斜纹夜蛾受翻译控制肿瘤蛋白基因的克隆及表达

【目的】克隆斜纹夜蛾受翻译控制肿瘤蛋白（TCTP）基因，分析其序列特征，为深入研究该基因奠定基础。【方法】利用RACE末端快速扩增技术，在斜纹夜蛾卵巢细胞中克隆TCTP基因的全长。根据不同物种间TCTP基因的同源性来构建进化树，并在原核细胞中对斜纹夜蛾TCTP进行表达。【结果】克隆得到斜纹夜蛾TCTP基因全长cDNA，登录号为HQ896486.1。序列分析表明，该基因cDNA全长829 bp，开放阅读框长519 bp，编码含172个氨基酸的蛋白，蛋白分子质量19.67 kD。生物信息学分析表明，斜纹夜蛾TCTP基因属于受翻译控制肿瘤蛋白家族，与其它物种TCTP基因有很高相似性。构建重组质粒 pET32a(+)-TCTP，在大肠杆菌中表达重组蛋白，确定了1 mmol•L-1 IPTG诱导4 h为重组蛋白表达的最佳条件，在短期内能得到大量稳定性好的TCTP蛋白。【结论】从斜纹夜蛾卵巢细胞中克隆得到斜纹夜蛾TCTP基因全长cDNA，并且成功在原核细胞中进行表达。本研究为深入研究斜纹夜蛾TCTP的功能以及针对斜纹夜蛾TCTP基因RNAi干扰的生物防控提供了理论依据和技术支持。

Molecular Cloning and Expression of Translationally Controlled Tumor Protein of Spodoptera litura (Fab.)

【Objective】 The objective of this study is to clone the full-length cDNA of TCTP from Spodoptera litura cells, as well as to analyze its sequence.【Method】The full-length cDNA of TCTP was obtained by RACE-PCR. A phylogenetic tree of TCTP protein in different species was constructed. TCTP was cloned in pET-32a (+) and was expressed as fusion protein in Escherichia coli BL21(DE3) cells.【Result】The full-length cDNA of SLTCTP (GenBank accession number: HQ896486.1) was 829 bp with an open reading frame of 519 and encodes 172 amino acids. According to Protparam on-line tools, the deduced protein was predicted to have a computed molecular mass of 19.67 kD. TCTP cloned in pET-32a (+) was expressed as fusion protein in E. coli BL21 (DE3) cells. In order to obtain much target protein of TCTP, optimal culture conditions of pET-32a (+)-TCTP were studied. Induced by 1mmol•L-1 IPTG for 4 h was the best optimized condition for recombinant protein expression. TCTP gene belonged to the TCTP protein family and had a high similarity with other species.【Conclusion】This study has provided a basis of method for further research on the function of SLTCTP and pest control.