原位ELISA法在抗巨细胞病毒药物筛选中的应用

建立一种体外筛选抗 CMV药物的 is ELISA法。方法 采用 ELISA测定固定后培养细胞上的病毒抗原 ,并与空斑减少试验 ( PRA)法进行比较。结果 is ELISA法病毒接种量范围宽 ,重复性好 ,结果客观可靠 ,操作简便 ,只需 5 d即可观察结果。结论 ELISA可代替 PRA法用于体外抗 CMV药物的筛选。

Establishment of an Indirect ELISA with the Major Epitope Domain of ApxⅡ of Actinobacillus pleuropneumoniae Expressed in Prokaryotic Cells

[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae （APP） using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a （＋） to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 （DE3） for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1：100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

鼻息肉组织中固生蛋白C的表达与转化生长因子β1的关系

目的探讨细胞外基质(extracellularmatrix,ECM)成分固生蛋白C(tenascinC,TNC)在鼻息肉组织中异常表达的原因。方法采用免疫组化方法检测TNC和转化生长因子β1(transforminggrowthfactor-β1,TGF-β1)在鼻息肉组织中的表达和关系。进一步采用细胞培养、实时定量RTPCR和原位ELISA技术研究TGFβ1及嗜酸粒细胞对人呼吸道上皮细胞系BEAS2B细胞TNC表达的调控作用。结果①TNC和TGF-β1蛋白在鼻息肉组织中的表达显著上调,TNC表达强度与TGFβ1阳性细胞总数(r=-0.58,P<0.01)和TGFβ1阳性的嗜酸粒细胞数显著相关(r=-0.61,P<0.01);②浓度为1ng/ml和10ng/ml的TGFβ1刺激4h后BEAS2B细胞TNCmRNA的表达分别为未刺激状态下的(7.20±3.43,x±s,下同)倍和(22.48±5.35)倍,与未刺激状态下的表达水平相比差异有统计学意义(P值<0.01);与刺激24h后TNC蛋白表达的荧光强度相比差异亦有统计学意义(P<0.05);③BEAS2B细胞和嗜酸粒细胞以2∶1、1∶1和1∶2的数量比例进行共培养,4h后BEAS2B细胞TNCmRNA的表达与未刺激状态下的表达水平相比,差异均有统计学意义(P值均<0.01);24h后TNC蛋白表达的荧光强度与未刺激状态下的荧光强度相比差异亦均有统计学意义(P值均<0.05);嗜酸粒细胞的这种诱导作用可以被抗TGFβ1的中和抗体显著抑制(P<0.05)。结论TGF-β1和嗜酸粒细胞可以诱导呼吸道上皮细胞对TNC的表达,嗜酸粒细胞的作用部分通过TGFβ1介导,鼻息肉组织中TNC表达的增高同嗜酸粒细胞来源的TGF-β1有关。