牛肝提取物提高鸡免疫力的研究

将牛肝提取物注射到第１３日龄第１５日龄鸡胚中能显著提高孵出后３仔鸡的抗ＳＲＢＣ血清抗体的效价,促进脾和法氏囊淋巴细胞增殖、分化。同时,牛肝提取物与雏鸡法氏囊粗提取液,抗鸡法氏囊病毒抗体均有增强仔鸡抵抗传染性法氏囊病的能力。实验结果表明牛肝提取物中可能含有类似法氏囊素的物质。     

禽呼肠孤病毒感染对鸡法氏囊及免疫应答的影响

研究LY株禽呼肠孤病毒(ARV)感染1日龄SPF鸡后对法氏囊发育影响,对传染性法氏囊病毒(IBDV)、禽流感病毒(AIV)、新城疫病毒(NDV)疫苗免疫诱发的抗体的影响,及对强毒株IBDV致病作用的影响。结果表明,LY株ARV感染1日龄SPF鸡可引起法氏囊萎缩和部分淋巴细胞减少,但对增重及AIV和NDV疫苗免疫后抗体滴度却没有显著影响。ARV感染可降低弱毒IBDV疫苗免疫后的抗体反应,但对随后IBDV强毒株攻毒的抵抗力却与对照鸡无显著差异。经IBDV弱毒疫苗免疫后,再接种强毒株IBDV,不会引起死亡,但却仍能显著抑制对AIV、NDV疫苗免疫后的抗体滴度。然而,对于1~7日龄经ARV感染的鸡,IBDV强毒的这种免疫抑制作用又显著低于未经ARV感染的对照鸡。     

鸡传染性法氏囊病病毒超强毒株GX8/99株的致病性

鸡传染性法氏囊病病毒(IBDV)超强毒株GX8/99,系1999年从广西一自然发病鸡群采集到.用原始病鸡法氏囊悬液连续3次人工感染SPF鸡后,再取其法氏囊制备悬液,分装,在-70℃保存.以此悬液经卵黄囊接种10日龄SPF鸡胚,测定鸡胚的半数致死量(ELD50).随着鸡的日龄和接种剂量的不同,其致死率有很大差异.对28～30日龄SPF鸡,病毒接种量为200个ELD50时,感染后7日内死亡率最高可达73%～90.5%(11/15和19/21);感染量为20个ELD50时,死亡率亦可达53%～92%(8/15和23/25).以500个ELD50感染28～104日龄的SPF鸡,死亡率均在55.1%～67.2%;甚至113～120日龄的SPF鸡,感染后仍有10%～15%致死率.但128日龄的SPF鸡感染后既不引起死亡也不表现任何症状,但抗体全部转阳.人工接种发病死亡的鸡,其法氏囊的出血程度也随感染量和年龄而异.50日龄鸡接种2000个ELD50后,死亡的鸡100%(12/12)法氏囊严重出血;而40日龄鸡感染200个ELD50后,死亡鸡中仅17%(3/18)发生出血.在2、3、4周龄带有母源抗体的商品代蛋鸡,以2000个ELD50病毒接种后,只引起10%(2/20)、35%(7/20)和35%(7/20)的死亡率.但在5周龄商品代蛋鸡,仅接触感染的致死率可达61.3%(98/160).另一批商品代蛋鸡,在4周龄和5周龄人工接种200个ELD50病毒后,死亡率分别是81.6%～94.3%(62/76～33/35)和93.9%～94%(31/33～47/50).通过总共1200多羽鸡的试验表明,GX-8/99株是一个超强毒IBDV毒株,表现为高死亡率(最高可达94%),易感年龄延长至4月龄,中枢性免疫器官法氏囊出血严重和胸腺明显萎缩.     

传染性法氏囊病不同代次细胞毒免疫效果的研究

将从山东省东营分离到的1株传染性法氏囊病病毒(IBDV)野毒(暂命名 IBDV SDDY株,经鉴定该株与 IB DV STC株具有较高的同源性)经 SPF 鸡胚传代,然后转为细胞培养,取第 20 代、21 代、25 代毒,以 1 倍剂量(3000TCID50/0.2mL )和5倍剂量不同的免疫剂量,分别在7日龄、14日龄对商品代海蓝褐蛋鸡进行免疫,并于免疫前用 IBD ELISA试剂盒检测 IBDV母源抗体水平,于 35 日龄用传染性法氏囊病病毒超强毒 (very virulent In fectious Bursal Disease Virus, vvIBDV )GX 8/99攻毒,在攻毒前再次检测IBDV的抗体水平,攻毒后观察记录各分组鸡的致病率和死亡率,并计算免疫器官体重指数,观察免疫器官的组织损伤情况。试验结果表明:3 代毒都具有较高免疫原性,但是20代毒仍具有较大的毒性,7 日龄接种会引起法氏囊的萎缩,造成持续的组织损伤;21 代毒、25代毒保护率高,无免疫抑制,是比较理想的疫苗来源;7日龄免疫较14日龄免疫更易造成组织损伤和免疫抑制,14日龄免疫较为合适。     

传染性法氏囊病不同代次细胞毒免疫效果的研究

将从山东省东营分离到的1株传染性法氏囊病病毒(IBDV)野毒(暂命名IBDV SDDY株,经鉴定该株与IBDV STC株具有较高的同源性)经SPF鸡胚传代,然后转为细胞培养,取第20代、21代、25代毒,以1倍剂量(3000TCID50/0.2mL)和5倍剂量不同的免疫剂量,分别在7日龄、14日龄对商品代海蓝褐蛋鸡进行免疫,并于免疫前用IBD-ELISA试剂盒检测IBDV母源抗体水平,于35日龄用传染性法氏囊病病毒超强毒(very virulent Infectious Bursal Disease Virus,vvIBDV)GX 8/99攻毒,在攻毒前再次检测IBDV的抗体水平,攻毒后观察记录各分组鸡的致病率和死亡率,并计算免疫器官体重指数,观察免疫器官的组织损伤情况.试验结果表明3代毒都具有较高免疫原性,但是20代毒仍具有较大的毒性,7日龄接种会引起法氏囊的萎缩,造成持续的组织损伤;21代毒、25代毒保护率高,无免疫抑制,是比较理想的疫苗来源;7日龄免疫较14日龄免疫更易造成组织损伤和免疫抑制,14日龄免疫较为合适.     

鸡传染性法氏囊病上海超强毒株NH99的分离与鉴定

通过对上海近郊某鸡场数群 10日龄左右的病鸡的临床症状、病理变化、病毒分离、纯化、动物回归、血清学检测及病毒核酸纯化、VP2基因序列的测定与分析 ,确认上海地区以发病日龄早 ,发病率、死亡率高为特征的鸡传染病为超强毒型鸡传染性法氏囊病 ,病原具有鸡传染性法氏囊病病毒超强毒 (vvIBDV)的分子特征 ,为vvIBDV。     

鸡传染性法氏囊病上海超强毒株NH99的分离与鉴定

通过对上海近郊某鸡场数群10日龄左右的病鸡的临床症状、病理变化、病毒分离、纯化、动物回归、血清学检测及病毒核酸纯化、VP2基因序列的测定与分析,确认上海地区以发病日龄早,发病率、死亡率高为特征的鸡传染病为超强毒型鸡传染性法氏囊病,病原具有鸡传染性法氏囊病病毒超强毒 (vvIBDV)的分子特征,为vvIBDV.     

鸡的法氏囊表面上皮的扫描电镜观察

扫描电镜观察鸡的法氏囊表面上皮形态变化的结果如下: 孵化第十三天的鸡胚,褶表面开始突起,法氏囊表面上皮起始分化成FAE和ISE两种不同的上皮细胞。两种表面上皮细胞的质膜上,均有分布均匀的微绒毛,FAE的微绒毛较粗大,ISE细胞的较细长。初生雏的法氏囊淋巴泸泡发达,FAE和ISE之间有明显的凹沟。前者的微绒毛较粗大而稀疏,后者的较细长而集密。一月龄的仔鸡法氏囊淋巴泸泡最发达,皮部的ISE与周围的ISE之间又形成一个凹沟。二月龄仔鸡法氏囊淋巴泸泡开始萎缩FAE也开始下陷,有的淋巴泸泡陷入间质中。 扫描电镜的观察,符合并证实了透射电镜观察表面上皮的形态变化。     

表达IBDV VP2融合蛋白的重组MDV的构建及其免疫特性

将增强型绿色荧光蛋白基因(eGFP)与鸡传染性法氏囊病病毒(IBDV)的VP2基因融合,插入马立克氏病毒(MDV)CVI988/Rispens的非必需区US10片段中,成功构建表达VP2融合蛋白的MDVCVI988转移载体pUC18-US10-VP2。将转移载体质粒与CVI988/Rispens疫苗毒共转染鸡胚成纤维细胞(CEF),筛选获得表达VP2融合蛋白的重组MDV(rMDV)。聚合酶链式反应(PCR)和间接免疫荧光实验(IFA)证明,rMDV传至第31代仍能稳定表达VP2融合蛋白。用rMDV免疫SPF鸡,进行IBDV攻毒保护试验,1日龄SPF鸡分别用1000PFU、2000PFU、5000PFU的rMDV进行免疫,33日龄用100LD50的IBDVJS超强毒进行攻毒,鸡的免疫保护率分别为50%、60%、80%。值得注意的是,5000PFU的rMDV一次免疫1日龄SPF鸡,其法氏囊组织病理损伤等级与IBD中等毒力活疫苗常规二次免疫相当(2·0/1·5),其保护效果无显著差异(p>0·05),而与非重组病毒免疫组相比较,保护效果差异显著(P<0·01),这表明构建的表达IBDVVP2融合蛋白的rMDV可以有效地为SPF鸡提供免疫保护作用。     

无法氏囊仔鸡脾脏发育的观察

用手术摘除法氏囊，酶标技术和生物统计学等方法，研究无法氏囊后仔鸡脾脏Ｂ淋巴细胞的发育，证明鸟类脾脏Ｂ淋巴细胞主要来源于法氏囊，但同时也有其它的来源。     

Immunological evidence for an H1° type of histone protein in chicken liver

We prepared monoclonal antibodies against chicken histone H5. These antibodies could be divided into two classes, and we present the results obtained with one representative antibody of each class. One class reacted exclusively with chicken H5, whereas the other additionally cross-reacted with rat H1(0) and with material present in adult but not embryonic chicken liver. The cross-reacting material in adult liver was identified by Western blotting as representing a minor band in histone preparations. The protein was not present in histone extracts from chicken erythrocytes. It is likely that this newly identified protein is a chicken H1(0) histone.     

Characterization of bovine plasma retinol-binding protein and evidence for lack of binding between it and other bovine plasma proteins.

Bovine retinol-retinol-binding protein (RBP) was isolated from serum as a free, uncomplexed protein under experimental conditions in which human, rabbit, and chicken retinol-RBP are present as tight complexes with prealbumin (thyroxine-binding protein). Purified bovine retinol-RBP formed tight complexes with purified human and chicken prealbumin in physiological ionic strength buffers as judged by gel filtration chromatography, hyperchromic effect on the absorption spectrum of retinol-RBP, and changes in the circular dichroism spectrum. Addition of purified human prealbumin to whole bovine serum shifted the elution position of the specific retinol-RBP fluorescence from a gel filtration column, indicating complex formation in the whole bovine serum. It was concluded from this series of experiments that bovine serum lacks a protein with the binding properties of prealbumin and that bovine retinol-RBP has the normal potential binding to human, chicken, and presumably other prealbumins. Bovine retinol-RBP has a molecular weight, amino acid composition, absorption, and fluorescence spectra which are indistinguishable from that of human retinol-RBP, although the magnitude of the optical rotatory strength of the induced circular dichroism signal at 330 nm was 50% larger in the bovine than in the human material (1.65 and 1.1 Debye-Bohr magnetons, respectively). About 12 liters of bovine and human urine were concentrated by pressure dialysis and a search was made for retinol-RBP using gel filtration and ion exchange chromatography. No retinol-RBP was found in either of these species. This suggested that if, indeed, bovine retinol-RBP is filtered through the kidney's glomeruli due to small molecular size (molecular weight 21,000), there are efficient mechanisms of tubular reabsorption.     

Distribution of Injected 14C-Diethylstilbestrol in the Chicken and Laying Hen

Distribution of intramuscularly administered 14C-diethylstilbestrol(DES)in the chicken and laying hen was studied by whole body autoradiography and impulse counting techniques. In both groups the liver showed the highest concentration of 14C. The 14C-level of the chicken liver and bile appeared greater than the same level in the hen. The 14C-content of the hen kidney was twice that found in the chicken. These findings could mean that the metabolic and excretory function of the kidney is less developed in the young chicken than in the adult bird and that the chicken liver may compensate for insufficient kidney function. The lower l4G-concentration of the chicken adrenal as compared to the hen, could indicate a reduced physiological activity of the young adrenal. The accumulation of radioactivity in the membrane of the follicular yolk should represent excretion. Low 14C-content was found in the skeletal muscle. It is concluded that consumer products based mainly on the liver from DES-treated chickens should not be used for consumption.     

Amino-terminal sequence of chicken preproalbumin

Poly(A)-containing RNA was isolated from chicken liver and translated in a reticulocyte lysate protein-synthesizing system in the presence of radiolabeled amino acids. Chicken albumin was isolated from the translation products by immunoprecipitation and subjected to automated Edman radiosequencing. Comparison with the sequence of proalbumin showed that the translocation product (preproalbumin) contains an NH2-terminal extension of 18 amino acid residues. The NH2-terminal sequence of chicken preproalbumin was as follows: Met-18-Lys-Asn-Val-15-Thr-Leu-Ile-Ser-Phe-10-Ile-Phe-Leu-Phe-Ser-5-Ser-Ala-Thr- Ser-1-Arg1, where Arg1 represents the NH2-terminal residue of proalbumin. This NH2-terminal extension is very rich in hydrophobic amino acid residues and is similar to the signal sequences found in other secreted proteins. The signal sequence of chicken preproalbumin shows considerable homology with the signal sequences of rat and bovine preproalbumins, but little homology with the signal sequences of other chicken preproteins.     

Isolation and characterization of cDNA clones for chicken major histocompatibility complex class II molecules

The chicken major histocompatibility complex (MHC), the B complex, is being intensively analysed at the DNA level. To further probe the molecular structure of chicken MHC class II genes, cDNA clones coding for chicken MHC class II (B-L) p chain molecules were isolated from an inbred G-B2 Leghorn chicken spleen and liver. Twenty-nine cDNA clones were isolated from the spleen and eight cDNA clones were isolated from the liver. Based on restriction maps, most clones could be clustered into one family of genes. Four cDNA clones were sequenced (S7, S10 and S19 from the spleen and L1, which was identical to S19, from the liver). Complete amino acid sequences of B-Lβ chain molecules were predicted from the nucleotide sequences of the cDNA clones. Although both the nature and the location of the conserved residues were similar in chicken and mammalian sequences, some species-specific differences were found, suggesting that the structures of the B-L molecules of this haplotype are similar, but not identical, to their mammalian counterparts.     

染色质在非洲爪蟾卵的无细胞系统中形成组装核的研究

从大鼠肝、鸡肝提取了染色质并从受精未孵育鸡蛋胚下表层卵黄颗粒提取了染色质,在荧光显微镜和电子显微镜下观察了这些染色质在无细胞系统中形成的组装核;研究二阶钙镁离子对形成组装核的影响。在有ＡＴＰ再生系统存在的无细胞系统中,围绕染色质能形成与间期细胞核相似的组装核;二价钙离子不利于形成组装核,而二价镁离子是形成组装核所必须的,但过高浓度的镁离子对形成组装核有抑制作用。     

The binding of calmodulin to myelin basic protein and histone H2B.

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.     

Comparison of calregulins from vertebrate livers.

Calregulins were purified from bovine, rabbit and chicken liver, and their structural properties were compared. Significant differences between the three calregulins include a lower Mr for chicken calregulin (57,000) than for rabbit and bovine calregulin (63,000), and the glycosylation of only bovine calregulin. Amino acid composition and peptide maps of the three calregulins were very similar. No major differences were detected in the Ca2+-binding properties of the three proteins. Zn2+-induced changes in calregulin conformation and hydrophobicity monitored by intrinsic protein fluorescence and the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulphonate were very similar, suggesting that the Zn2+-dependent increase in the hydrophobicity of bovine, rabbit and chicken calregulin was conserved. These studies more fully define what is a calregulin, demonstrate that calregulin is a relatively invariant constituent of vertebrate liver, and indicate that calregulin structure has been highly conserved in bovine, chicken and rabbit liver.     

Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine enzyme.

The glutamate dehydrogenase from a single human liver has been studied. The subunit size was found to be 55,200 +/- 1,500 by sedimentation equilibrium. The partial specific volume is 0.732 as calculated from the amino acid composition. The sequence was determined by isolation of peptides after cyanogen bromide (CNBr) cleavage; the fraction containing the largest peptides was hydrolyzed by trypsin after maleylation. Studies on these peptides accounted for 454 residues of the 505 residues that are presumably present in the protein. For the 51 residues that were not represented in isolated peptides, we have tentatively assumed that the sequence is the same as that of the bovine enzyme. Methionine and arginine residues in these peptides could be placed on the basis of the specificity of cleavage by CNBr or trypsin. In all, 349 residues were placed in sequence, and were aligned by homology with the corresponding peptides of the bovine and chicken enzymes. From the present information, there are 24 known differences in sequence between the human and bovine enzymes and 41 between the human and chicken enzymes. In addition, the human enzyme contains 4 additional residues at the NH2 terminus as compared to the bovine enzyme. In a peptide from the human enzyme, an additional residue, isoleucine 385, was detected by automated Edman degradation. Reinvestigation of the bovine sequence demonstrated that this residue is also present in the bovine enzyme (and presumably in the chicken enzyme also). Residue 384 of the bovine enzyme, previously reported as Glx has now been shown to be glutamine.     

Chicken microsomal albumin: Amino terminal sequence of chicken proalbumin

Chicken liver microsomes contain an albumin having an isoelectric point approximately 0.2 pH unit in excess of that of chicken serum albumin. Although the serum protein is also present in microsomes, only the basic albumin there becomes labelled and undergoes turnover $\text{in vivo}$. Sequence analysis of the purified basic microsomal albumin indicates that the first twelve residues are: $\text{Arg-Asn-Leu-Gln-Arg-Met-Ala-Arg}\text{-Asp-Ala-Glu-His}$. The data suggest that the octapeptide (underlined) is attached to the amino terminus of chicken serum albumin (the last four residues). The amino terminal sequence of the serum albumin precursor in chicken liver is thus markedly different from that of the rat and bovine proalbumins.