新城疫病毒核衣壳蛋白基因在大肠埃希菌中的表达、纯化及其活性分析

Prokaryotic Expression and Purification of NDV NPGene in Escherichia coli and its Activity Analysis

构建新城疫病毒(Newcastle disease virus,NDV)核衣壳蛋白(nucleocapsid protein,NP)基因的原核表达载体,并将其在宿主菌E.coli BL21(DE3)感受态细胞中表达。以NDV La Sota株NP基因序列为模板,设计特异性引物,通过PCR扩增获得NP蛋白全长基因片段,定向插入原核表达载体pET-30a,构建重组质粒pET-NDV NP;将构建的重组质粒转化宿主菌E.coli BL21(DE3)感受态细胞,经IPTG诱导,表达出目的蛋白,并采用亲和层析的方法纯化表达蛋白;表达蛋白采用Western blotting方法检测其反应原性。结果显示,成功克隆了新城疫病毒NP基因全长,片段序列1470bp;构建的pET-NDV NP载体经PCR、双酶切、测序鉴定均无误;转化表达宿主菌后经SDS-PAGE检测结果显示目的基因得到成功表达,且表达蛋白经Ni-NTA镍离子亲和层析法被成功纯化,蛋白质浓度为3mg/mL;Western blotting检测结果显示表达蛋白具有良好的反应原性。试验结果表明,成功构建了含有新城疫病毒核衣壳蛋白基因的原核表达载体,成功表达、纯化得到了NDV NP蛋白。

The nucleocapsid protein （NP） gene of Newcastle disease virus （NDV） was cloned into a prokaryotic expression vector, and then using E. coli BL21（DE3） to express this protein. NP gene was amplified from cRNA which was reverse tran scription of RNA of NDV Ira Sota strain, then the gene was cloned into pET-30a. PCR,digestion and sequencing were used to identify positive plasmid, which named pET-NDV NP; the identified recombined plasmid was expressed by E. coli BL21（DE3） and induced by IPTG, the expressed products were purified by Ni-NTA Ni＋ affinity column; SDS-PAGE was used to analyze the expressed protein; Western blotting was used to identify the reaction activity. The results showed that the sequence of NP gene was consistent with predicted size, 1470 bp. PCR, digestion and sequencing results showed that the recombinant plasmid pET NDV NP was correct. NP protein was successfully expressed by E. coli BL21（DE3）, then the recombinant protein was purified by Ni-NTA Ni＋ affinity column and the concentration of the purified protein was 3 mg/mL. The recombinant protein could react with the antibody of NDV NP protein using the method of Western blotting assay. In summary, the recombinant plasmid pET NDV NP containing NP gene was constructed and recombinant protein was successfully expressed in E. coli BL21 （DE3） and purified.