猪繁殖与呼吸综合征病毒A06株单克隆抗体的制备及鉴定

用纯化的猪繁殖与呼吸综合征病毒(PRRSV)A06株病毒抗原免疫Balb/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合.经间接ELISA方法筛选,有限稀释法克隆,获得4株稳定分泌特异性单克隆抗体的杂交瘤细胞株,分别命名为3D12、4E10、6E2和7G6.其细胞培养上清效价分别为1:256、1:512、1:512和1:256,小鼠腹水效价分别为1:1.024×106、1:2.048×106、1:1.024×106和1:2.048×106.亚型鉴定表明,3D12、4E10、6E2和7G6分别为IgG2b、IgG2b、IgG1和IgG2a.ELISA检测结果显示,4种PRRSV单克隆抗体仅与PRRSV反应,不与其他病毒反应,表明均为抗PRRSV的型特异性单克隆抗体.对单克隆抗体腹水液进行IgG提取纯化,SDS-PAGE电泳验证,仅出现两条清晰条带,无杂带出现,证明纯化效果较好.这4种单克隆抗体的获得,为建立PRRSV检测方法提供了强有力的工具.     

血清中IBV特异性IgG及总IgG含量变化规律的研究

本研究采用间接酶联免疫吸附试验对人工感染鸡传染性支气管炎病毒的SPF鸡血清中特异性抗体及总IgG含量进行检测并比较其相关性.结果表明感染IBV-M41第1 d、3 d、5 d、7 d、10d、14d、21 d、28 d的血清中特异性抗体水平随感染天数增加而升高,于第14天达到最高,之后略呈降低趋势,与健康对照比较差异显著(P＜0.01);总IgG含量较健康对照组增幅小,差异显著(P＜0.01).同时,两者的消长趋势基本一致.     

Development of an Indirect ELISA for the Detection of Antibodies against Peste-des-petits-ruminants Virus in Small Ruminants

Peste des petits ruminants (PPR) is an acute, febrile, highly contagious and economically important viral disease of small ruminants. A polyclonal antibody based indirect ELISA was developed for detection of antibodies to PPR virus in the serum samples of goats and sheep using purified PPR viral antigen propogated in Vero cell culture. A threshold (cut-off) value was set as twice the mean of the negative population based on the distribution of known negative serum samples in respect of PPR virus antibodies in the test. A total of 1544 serum samples from goats and sheep were screened by indirect ELISA and competitive ELISA. The indirect ELISA compared very well with competitive ELISA, with a high degree of specificity (95.09%) and sensitivity (90.81%). When compared with virus neutralization test, the present assay had 100% specificity and 80% sensitivity. With serum samples, the assay could clearly differentiate animals from the infected population from uninfected ones. These results suggest that the indirect ELISA may be a good alternative tool to competitive ELISA for seroepidemiological surveys.     

猪伪狂犬病病毒gE抗体胶体金免疫层析检测方法的建立及应用

以纯化的抗人红细胞单链抗体(ScFv)-猪伪狂犬病病毒(PRV)gE蛋白双功能融合蛋白为诊断抗原和胶体金标记物,以羊抗猪IgG包被硝酸纤维膜作为质控带,制作检测猪伪狂犬病毒gE抗体的双抗原胶体金试纸条。利用方阵滴定试验筛选出金标抗原最佳工作浓度为17.6μg,检测线诊断抗原最佳标记量为1.76μg,血清最佳稀释度为1∶10,作用时间15 min,与猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙型脑炎病毒(JEV)、猪布鲁菌(Brucella)阳性血清和PRVgE缺失疫苗接种的猪免疫血清检测线均不出现红色条带,与PRV标准阳性血清反应检测线出现红色条带。试纸条操作简单,肉眼于15 min内可判定结果;试纸条在室温保存6个月,其特异性和敏感性没有明显变化;与美国IDEXX和法国LSI gE-ELISA抗体检测诊断试剂盒检测结果比较,1 164份猪血清的符合率均为90.55%。制备的胶体金试纸条具有操作简便、敏感性和特异性较高的特点,可用于PRV野毒感染的快速筛查。     

猪细小病毒NS基因原核表达与多克隆抗体制备

旨在制备抗猪细小病毒(PPV)非结构蛋白共有氨基酸的NS多肽多克隆抗体。根据GenBank(MK993540)公布的PPV基因组序列,克隆其非结构蛋白NS1、NS2共有基因序列(NS基因),并进行生物信息学分析。进而将NS基因克隆至原核表达载体pET-32a,构建重组质粒pET-32a-NS,转化至大肠埃希菌Rosetta(DE3)中进行诱导表达,利用镍柱亲和层析技术纯化表达的重组多肽,用重组多肽免疫Balb/c小鼠制备多克隆抗体。结果显示,PPV杨凌株NS基因长258bp,编码86个氨基酸的多肽,是具有亲水性的非跨膜NS多肽,具有大量的B细胞线性表位。NS多肽在37℃、0.8mol/L IPTG条件下,诱导6h有大量的可溶性表达。免疫印迹试验结果显示,该多肽具有较好的抗原性。用纯化NS多肽免疫小鼠后获得鼠抗NS多肽的血清抗体效价为1∶12800。用制备的NS多克隆抗体检测纯化的NS重组多肽及真核表达的NS1蛋白,均能检测出相应特异性条带,为进一步研究NS蛋白在PPV致病过程中的作用奠定了基础。     

羊胎盘转移因子注射液体液免疫调节作用的研究

[目的]通过研究羊胎盘转移因子注射液对实验动物免疫球蛋白、B淋巴细胞和抗体形成细胞数量变化的影响,探讨受试药品对实验动物体液免疫的调节作用,进而评估其临床体液免疫调节的使用价值。[方法]试验1:用小白鼠进行免疫球蛋白测定试验,比较给药前后小白鼠血清免疫球蛋白的变化;试验2:用大白兔进行EAC玫瑰花环试验,测定给药前后B淋巴细胞数量的变化;试验3:用小白鼠进行溶血空斑试验,比较试验组和对照组抗体形成细胞数的变化。[结果]试验1:试验组小白鼠血清免疫球蛋白含量显著提高(P<0.05);试验2:试验组受试动物EAC玫瑰花环的成环细胞数显著高于对照组(P<0.05);试验3:试验组小白鼠抗体生成细胞的数量极显著增加(P<0.01)。[结论]羊胎盘转移因子注射液对受试动物体液免疫有显著调节作用。     

Dot-ELISA检测兔疥螨抗体方法的建立和应用

将从病兔痂皮内收集的疥螨经研磨、冻融、离心后,制成可溶性抗原,作为诊断抗原,建立Dot-ELISA方法检测兔疥螨血清抗体.研究确定了该方法的最佳工作条件.制备的诊断膜片特异性强,不与兔瘟病毒、兔大肠埃希菌、兔附红细胞体等阳性血清反应.膜片具有良好的灵敏性,高免血清作1∶210稀释亦能检出;重复性试验表明该法重复性良好.诊断膜片在4 ℃保存5个月其检测活性不变.结果表明,建立的Dot-ELISA可用于免疥螨抗体的检测.     

Plantago ovata in broiler chicken nutrition: Performance,carcass criteria,intestinal morphology,immunity, and intestinal bacterial population

In this experiment, the effect of dietary Plantago ovata (PO) on performance, carcass criteria, intestinal morphology, immunity, and intestinal bacterial population of broiler chickens was evaluated. A total of 250 one‐day‐old male broiler chicks (Ross 308) were randomly assigned to five treatments containing 0, 5, 10, 15, or 20 g/kg of PO with five replicate pens and 10 birds in each replicate. Dietary PO increased body weight gain and decreased feed conversion ratio in the finisher period, improving the performance index (p < .05). Dietary treatments had no effects on carcass criteria, but breast meat percentage showed an increasing trend with incremental levels of PO in the diet (p = .069). The length of small intestine, especially jejunum section, as well as the villus height, villus width, villus area, and goblet cell numbers were significantly increased with supplemental PO (p < .05). Humoral and cellular immunity parameters, and oxidation stability of meat were improved due to use of dietary PO (p < .05). Dietary PO decreased the CFU of Escherichia coli, whereas the Lactobacilli population was increased (p = .001). Broken‐line regression revealed that dietary PO at the rate of 10 g/kg may results in the best performance in broiler chickens. This study showed that PO at the level of 10 g/kg could be considered as a beneficial feed additive in broiler diet.     

兔病毒性出血症母源抗体动态与抗病毒感染的关系和对策

兔病毒性出血症疫苗免疫母兔所产６、１５、３０、４５、６０、日龄共１２６只兔的母源抗体随日龄增长而下降，至３０￣４５日龄，４８￣８５％的兔为阴性，其平均值为２＾３。强毒攻击耐过母兔所产的未吃初乳、已吃初乳、１５、３０、４５、６０、９０及１１４日龄以上兔１８６只的母源抗体也随日龄增长而消失，但消失时间长，其平均值至１１４ｄ后才降至２＾３。已吃初乳仔兔抗体平均值比未吃初乳高，未吃初乳兔的抗体与母兔基本持     

高致病性猪繁殖与呼吸综合征病毒HuN4株GP3单克隆抗体的制备及其抗原表位的鉴定

为制备抗高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV) GP3株的单克隆抗体(MAb),本研究将HP-PRRSV HuN4株免疫BALB/c小鼠,以该病毒感染的细胞及真核表达的GP3蛋白为检测抗原,经间接免疫荧光(IFA)筛选获得了一株稳定分泌MAb的细胞株,命名为4G5.抗体亚类鉴定重链类型为IgG1,轻链类型为K.该MAb细胞培养上清及腹水IFA效价分别为1∶256和1∶1 280.IFA结果显示,MAb 4G5能够识别CH-1R、JXA1-R、HP-PRRSV HuN4株及其疫苗株HuN4-F112,而不识别RespPRRS MLV病毒株.Western blot结果显示,4G5与HP-PRRSV HuN4株及原核表达的GP3蛋白均可以反应,表明其针对的抗原表位为线性表位,中和试验结果显示该MAb无中和活性.通过截短表达GP3蛋白鉴定该MAb抗原表位识别序列为74WCRIGHDRCS83.本研究获得的MAb为进一步研究HP-PRRSV GP3蛋白的结构及功能奠定了基础.