乳酸菌微生态制剂对肉鸡生产性能及免疫机能的影响

本实验利用乳酸菌微生态制剂饲喂AA肉鸡,通过检测鸡增重、料重比、免疫器官指数、抗体效价以及盲肠内容物的乳酸菌和大肠杆菌数量,探索乳酸菌微生态制剂对肉鸡生产性能及免疫功能的影响。首先将150只肉鸡随机分成3组,第1组在饮水中添加0.3%的乳酸菌微生态制剂,第2组添加抗生素饮水,第3组为空白对照组,7日龄时接种新城疫疫苗;于每周末称取各组鸡的体重、耗料,计算增重和耗料量;在21日龄和35日龄时,采取各组鸡的脾脏、法氏囊和胸腺、分别计算免疫器官指数;用HI和微量平板滴种法分别检测抗体效价和盲肠内容物乳酸菌、大肠杆菌数量。结果显示,添加0.3%乳酸菌微生态制剂组与对照组和抗生素组鸡比较,分别提高鸡体重15.55%和8.57%(差异显著P<0.05),料重比分别降低5.11%和4.57%;于21日龄和35日龄时检测发现,添加0.3%乳酸菌微生态制剂组与对照组鸡比较,脾脏、法氏囊和胸腺指数分别提高了44.26%1、3.51%、33.96%和67.14%、16.47%3、8.16%;抗体水平分别升高了1.8log2和2 log2,差异显著(P<0.05),与抗生素组比较,抗体效价分别提高了1.4log2和1.75log2,差异显著(P<0.05);另外,21日龄和35日龄时,与对照组比较,添加0.3%乳酸菌微生态制剂鸡盲肠内容物中乳酸菌数量分别增加1.28log2和0.27log2,差异显著(P<0.05),同时分别使大肠杆菌数量减少1.88log2和0.84log2,差异显著(P<0.05)。本研究为合理开发利用乳酸菌微生态制剂提供了理论依据和技术支持。     

抗沙丁胺醇单克隆抗体制备与鉴定

[目的]制备特异性抗沙丁胺醇单克隆抗体,为其免疫学检测方法的建立奠定基础。[方法]用SAL-BSA抗原免疫BALB/c小鼠;使用细胞融合技术建立抗SAL的单克隆抗体（SAL mAb）杂交瘤细胞株;体内诱生腹水法制备SAL mAb,并鉴定其免疫学特性。[结果]筛选出2D9和1C4两株杂交瘤细胞,其细胞培养上清效价达1∶104,腹水效价达1∶106;免疫球蛋白亚型鉴定为IgG1;抗体对SAL的半数抑制浓度（IC50）为1.14 ng/ml;与沙丁胺醇和盐酸克伦特罗的交叉反应率（CR）分别为100.00%和26.09%,与其他化合物无交叉反应。[结论]获得了高效价、敏感且异性的抗SAL抗体,为沙丁胺醇残留的免疫学检测方法的建立奠定了基础。     

1株抗鸡CD8α链单克隆抗体的鉴定

[目的]获得研究鸡CD8分子的单克隆抗体。[方法]用自行设计的引物PCR扩增鸡CD8分子α链部分基因片段,构建2个原核重组质粒,该片段经原核表达和纯化后免疫BALb/c小鼠,最后将免疫小鼠脾细胞与骨髓瘤细胞融合,细胞上清液用ELISA检测。[结果]PCR扩增获得大小约为510 bp的基因片段。构建的pET-32a-CD8α导入大肠杆菌,诱导表达获得了大小为39 kD的融合蛋白H is-CD8α。融合细胞经亚克隆,其上清液用ELISA检测和筛选,获得1株稳定传代和分泌抗CD8α的抗体的杂交瘤细胞株。该株细胞被命名为C11。上清液的ELISA抗体效价达1：600。[结论]该研究以原核表达的CD8α为抗原制备了1株单克隆抗体。     

鸡新城疫病毒HN基因片段的原核表达及其单抗制备

[目的]研究NDV-HN蛋白抗原表位在动物免疫应答中的作用特点。[方法]根据HN基因和载体pGEX-4T-1的多克隆位点自行设计1对引物,以该实验室保存的重组质粒为模板,进行PCR扩增,再将其所得片段定向插入pGEX-4T-1,构建重组质粒,并对该重组质粒进行鉴定;再将构建的重组质粒在大肠杆菌中表达;最后,鉴定了相应单克隆抗体。[结果]PCR结果表明,得到了与预期结果一致的长度为850 bp的片段;重组质粒鉴定结果表明,成功构建了包含目的基因片段的重组表达质粒pGEX-4T-HN23;重组质粒经原核表达获得大小为57 kD的融合蛋白,免疫Balb/c小鼠试验表明,用免疫鼠脾细胞与骨髓瘤细胞融合,经ELISA筛选和3次亚克隆获得2株稳定传代（可传20代以上）和分泌抗NDV-HN抗体的杂交瘤细胞株。[结论]获得的鸡新城疫病毒HN单克隆抗体,为进一步研究HN在致病过程的作用及特异性诊断提供了重要试验材料。     

感染鸡贫血病毒雏鸡接种新城疫疫苗后免疫保护效应及其机理研究

 雏鸡 1日龄人工感染鸡贫血病毒 (CAV) ,于 8日龄接种 L asota疫苗 ,免疫后 2 8d进行新城疫强毒 (v NDV)攻击 ,以同龄未感染 CAV和用 L asota疫苗免疫并经 v NDV攻毒雏鸡为对照 ,检测其免疫保护效应 ,胸腺和脾脏 T细胞数量及 T细胞增殖反应 ,法氏囊和脾脏 Ig G+、Ig M+、Ig A+抗体生成细胞数量 ,血清免疫球蛋白 Ig G、Ig M、Ig A含量 ,血凝抑制抗体 (HI)滴度等。结果表明 ,CAV人工感染并用新城疫 (ND)疫苗免疫雏鸡经 v NDV攻击后的免疫保护率为 6 0 % ,对照组雏鸡 v NDV攻击后免疫保护率为 10 0 % ,胸腺和脾脏 T细胞数量和 T细胞增殖反应、法氏囊和脾脏Ig G+ 、Ig M+ 、Ig A+ 抗体生成细胞数量、血清 Ig G、Ig M、Ig A含量及 HI抗体滴度 ,均较对照组雏鸡显著降低。说明感染鸡贫血病毒雏鸡接种新城疫疫苗后的免疫保护效应及免疫功能明显降低。     

大豆主要过敏原Gly m Bd30K蛋白单克隆抗体的制备与应用

利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合.采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化.采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体...     

兔病毒性出血症首免时间的研究

针对生产上出现的从断奶育成这一年龄段多发兔病毒性出血症的实际情况，采用快速、敏感的Dot-ELISA方法，通过测定不同免疫状态下的母兔所产兔的母源抗体动态变化规律，并进一步测定不同抗体效价的成年兔攻毒后是否得到保护的结果，再结合生产实际，得出兔病毒性出血症首免时间为30日龄，且剂量加倍。     

Relation of Japanese <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">vesicatoria</Emphasis> with worldwide strains revealed with three specific monoclonal antibodies

Strains of Xanthomonas campestris pv. vesicatoria Dye 1978 (Xcv), the causal agent of bacterial spot, have been classified into two groups based on their ability to hydrolyze starch. Three monoclonal antibodies (MAbs), 7AH10, 5HB3, and 4AD2, were produced immunized against the living bacteria and were specific to and could distinguish Xcv strains able or unable to hydrolyze starch (Amy⁺ or Amy^–). The MAb 7AH10, obtained against strain UPB141(Amy^–) reacted in an enzyme-linked immunosorbent assay with all the Amy^– strains (n = 19) and 1 of 11 Amy⁺ strains. Against Xcv 2625, an Amy^– unusual phenotype strain, MAb 5HB3, recognized 97% of our worldwide collection of Xcvs (n = 30). Also against that strain, the MAb 4AD2 reacted with none of the homologous Amy^– phenotypes and with 90% (n = 11) of the heterologous Amy⁺ phenotypes. For all the MAbs, cross reactions with other pathovars or species were less than 4% (n = 67). By assaying a Japanese collection of strains against the three MAbs, the Amy⁺ strains were distinguished from the Amy^– strains, and their relation with other world strains could be demonstrated. All the MAbs reacted with the lipopolysaccharide fraction of the bacterial cell wall during immunoblotting.     

Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia

Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9–36.7%, but was eliminated by the use of the F(ab)₂ fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs) test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.     

Thrombolytic properties of a conjugate of liposomal urokinase with a monoclonal antibody specific for cross-linked fibrin in the model of rabbit artery thrombosis

AIM: To observe the targeting thrombolytic effect of a monoclonal antibody specific for cross-linked fibrin connected with liposomal urokinase（UK） in the model of rabbit artery thrombosis.METHODS: Preparation of thrombolytic solutions: with the method of controllable dialysis eradicator in liposomat,empty liposomes,liposomally entrapped urokinase and liposomally entrapped urokinase linked with D-dimer antibody were made.Experiment in vivo: a rabbit thrombosis model of abdominal aorta was induced by ferric chloride.When the blood pressure fall to the lowest,5 different solutions were separately imported (PBS,maximal-level UK,Ab/Lip/UK,Ab/UK-Lip,Ab-UK-Lip) and observed for 40 min continuously.RESULTS: The varieties of 5 groups blood pressures were analyzed with q-test.Significant differences were observed among Ab-UK-Lip group,maximal-level UK group and others (P<0.01).There were marked differences between Ab-UK-Lip group and others in wet weight of thrombuses (P<0.05).However,the quantity of UK in Ab-UK-Lip group was 1/3 of that in maximal-level of UK group.CONCLUSION: The thrombolytic effect on Ab-UK-Lip group is similar to that on maximal-level of UK group,but the dosage of UK is less,and the side-effect is light.This shows,as a homing-device of liposomal urokinase,the D-dimer antibody has targeting effect and the agent of Ab-UK-Lip will be attached importance by-and-by.