口蹄疫荧光抗体的制备与应用

用猪抗口蹄疫病毒IgG制备荧光抗体,检测口蹄疫病毒感染BHK-21细胞中的病毒抗原,结果表明,该方法可以检出口蹄疫病毒感染BHK-21细胞中的病毒抗原,荧光抗体的工作浓度确定为1：40,且这种荧光能被FMDV阳性血清特异性地抑制。用制备的荧光抗体检测感染口蹄疫病毒CS株和LB株的BHK～21细胞抹片和飞片,结果均为阳性,空白对照均为阴性。对于低代次的分离毒,即使感染细胞产生明显的CPE,采用反向间接血凝试验也不能检出收获细胞液中的病毒抗原,本试验弥补了这一缺陷。采用直接荧光抗体法可确定病毒在感染细胞中增殖的位置,可作为兽医临床诊断方法之一。     

鸡堆型艾美尔球虫3-1E抗原表位的生物信息学预测

应用生物信息学分析和预测鸡堆型艾美尔球虫3-1E蛋白二级结构和抗原表位,为确定和筛选优势表位,研制安全、高效表位疫苗奠定基础。以克隆获得的鸡堆型艾美尔球虫3-1E蛋白氨基酸一级结构为基础,采用DNAStar软件和生物信息学在线分析程序Prot Param、SOSUI、IEDB、SYFPEITHI等预测3-1E蛋白理化性质和二级结构,并分析其序列跨膜区、可溶性、亲水性、可及性、柔韧性参数以及抗原指数,预测B细胞表位和T细胞表位的可能区域。3-1E蛋白由170个氨基酸组成,分子式C818H1257N213O272S3,分子质量18.5234 ku,理论等电点为4.25,半衰期为10 h;无跨膜区均为膜外区,是一种可溶性蛋白。其二级结构中α螺旋占31.76%,β转角为12.35%。B细胞表位预测区域:44-49、65-67、86-87、111-116;分值较高的T细胞表位区域:52-60、94-102、97-105、154-162、157-165。运用生物信息学方法确定3-1E存在多个抗原表位,对进一步研究3-1E的抗原性和研发优势表位疫苗具有重要意义。     

猪流行性腹泻病毒血清IgG间接ELISA检测方法的建立

以大肠杆菌表达系统制备的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)中和抗原S1D(1μg/mL)每孔100μL进行包被,利用HRP-兔抗猪IgG(1∶20 000)建立了检测猪血清中抗PEDV IgG的间接ELISA方法。阴性判断临界值(M+2SD)为0.632,批内和批间检测的平均变异系数分别为2.3%和3.1%,该方法与商品化的PEDV抗体间接ELISA检测试剂盒同时检测50份临床血清样品,均为阳性,上述结果表明该方法可用于PEDV血清IgG的临床检测。     

Expression and clinical significance of RhoC and Ki-67 in human esophageal squamous cell carcinoma tissues

AIM: To investigate the expression and significance of RhoC and Ki-67 in human esophageal squamous cell carcinoma (ESCC) tissues.METHODS: The expression of RhoC and Ki-67 was detected in 52 specimens of ESCC by the method of immunohistochemistry. The clinicopathological features were also analyzed.RESULTS: The expression of RhoC was detected in 32 of the total 52 (61.5%) cases of human ESCC tissues, significantly higher than that in the adjacent histologically normal epithelium, which was only in 11 of 37 cases (29.7%, P<0.05). RhoC expression was closely correlated with clinical tumor-node-metastasis (TNM) stage (P<0.05) and lymph node metastasis (P<0.05) in ESCC. The over-expression of RhoC was positively correlated with Ki-67 in ESCC (r=0.322, P<0.05).CONCLUSION: The over-expression of RhoC protein significantly correlates with advanced TNM stage, lymph node metastasis and cell proliferation ability of ESCC. Therefore, RhoC may be a new auxiliary parameter for early diagnosis and prognostic evaluation of ESCC.     

Intrathymic inoculation of liver specific antigen protects hepatocytes from apoptosis after liver allotransplantation

AIM: To study the effects of intrathymic inoculation of liver specific antigen (LSA) on hepatocyte apoptosis after liver allotransplantation. METHODS: Orthotopic liver transplantation was used in this study. Group Ⅰ: syngenic control (Wistar-to-Wistar); Group Ⅱ: acute rejection (SD-to-Wistar); Group Ⅲ: thymus inoculation of SD rat LSA day 7 before transplantation. The observation of general situation and survival time, hepatocyte apoptosis and LAT expression in liver transplants were used to analyze immune state of animals in different groups. RESULTS: The general situation of group Ⅰ was very well after transplantation. Recipients of groupⅡ lost body weight progressively and all died within day 9 to day 13 post transplantation. As for group Ⅲ, the general situation of recipients was remarkably better than that in group Ⅱ. The positive cells of apoptosis in group Ⅲ detected by TUNEL were not significantly different from that in group Ⅰ, but was significantly lower than that in group Ⅱ. LAT was detected at any time in group Ⅱ with peak expression at day 5 and day 7 post transplantation. In contrast, LAT was not detected in any other groups. CONCLUSION: Intrathymic inoculation of LSA protects hepatocytes from apoptosis after liver allotransplantation.     

Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd

OBJECTIVE: To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. PROCEDURE: Sera from the whole herd (n = 266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. RESULTS: The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre > or = 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT > or = 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT > or = 160 were immunoblot +ve. The association between seropositivity (IFAT > or = 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). CONCLUSIONS: The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests.     

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.     

鸭疫里默氏杆菌基因组文库的构建及免疫原性基因初步筛选

以血清1型鸭疫里默氏杆菌(RA)CH-1株为材料提取基因组DNA,采用限制性内切酶Sau3A进行部分酶切消化,纯化回收1.0~6.5 kb片段,用T4DNA连接酶将回收片段与经BamH酶切并去磷酸化的ZAP Express载体进行连接,用噬菌体包装蛋白包装。经测定包装滴度为5.23×106pfu/mL,蓝白斑筛选重组率为96.8%,表明已成功构建血清1型RA CH-1株部分基因组文库。在此基础上以RA抗血清为抗体探针进行免疫筛选,获得3个阳性克隆,经多次重复筛选后再体内删除,得到含有插入片段的质粒并测序。生物信息学分析结果显示,该序列(GenBank登录号:DQ151838)含有1个编码369个氨基酸的完整开放阅读框架,是尚未见报道的RA基因序列,并且存在多个预测的抗原表位。     

柔嫩艾美耳球虫表面抗原SAG2基因的克隆与表达

根据生物信息学预测的基因序列设计引物,应用RT-PCR方法从柔嫩艾美耳球虫第二代裂殖子总RNA扩增获得了鸡柔嫩艾美耳球虫表面抗原2(surface antigen 2,SAG2)基因序列,将其与pGEM-T easy载体连接后转化E.coliDH5α,筛选阳性克隆,以带有限制酶切位点的特异性引物用PCR方法扩增不含SAG2 N端信号肽序列的ORF序列后克隆至表达载体pET-32 a(+),构建了重组表达质粒pET-32 a(+)-SAG2,并将其转化至E.coliBL21(DE3)。经IPTG诱导,获得了SAG2重组抗原在大肠杆菌的高效表达,重组蛋白的表达量约占菌体总蛋白的35%,融合蛋白的分子量约为47 ku。菌体经超声处理后进行SDS-PAGE分析表明,表达蛋白以包涵体的形式存在。     

不同佐剂和免疫途径对柔嫩艾美耳球虫SO7抗原免疫保护效果的影响

利用PGEX-6P-1融合表达系统将SO7基因在大肠杆菌中进行融合表达,对表达产物进行初步纯化和复性,制备免疫原。分别使用不同剂量的人参总皂甙和重组γ-干扰素以及弗氏完全佐剂作为免疫佐剂,于5日龄、12日龄和19日龄对雏鸡进行3次免疫,同时设立蛋白口服免疫组、蛋白皮下注射免疫组、卵囊口服免疫组、未免疫未攻毒组和未免疫攻毒组作对照,于26日龄用105个柔嫩艾美耳球虫卵囊进行攻毒,8d后扑杀,对各组的存活率、相对增重率、病变减少率、相对卵囊产量和抗球虫指数ACI等指标进行统计分析,比较免疫保护效果。与非免疫攻毒组以及不加佐剂的免疫攻毒组相比,佐剂使用后各组的增重、病变记分、相对卵囊产量、ACI等指标均有一定的改善,对柔嫩艾美耳球虫的人工感染可以提供部分保护。3种佐剂中,γ-干扰素和人参总皂甙能增强重组蛋白的免疫保护效果,且佐剂的剂量对免疫保护的效果有一定的影响,以5000Uγ-干扰素效果最佳,效果与E.tenella活卵囊免疫相当,弗氏完全佐剂的免疫增强效果不明显。单独使用重组蛋白皮下注射或口服免疫,虽有一定的保护作用,但不明显。一定剂量的γ-干扰素对SO7抗原的免疫保护效果起明显的增强作用,是一个具有很好临床应用前景的新型佐剂。