摘要
目的构建旋毛虫(Trichinella spirais)新生幼虫期特异性T314基因真核表达质粒,并在BHK细胞中进行表达。方法从旋毛虫新生幼虫RNA中通过RT-PCR技术扩增无信号肽T314全长基因,定向克隆至真核表达载体pEGFP-N1中,构建重组真核表达质粒T314-pEGFP-N1,脂质体法转染BHK细胞,荧光显微镜观察EGFP的表达,Western blot检测T314融合蛋白的表达。结果重组真核表达质粒T314-pEGFP-N1经双酶切及测序证实构建正确;转染的BHK细胞48 h转染效率最高;表达的T314融合蛋白可与旋毛虫感染的猪血清发生特异性反应。结论已成功构建了T314基因重组真核表达质粒T314-pEGFP-N1,并在BHK细胞中融合表达,为进一步研究旋毛虫包囊形成机制奠定了基础。
Objective To construct a eukaryotic expression vector for stage-specific gene T314 from newborn larvae of Trichinella spiralis and express in BHK cells.Methods Signal peptide-free full-length T314 gene was amplified from RNA of newborn larvae of T.spiralis by RT-PCR and cloned into eukaryotic expression vector pEGFP-N1.BHK cells were transfected with the constructed recombinant plasmid T314-pEGFP-N1 in mediation of liposome,then observed for expression of EGFP by fluorescent microscopy,and for that of T314 fusion protein by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid T314-pEGFP-N1 was constructed correctly.The transfection efficacy of BHK cells reached the maximum 48 h after transfection.The expressed T314 fusion protein showed specific reaction with porcine serum infected with T.spiralis.Conclusion The recombinant eukaryotic expression vector T314-pEGFP-N1 for T314 gene was successfully constructed,and fusion protein was expressed in BHK cells,which laid a foundation of further study on mechanism of nurse cell formation of T.spiralis.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第3期281-284,共4页
Chinese Journal of Biologicals
关键词
毛线虫
旋
新生幼虫
T314基因
真核细胞
基因表达
Trichinella
spiralis
Newborn larvae
T314 gene
Eukaryotic cells
Gene expression
