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目的对犬恶丝虫酪蛋白激酶2β亚基(Csnk2b)部分基因片段进行克隆、原核表达及免疫反应性分析。方法根据犬恶丝虫cDNA文库中筛选出的Csnk2b部分基因片段设计引物,以含有插入Csnk2b部分基因片段的噬菌体DNA为模板,进行PCR扩增。产物亚克隆至原核表达载体,构建重组载体pGEX-4T-1-Csnk2b,双酶切鉴定。将其转入大肠埃希菌(E.coli)Rosetta(DE3)中,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达的重组蛋白,蛋白质印迹(Western blotting)以小鼠抗犬恶丝虫阳性血清为一抗,分析重组蛋白免疫反应性。结果Csnk2b部分基因片段PCR扩增产物约为700 bp,测序结果与cDNA文库中筛选得到的序列一致。重组表达载体pGEX-4T-1-Csnk2b双酶切鉴定正确。SDS-PAGE结果显示,IPTG诱导后重组蛋白GST-Csnk2b表达,相对分子质量(M r)约为45 000,与预期大小一致。Western blotting分析表明,重组蛋白能被小鼠抗犬恶丝虫阳性血清识别。结论克隆并表达了犬恶丝虫Csnk2b重组蛋白,且该蛋白具有免疫反应性。
Objective To clone, prokaryotic expression and immunoreactivity analysis of some gene fragments of casein kinase 2β subunit (Csnk2b) of canine filariasis. Methods Based on the Csnk2b partial gene fragments selected from the cDNA library of D. canadensis, primers were designed. The phage DNA containing partial fragment of Csnk2b gene was used as a template for PCR amplification. The product was subcloned into the prokaryotic expression vector, and the recombinant vector pGEX-4T-1-Csnk2b was constructed and double digested. This was transfected into E. coli Rosetta (DE3) and induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polypropylene The expressed recombinant protein was analyzed by gel electrophoresis (SDS-PAGE). Western blotting was used to detect the immunological reactivity of the recombinant protein with anti-Dirofilaria-positive serum as the primary antibody. Results The PCR products of some Csnk2b gene fragments were about 700 bp. The sequencing results were consistent with those screened in the cDNA library. Recombinant expression vector pGEX-4T-1-Csnk2b double digestion correct identification. The results of SDS-PAGE showed that the recombinant protein GST-Csnk2b was induced by IPTG. The relative molecular mass (M r) was about 45 000, consistent with the expected size. Western blotting analysis showed that the recombinant protein could be recognized by the positive anti-Sphaerotheca fulminanthera antibody in mice. Conclusion The recombinant protein of Csnk2b was cloned and expressed, and the protein was immunoreactive.