发展中的作用奠定了基础。     

Oral Passive Immunization Effect of Anti-Human Rotavirus IgY and Its Behavior against Proteolytic Enzymes

The neutralization titer of anti-human rotavirus (HRV) IgY was completely inactivated by pepsin at pH 2.0. However, it was not significantly affected by trypsin or chymotrypsin under certain conditions. The immunological activity of the IgY was observed in the intestine of suckling mice for 2 h after oral administration and the activity rapidly decreased thereafter. The effects of oral supply of IgY were thus estimated for HRV-induced diarrhea in suckling mice and it was found that a previous supply of the IgY, (1 h before HRV infection) completely prevented the HRV-induced diarrhea. The preventive effect was decreased as the time gap between IgY administration and HRV infection was longer. However, the oral supply of the IgY within 24h after HRV infection was still effective and decreased the incidence of HRV diarrhea in suckling mice.     

人脾DNA拓扑异构酶Ⅰ的提纯及其性质的研究

 经三个步骤从人脾提纯了拓扑酶Ⅰ。SDS-PAGE和等电聚焦均显示单一蛋白带。分子量77kD,pI7.3。新生霉素和香豆霉素A_1不抑制酶活性。本实验表明,反应最适K~(+)或Na~(+)浓度为180mmol/L。反应活性不要求Mg~(2+),但5mmol/L存在时,活性增加90％。在pH5.0～9.0均有活性,但在7.5～8.0时活性较大。30℃放置24小时或50℃处理30分钟活性基本丧失。最适反应温度为37℃。对-氯汞苯甲酸强烈抑制酶活性。     

类产碱假单胞菌杀虫蛋白的N端鉴定及抗体制备

The insecticidal protein from Pseudomonas pseudoaligenes was and exotoxin which had toxicity on locusts.In order to elucidate its molecular properties an amino acid sequence,the insecticidal protein was purified from the culture supernatant by ultrafiltration,ion\|exchange chromatography and gel filtration,and showed a single band on SDS\|PAGE.Analysis of the purified insecticidal protein dentified N\|terminal sequence of ten amino acid residues.Its polyclonal antibody was also obtained by immunizing rabb…     

In Vitro Inhibition of Oral Candida albicans by Chicken Egg Yolk Antibody (IgY)

This study was conducted to measure Candida albicans-specific chicken egg yolk antibody (IgY) inhibition of fluconazole-sensitive and resistant strains of C. albicans in order to assess potential use in the prevention and treatment of oral candidiasis. In this study, laying hens were immunized, and IgY was extracted by water dilution. The Minimal Inhibitory Concentrations (MICs) of IgY for inhibiting C. albicans growth were determined using the broth microdilution method from the CLSI M27-A2 protocol. Fluconazole (FLC) was used as the control. The results were analyzed with the chi(2) test. The anti-Candida titer of anti-C. albicans IgY was 1:12,000. The concentration of the IgY extract that effectively inhibited the growth of C. albicans was between 1.25 g/l and 5.0 g/l, and the efficacy rate was 82.98% during the observed 24-48 h time period. No correlation was recorded between the drug resistance of FLC and growth inhibition by IgY. It was concluded that anti-C. albicans IgY inhibited the growth of C. albicans in vitro and there was no correlation between the drug resistance of FLC and the growth inhibition by IgY (P > 0.99).     

Preparation and characterization of immunoglobulin yolk against the venom of Naja naja atra

Chinese Cobra (Naja naja atra) bite is one of the leading causes of snake-bite mortality in China. The traditional anti-cobra venom serum therapy was found to be expensive and with high frequency of side effects. Therefore attempts were made to generate a high titer immunoglobulin from egg yolk (IgY) of crude cobra-venom immunized Leghorn hens, and to standardize an effective method for producing avian antivenom in relatively pure form. The IgY was isolated first by water dilution method to remove the lipid, then extracted by ammonium sulfate precipitation, and purified through anion exchange chromatogram. The different purities of IgY from different isolating stages were submitted to enzyme-linked immunosorbent assay and SDS-PAGE to determine their titers. Immunoblotting showed that the purified IgY (ion exchange chromatography fraction, IECF) recognized several antigenic fractions of cobra venom, and presented with the character of polyclonal antibody. IECF on SDS-PAGE under reducing conditions migrated as a 65 kDa heavy chain and a 35 kDa light chain, respectively. The LD50 of the N. naja atra venom was 0.62 mg/kg body weight in mice. Four times the LD50 dose of venom was selected as challenge dose, and the ED50 of IgY was 3.04 mg IECF/mg venom. The results indicate that the activity of anti-snake venom IgY could be obviously elevated by ion exchange chromatography, thus possessing therapeutic significance for snakebite envenomation.