猪繁殖与呼吸综合征病毒BJ-4株GP4蛋白的原核表达与纯化

发布时间：2018-01-23 17:30

本文关键词： 猪繁殖与呼吸综合征病毒 GP 原核表达蛋白纯化　出处：《西北农林科技大学学报(自然科学版)》2017年05期 　论文类型：期刊论文

【摘要】：【目的】原核表达和纯化猪繁殖与呼吸综合征病毒(PRRSV)的结构蛋白GP4,为深入了解其结构和功能奠定基础。【方法】采用RT-PCR方法扩增PRRSV BJ-4株ORF4基因片段,将其克隆到pMD19-T载体中并进行测序。从测序正确的重组质粒pMD19-T-ORF4中扩增缺失N端及C端疏水序列的基因片段tGP4(truncated GP4),再将其亚克隆到pET-32a载体并转化到大肠杆菌BL21,IPTG进行诱导表达。对表达的重组蛋白进行可溶性分析,使用尿素梯度法进行蛋白纯化,并通过ELISA法检测重组蛋白的免疫活性。【结果】RT-PCR获得了537bp的ORF4。成功构建了原核表达载体pET-32a-tGP4,在IPTG诱导后重组蛋白以包涵体的形式大量表达。使用尿素梯度法获得了纯度较高的目的蛋白,纯化的目的蛋白能与PRRSV阳性血清特异性结合。【结论】使用原核细胞成功表达了PRRSV的结构蛋白GP4,纯化后的重组蛋白具有很好的免疫原性。
[Abstract]:[objective] to express and purify the structural protein GP4 of porcine reproductive and respiratory syndrome virus (PRRSVV). [methods] the ORF4 gene fragment of PRRSV BJ-4 strain was amplified by RT-PCR. It was cloned into pMD19-T vector and sequenced. The deletion of N-terminal and C-terminal hydrophobic sequence tGP4 (TGP4) was amplified from the correctly sequenced recombinant plasmid pMD19-T-ORF4. Truncated GP4. The recombinant protein was subcloned into pET-32a vector and transformed into E. coli BL21. The recombinant protein was analyzed by soluble analysis and purified by urea gradient method. The immunological activity of the recombinant protein was detected by ELISA method. [results] 537 BP ORF4 was obtained by RT-PCR. The prokaryotic expression vector pET-32a-tGP4 was successfully constructed. After induction of IPTG, the recombinant protein was expressed in the form of inclusion body, and the target protein with high purity was obtained by urea gradient method. The purified target protein can specifically bind to PRRSV positive serum. [conclusion] GP4, the structural protein of PRRSV, was successfully expressed using prokaryotic cells. The purified recombinant protein has good immunogenicity.
【作者单位】：郑州大学生命科学学院;河南师范大学生命科学学院;河南农业大学牧医工程学院;
【基金】：国家自然科学基金面上项目(31472177)
【分类号】：S852.651
【正文快照】： 猪繁殖与呼吸综合征是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndromevirus,PRRSV)引起的一种高度接触性传染病,能引起妊娠母猪后期发生流产、死胎、木乃伊胎[1-2]。1987年美国首先发生此病,并于1992年分离获得PRRSV,命名为VR-2332株;1990年欧洲也

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