Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.     

倍他米松ELISA检测方法的建立

为建立倍他米松(BET)ELISA检测方法,先使BET与琥珀酸酐反应,再采用活化酯法与牛血清白蛋白(BSA)偶联制备免疫原;其次,利用免疫动物获得多克隆抗体,经Sephrose 4B-proteinA方法对抗体进行纯化;最后,建立倍他米松ELISA检测方法。结果表明,免疫抗原偶联成功,获得了亲和力较高的多克隆抗血清,间接竞争ELISA测定其滴度为102 400、IC15为6.60×10-5 ng/mL和IC50为0.97ng/mL。该方法适用于残留在动物性食品中的BET现场大批量检测。     

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.     

重氮化法合成全抗原的研究

[目的]制备氯霉素全抗原,探索氯霉素合成的方法,为动物免疫试验作准备。[方法]采用重氮化法合成氯霉素卵清蛋白全抗原,通过紫外法和红外光谱法测定合成的偶联效果。[结果]重氮化法可用于制备较高偶联率的氯霉素全抗原,用于免疫动物制备抗体。[结论]该研究为合成高效价的抗体奠定了基础。     

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.     

H5亚型禽流感病毒血凝素基因的克隆及表达

研究以RT-PCR方法从H5N1亚型禽流感病毒A/DKZJ/12/00(H5N1)中获得禽流感病毒HA部分基因,将目的基因定向克隆到原核表达载体pET-32a,将测序和酶切验证正确的阳性重组质粒转化大肠杆菌BL21,经IPTG诱导表达。结果表明该蛋白得到了可溶性的表达,表达蛋白的分子质量为25ku;Western-blot分析结果表明,该蛋白可以与禽流感H5阳性血清反应(禽流感的单克隆抗体所识别),检测HA的抗体时具有良好的免疫原性。     

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。