非洲猪瘟多基因家族MGF-505-3R基因重组原核表达质粒的构建及表达

Construction of prokaryotic expression vector for MGF-505-3R gene in multi gene family of African swine fever virus

目的构建非洲猪瘟多基因家族MGF-505-3R基因重组原核表达质粒,并进行表达。方法根据非洲猪瘟病毒(African swine fever virus,ASFV)MGF-505-3R基因CDS区序列设计特异性引物并进行PCR扩增,将目的基因片段与原核表达载体pET-32A(+)连接,构建重组原核表达质粒pET-32A(+)-MGF-505-3R。对蛋白进行生物信息学分析后,将重组原核表达质粒转化大肠埃希菌BL21(DE3)感受态细胞中,用1 mmol/L IPTG于37℃诱导表达6 h后,超声破碎菌体,获得的上清液经Ni-NTA亲和层析和CMSepharose FF阳离子交换层析纯化,BCA法测定蛋白产量。结果重组原核表达质粒经菌液PCR、双酶切及测序证明构建正确。MGF-505-3R蛋白为不稳定的疏水蛋白,为跨膜蛋白,蛋白二级结构由无规卷曲、α螺旋和β折叠组成为主要结构,三级结构预测结果与二级结构预测相符。表达的重组蛋白相对分子质量约为33000,BCA法测定重组蛋白产量可达19.8 mg/L。结论成功构建了MGF-505-3R基因重组原核表达质粒,表达并纯化了重组蛋白,为我国非洲猪瘟免疫血清学诊断试剂的制备及预防控制奠定了基础。

Objective To construct a prokaryotic expression vector for MGF-505-3R gene in multi gene family of African swine fever virus(ASFV).Methods Specific primers were designed according to the sequence of CDS region of MGF-505-3R gene of ASFV and used for PCR amplification.The target fragment was inserted into vector pET-32A(+),and the constructed recombinant plasmid pET-32A-MGF-505-3R was analyzed for bioinformatics,transformed to competent E.coli BL21(DE3)and induced with 1 mmol/L IPTG at 37℃for 6 h,then subjected to ultrasonication.SDS-PAGE was used to identify the protein expression.The obtained supernatant was purified by Ni-NTA chromatography and CM Sepharose FF cation exchange chromgatography,and determined for yield of protein by BCA method.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-32A-MGF-505-3R was constructed correctly.MFG-505-3R was a unstable hydrophobic transmembrane protein,of which the secondary structure consisted of random coli,αhelix andβsheet.The prediction result of tertiary structure was consistent with that of secondary stru-cture.The yield of expressed recombinant protein,with a relative molecular mass of about 33000,reached 19.8 mg/L as proved by BCA method.Conclusion Recombinant prokaryotic expression vector for MGF-505-3R gene in multi gene family of ASFV was successfully constructed,which laid a foundation of preparation of serological diagnostic kit as well as prevention and control of ASFV.