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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 eukaryotic expression plasmid of T314 gene of neonatal larva of Trichinella spirais and express it in BHK cells. Methods The T314 full-length gene without signal peptide was amplified from the RNA of Trichinella spiralis by RT-PCR and cloned into eukaryotic expression vector pEGFP-N1 to construct recombinant eukaryotic expression plasmid T314-pEGFP-N1. BHK cells were transfected by MTT method. The expression of EGFP was observed by fluorescence microscopy. The expression of T314 fusion protein was detected by Western blot. Results The recombinant eukaryotic expression plasmid T314-pEGFP-N1 was confirmed by double enzyme digestion and sequencing. The transfected BHK cells were the most efficient at 48 h, and the expressed T314 fusion protein reacted specifically with Trichinella infected pig serum . Conclusion The recombinant eukaryotic expression vector T314-pEGFP-N1 of T314 gene has been successfully constructed and fused in BHK cells, which laid the foundation for further study on the cyst formation mechanism of Trichinella spiralis.