摘要
本实验通过将质粒PGEX-ORF2转化大肠埃氏菌BL21(DE3)的方法,经IPTG诱导,成功表达了重组猪圆环病毒Ⅱ型ORF2(PCV-ORF2)的融合蛋白。通过对以包涵体形式表达的融合蛋白进行复性、透析得到重组融合蛋白,并以此为抗原免疫家兔,制备兔抗PCV-ORF2的IgG抗体,再以此IgG为第一抗体,选择最佳工作浓度,采用免疫组织化学Envision二步法,对肺组织中的病毒抗原进行定位、定性和定量检测,确立了诊断标准,构建了PCV2的Envision法诊断体系。试验采用RT-PCR诊断方法作为印证,并与间接ELISA诊断方法进行对比。试验结果证明,本试验制备的兔抗PCV-ORF2纯度高,特异性强,效价可达到1:10240(2~(10))。肺组织阳性表达主要出现在肺巨噬细胞、肺泡上皮细胞和支气管粘膜上皮细胞的胞浆内。本研究构建的免疫组织化学诊断体系,不但可以对PCV2进行实验室鉴别诊断,也能广泛应用于基层兽医单位,及时明确诊断抗原,提高临床确诊率,更快速有效地防制该病、减少经济损失。同时本研究成果也将为其他多种畜禽病毒性疾病的诊断提供新的思路和手段。
To build the diagnosis system for the porcine circovirus type 2 by the two steps of immnuohistochemistry assay(Envision assay),the plasmid of PGEX-ORF2 was transformed to E.coli BL21,and PCV-ORF2 fusion protein was expressed after induction by IPTG.Prepared anti-PCV2-ORF2 antibody and choosen the best working concentration,then the rabbits were immunized by the renaturated of PCV-ORF2 fusion protein.Used the two steps of immunohistochemistry assay to detect the lung of Porcine circovirus that doubtful illness tiers in the clinical,considered the anti-PCV2-ORF2 antibody was the first IgG,and then used the diagnosis assay of RT-PCR and an indirect enzyme-linked immunosorbent(iELISA)assay to confirm the consequence.The results of the two steps of immnuohistochemistry assay(Envision assay)directed exactly from localization,qualitation and quantitation,so that completed the standard of the diagnosis system.The experiment consequence certificated that the purity and the specificity of PCV-ORF2 fusion protein were very hight,the titer of the antiserum obtained in this experiment was up to 1:10240(2~(10)).The masculine cell of lung tissue where existed in the intracytoplasm from the porcine alveolar macrophage,alveolar epithilal cell and bronch epithelium mucosae.The diagnosis system of immunohisochemistry assay did not only use in the laboratory differential diagnosis but also used in the basement layer veterinary surgeon generally, it identified the diagnostic antigen in time and improved the rate of clinical definite,it prevented the disease and reduced the encomical loss effectually.The research offered the new ideas and new methods for the other animals viral disease.
引文
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