密码子优化的猪瘟病毒荧光抗体的表达及初步应用

发布时间：2018-06-08 06:53

本文选题：CSFV + 荧光抗体　；参考：《南京农业大学学报》2017年02期

【摘要】：[目的]本文旨在研究大肠杆菌表达的猪瘟病毒(CSFV)荧光抗体用于病毒直接免疫荧光检测的可行性。[方法]在基因水平上对编码猪瘟病毒纳米抗体(VHH)与绿色荧光蛋白(EGFP)的融合基因进行密码子优化,优化后的融合基因插入p ET32a(+)载体并在大肠杆菌BL21(DE3)中融合表达。表达的融合蛋白经过SDS-PAGE、Western blot进行表达及可溶性鉴定。利用镍柱亲和层析获得纯化的重组蛋白,经浓缩后用于猪瘟病毒直接免疫荧光检测并与CSFV间接免疫荧光试验进行效果比较,将该荧光抗体孵育CSFV、伪狂犬病病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒2型(PCV-2)感染的PK15细胞从而鉴定荧光抗体的特异性。[结果]SDS-PAGE结果表明荧光抗体在大肠杆菌中实现部分可溶性表达。特异性试验结果表明,只有接种CSFV的PK15细胞组出现明显的绿色荧光,而其他组不出现或只出现少量非特异性荧光。与用商品化单抗建立的间接免疫荧光试验结果相比,该荧光抗体建立的直接免疫荧光方法具有更强的清晰度。[结论]猪瘟荧光抗体可在大肠杆菌中高效表达,纯化后的抗体具有良好的特异性,且该抗体具有生产操作简单、制备周期短和成本低等特点。
[Abstract]:[objective] to study the feasibility of direct immunofluorescence detection of CSFV antibody expressed by Escherichia coli. [methods] the codon of the fusion gene encoding CSFV nano-antibody (VHHHHH) and green fluorescent protein (EGFP) was optimized at the gene level. The optimized fusion gene was inserted into pET32a() vector and expressed in E. coli BL21DE3. The expressed fusion protein was expressed and identified by SDS-PAGEG Western blot. The purified recombinant protein was obtained by nickel column affinity chromatography and then concentrated for direct immunofluorescence detection of CSFV and compared with CSFV indirect immunofluorescence test. The PK15 cells infected with CSFV, pseudorabies virus (PRV), porcine parvovirus (PPVV) and porcine circovirus type 2 (PCV-2) were incubated with the fluorescent antibody to identify the specificity of the fluorescent antibody. [results] SDS-PAGE showed that the fluorescent antibody was partially expressed in Escherichia coli. The results of specificity test showed that only PK15 cells inoculated with CSFV showed obvious green fluorescence, while no or only a small amount of non-specific fluorescence appeared in other groups. Compared with the results of indirect immunofluorescence assay established by commercial monoclonal antibody, the method of direct immunofluorescence established by this antibody has more clarity than that of the indirect immunofluorescence assay established by commercial monoclonal antibody. [conclusion] the fluorescent antibody against swine fever can be expressed in Escherichia coli, the purified antibody has good specificity, and the antibody has the characteristics of simple production, short preparation period and low cost.
【作者单位】：南京农业大学动物医学院;国家兽用生物制品工程技术研究中心;
【基金】：江苏省农业自主创新资金专项(CX(15)1026)
【分类号】：S852.651

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