本文選題：CSFV + 熒光抗體��； 參考：《南京農(nóng)業(yè)大學(xué)學(xué)報(bào)》2017年02期

【摘要】：[目的]本文旨在研究大腸桿菌表達(dá)的豬瘟病毒(CSFV)熒光抗體用于病毒直接免疫熒光檢測的可行性。[方法]在基因水平上對(duì)編碼豬瘟病毒納米抗體(VHH)與綠色熒光蛋白(EGFP)的融合基因進(jìn)行密碼子優(yōu)化,優(yōu)化后的融合基因插入p ET32a(+)載體并在大腸桿菌BL21(DE3)中融合表達(dá)。表達(dá)的融合蛋白經(jīng)過SDS-PAGE、Western blot進(jìn)行表達(dá)及可溶性鑒定。利用鎳柱親和層析獲得純化的重組蛋白,經(jīng)濃縮后用于豬瘟病毒直接免疫熒光檢測并與CSFV間接免疫熒光試驗(yàn)進(jìn)行效果比較,將該熒光抗體孵育CSFV、偽狂犬病病毒(PRV)、豬細(xì)小病毒(PPV)和豬圓環(huán)病毒2型(PCV-2)感染的PK15細(xì)胞從而鑒定熒光抗體的特異性。[結(jié)果]SDS-PAGE結(jié)果表明熒光抗體在大腸桿菌中實(shí)現(xiàn)部分可溶性表達(dá)。特異性試驗(yàn)結(jié)果表明,只有接種CSFV的PK15細(xì)胞組出現(xiàn)明顯的綠色熒光,而其他組不出現(xiàn)或只出現(xiàn)少量非特異性熒光。與用商品化單抗建立的間接免疫熒光試驗(yàn)結(jié)果相比,該熒光抗體建立的直接免疫熒光方法具有更強(qiáng)的清晰度。[結(jié)論]豬瘟熒光抗體可在大腸桿菌中高效表達(dá),純化后的抗體具有良好的特異性,且該抗體具有生產(chǎn)操作簡單、制備周期短和成本低等特點(diǎn)。
[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.
【作者單位】： 南京農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院;國家獸用生物制品工程技術(shù)研究中心;
【基金】：江蘇省農(nóng)業(yè)自主創(chuàng)新資金專項(xiàng)(CX(15)1026)
【分類號(hào)】：S852.651

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本文編號(hào)：1995010