摘要
目的原核表达猪轮状病毒(porcine rotavirus,PRV)Gottfried株和SB-1A株截短的VP8*基因,纯化重组蛋白,并检测其免疫原性。方法经PCR从含有全长VP4基因片段的重组质粒pCR4-Gottfried VP4和pCR4-SB-1A VP4中分别扩增截短的VP8*基因(△VP8*,64～223位氨基酸),分别插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-Gottfried-△VP8*和pET-28a-SB-1A-△VP8*,分别转化大肠埃希菌BL21(DE3),IPTG诱导表达,SDS-PAGE和Western blot分析表达产物。应用ProBondTMResin纯化两种重组蛋白,重组蛋白与A(lOH)3佐剂混合后,经肌肉注射免疫BALB/c小鼠,ELISA法检测免疫小鼠的血清抗体水平。结果两种重组表达质粒经双酶切和测序证实构建正确;表达的两种目的蛋白相对分子质量约25 000,均以包涵体和可溶性两种形式存在,均可与鼠源抗Histag单克隆抗体特异性结合;纯化的两种重组蛋白纯度均在95%以上,免疫BALB/c小鼠可产生较高的血清抗体水平。结论成功在大肠埃希菌中表达了PRV Gottfried株和SB-1A株截短的VP8*基因,纯化的两种蛋白免疫原性较好,为进一步研制安全、高效的PRV亚单位疫苗奠定了基础。
Objective To express the truncated VP8＊ genes（△VP8＊,64-223 amino acids）of porcine rotavirus（PRV） Gottfried and SB-1A strains in prokaryotic cells,purify the expressed protein and determine its immunogenicity.Methods △VP8＊ genes were amplified from recombinant plasmids pCR4-Gottfried VP4 and pCR4-SB-1A VP4,containing full length VP4 gene,respectively by PCR,and inserted into the expression vector pET28a（＋）.The constructed recombinant plasmids pET-28a-Gottfried△VP8＊ and pET-28a-SB-1A-△VP8＊ were transformed to E.coli BL21（DE3） and induced with IPTG.The expressed products were identified by SDS-PAGE and Western blot,purified with ProBondTM Resin and mixed with aluminum hydroxide adjuvant.BALB / c mice were immunized i.m.with the purified proteins and determined for serum antibody levels by ELISA.Results Recombinant plasmids pET-28a-Gottfried-△VP8＊ and pET-28a-SB-1A△VP8＊ were constructed correctly as proved by restriction analysis and sequencing.The expressed recombinant △VP8＊ protein,with a relative molecular mass of about 25 000,existed in both soluble and inclusion body forms,showed specific binding to murine monoclonal antibody against His tag,reached a purity of more than 95% after purification,and induced high serum antibody level in BALB / c mice.Conclusion The truncated VP8＊ proteins of porcine rotavirus Gottfried and SB-1A strains were successfully expressed in E.coli and showed good immunogenicity after purification,which laid a foundation of further preparation of safe and effective PRV subunit vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第8期1049-1053,共5页
Chinese Journal of Biologicals
基金
国家自然科学青年基金项目资助(31201909)
关键词
猪轮状病毒
截短VP8＊基因
原核细胞
基因表达
纯化
免疫原性
Porcine rotavirus（PRV）
Truncated VP8＊ gene
Prokaryotic cells
Gene expression
Purification
Immunogenicity
