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.     

侵染节瓜的小西葫芦黄花叶病毒CP基因的克隆、表达及其抗血清的制备

通过鉴别寄主反应、病毒部分序列测定确定了采自广州白云区表现花叶、斑驳症状的节瓜上的病毒为ZYMV。采用RT PCR方法扩增和克隆了该病毒的外壳蛋白基因 ,连接到原核表达载体pET 2 2b( )上。获得的重组子pET ZCP转化大肠杆菌BL2 1(DE3)后 ,用IPTG进行诱导表达。SDS PAGE和Westernblot分析表明 ,CP基因在大肠杆菌中获得了高效表达 ,融合蛋白分子量约为 33 0kD。将融合蛋白纯化后免疫兔子 ,获得了特异性较高的抗血清。ELISA测定其效价为 1 4 0 96     

肺炎支原体抗独特型卵黄抗体的制备与鉴定

目的制备肺炎支原体(M.pneumonia)抗独特型卵黄抗体(vitelline Ab2),鉴定其生物学特性,探讨其作为动物疫苗的潜在应用价值。方法首先制备兔、小鼠抗肺炎支原体抗体(Ab1),筛选出对肺炎支原体有免疫中和性的兔抗体和小鼠抗体,再用兔抗体免疫产蛋鸡,筛选并收集有高效价抗独特型卵黄抗体的鸡蛋,分离蛋黄,破碎蛋黄组织,用蒸馏水稀释和酸化提取抗独特型卵黄抗体。用冷酒精沉淀法纯化抗独特型卵黄抗体。用ELISA检测肺炎支原体抗独特型卵黄抗体(Ab2)的效价、质量浓度和特异性。用竞争抑制试验和动物免疫检测卵黄Ab2β。结果兔抗肺炎支原体抗体的效价为1×10~(-6),Ab1的效价为1×10~(-4)～1×10~(-5);以上抗体均只和肺炎支原体发生免疫反应,不和解脲支原体发生免疫反应;它们和肺炎支原体混合后能中和肺炎支原体的活性,使其不能在条件培养基生长。Ab2效价为1×10~(-5),质量浓度为8 mg/mL。该抗独特型卵黄抗体只和Ab1发生免疫反应,不和小鼠抗解脲支原体抗体发生免疫反应。该Ab2和Ab1结合能被肺炎支原体竞争抑制,且Ab2能诱导小鼠产生肺炎支原体抗体(Ab3),该Ab3能和肺炎支原体发生免疫反应。结论成功制备了特异性强、效价高的β型Ab2,具有作为动物肺炎支原体疫苗的潜在应用价值。     

MUC1粘蛋白的分离纯化及其抗体的制备

经高速离心从正常人乳中获得人乳汁颗粒膜(HMFGM),产量约0．4g／L。经进一步破碎、脱脂及sepharose CL-4B柱纯化,获得含MUC1粘蛋白的组分,并经SDS—PAGE、Western—blot及ELISA鉴定后,免疫家兔制备多抗。结果表明,进一步凝胶过滤获得MUC1粘蛋白,行SDS—PAGE后经希夫试剂和考马斯亮蓝染色呈单一条带,表观相对分子质量大干205000。Western—blot及ELISA结果表明可与MUC1特异性抗体结合。制备获得的多抗经ELISA测定效价为1：64000～1：128000。表明建立了MUC1粘蛋白的纯化方法,获得的MUC1粘蛋白及其抗体可进一步用于MUC1检测及其功能的研究。     

艰难梭菌毒素B羧基端的表达与卵黄抗体制备简

目的：在大肠杆菌中可溶性表达艰难梭菌毒素B羧基端（TcdB-e）,免疫产蛋鸡,获得针对TcdB-c的卵黄抗体（IgY）。方法：人工合成TcdB-c的基因,将其克隆至pET32b（＋）载体中,转化大肠杆菌BL21（DE3）,诱导表达产物经金属螯合层析纯化,凝血酶酶切后得到目的蛋白TcdB-c;利用兔红细胞凝集和兔肠袢实验检测目的蛋白活性,用TcdB-c免疫产蛋鸡制备鸡卵黄抗体,分离纯化卵黄抗体并经ELISA测定抗体效价,用兔肠袢实验检测抗体的中和活性。结果：构建了TcdB-c的重组表达载体,诱导表达的融合蛋白相对分子质量约为79000,经凝血酶酶切后的相对分子质量约65000;目的蛋白免疫产蛋鸡后获得效价为1：20000的抗TcdB-C卵黄抗体,且该抗体可以中和TcdB-c对兔小肠的毒性作用。结论：获得了具有生物学活性的TcdB-C,并制备了针对TcdB-c的鸡卵黄抗体,为利用基因工程方法防治艰难梭菌感染打下了基础。     

艰难梭菌毒素B羧基端的表达与卵黄抗体制备

目的:在大肠杆菌中可溶性表达艰难梭菌毒素B羧基端(TcdB-c),免疫产蛋鸡,获得针对TcdB-c的卵黄抗体(IgY)。方法:人工合成TcdB-c的基因,将其克隆至pET32b(+)载体中,转化大肠杆菌BL21(DE3),诱导表达产物经金属螯合层析纯化,凝血酶酶切后得到目的蛋白TcdB-c;利用兔红细胞凝集和兔肠袢实验检测目的蛋白活性,用TcdB-c免疫产蛋鸡制备鸡卵黄抗体,分离纯化卵黄抗体并经ELISA测定抗体效价,用兔肠袢实验检测抗体的中和活性。结果:构建了TcdB-c的重组表达载体,诱导表达的融合蛋白相对分子质量约为79 000,经凝血酶酶切后的相对分子质量约65 000;目的蛋白免疫产蛋鸡后获得效价为1∶20 000的抗TcdB-c卵黄抗体,且该抗体可以中和TcdB-c对兔小肠的毒性作用。结论:获得了具有生物学活性的TcdB-c,并制备了针对TcdB-c的鸡卵黄抗体,为利用基因工程方法防治艰难梭菌感染打下了基础。     

完整弓形虫P35重组蛋白IgM-酶联免疫吸附测定法检测弓形虫急性感染

为利用重组的完整弓形虫表面抗原P35 GST蛋白对弓形虫感染进行血清学诊断 ,构建了可表达P35 GST的JM10 9细胞株 .采用亲和层析对融合蛋白进行分离和纯化 ,用SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)和蛋白质印迹 (Westernblot)分析所表达的蛋白质 ,并用纯化的重组蛋白进行IgM 酶联免疫吸附测定法 (IgM ELISA)检测不同病人血清中的抗 P35抗体 .SDS PAGE分析发现P35 GST重组蛋白的大小约 6 0ku ,为一亲水性蛋白 ,蛋白质印迹分析表明该蛋白与弓形虫阳性感染病人的血清有特异性反应 .利用P35 GST为抗原 ,对 6 0例血清进行IgM ELISA分析 ,发现P35 GST可明显区分近期感染和既往感染 ,在弓形虫的诊断上有很大的应用前景 .     

灌胃和皮下注射脂肪膜蛋白卵黄抗体对大鼠生长和脂肪沉积的影响

目的：研究脂肪膜卵黄抗体不同处理对大鼠生长和脂肪沉积的影响。方法：选用140 g左右雌性SD大鼠96只,随机分成4组,分别灌胃阴性卵黄和含脂肪细胞膜蛋白抗体的阳性卵黄;皮下注射阴性卵黄和含脂肪细胞膜蛋白抗体的阳性卵黄。灌胃每3 d给予1 ml卵黄,皮下注射连续4 d经背部皮下多点注射1 ml卵黄,1月后同方式加强1次。75 d后屠宰并采集血样测定。结果：阳性卵黄处理后大鼠体重和摄食量无显著差异。灌胃阳性卵黄降低肠系膜脂指数、子宫周脂指数和肾脂肪囊指数（P〈0.05）;降低血清甘油三酯（P〈0.05）,升高血清游离脂肪酸（P〈0.01）;降低血清Leptin、胰岛素和TNF-α水平（P〈0.01或P〈0.05）,但对腓肠肌生长、血清总胆固醇无显著影响。皮下注射阳性卵黄提高腓肠肌指数（P〈0.05）;降低血清甘油三脂（P〈0.01）;降低血清Leptin（P〈0.01）,升高血清TNF-α（P〈0.05）;而对脂肪沉积、血清游离脂肪酸、总胆固醇和胰岛素无显著影响。结论：脂肪细胞膜蛋白卵黄抗体能有效改善机体组成,灌胃的效果优于皮下注射。     

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。     

抗肺炎支原体卵黄抗体的制备及其免疫特性

目的制备抗肺炎支原体卵黄抗体,并研究其免疫特异性。方法以超声粉碎法制备肺炎支原体抗原;以ELISA法测定卵黄抗体的效价及免疫特异性;以水稀释法联合疏水层析的方法分离纯化卵黄抗体;应用SDS-PAGE法测定分子量及鉴定抗体纯度;改良Lowry法测定蛋白含量。结果低、高剂量组均诱导母鸡产生有效免疫应答,高剂量组免疫效价高于低剂量组。高剂量组于初免疫后约50d抗体效价达高峰,持续约2个月;而低剂量组在初免疫后约60d抗体效价达高峰,持续约1个月。之后效价逐渐下降,在免疫约120d,高剂量组由13log2下降到10log2;而低剂量组则由11log2下降到7log2。以水稀释法联合疏水层析法制备了电泳纯抗肺炎支原体IgY,分子量约178KD,平均每1ml卵黄液可获得较纯抗体6.4mg。制备的IgY与肺炎支原体具有较高特异性,与解脲支原体和人型支原体无明显交叉反应,与生殖支原体有轻度的交叉反应。结论本研究初步制备了抗肺炎支原体卵黄抗体,为肺炎支原体的防治与检测提供新的途径。     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418     

Separation of the pineal vesicle from the wall of the third ventricle during the post-hatching development of the chicken

Summary According to light- and electron-microscopic observations the pineal organ of the 3-day-old chicken consists of a prominent end vesicle and a tapering parenchymal stalk. During this stage the pineal lumen is in open communication with the third ventricle. However, in the 40-day-old chicken, which still possesses a well-developed end vesicle, the proximal portion of the pineal stalk displays regressive changes leading to local fragmentation. At this stage the pineal stalk is reduced, and the pineal lumen is missing. In 1-year-old chickens the parenchyma of the proximal portion of the stalk is further diminished, and in 3-year-old domestic fowl is completely displaced by bundles of collagenous fibers, only some nerve fibers being present. This post-hatching pineal development may reflect the sequence of changes leading from pineal sense organs to pineal glands.This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan