绿盲蝽超气门蛋白ALUSP的克隆、原核表达及纯化

doi:10.3864/j.issn.0578-1752.2014.11.010

摘要/Abstract

摘要： 【目的】克隆绿盲蝽超气门蛋白基因（ALUSP），获得原核表达的重组蛋白，为ALUSP的功能研究奠定基础，也为进一步解析绿盲蝽蜕皮及变态发育的机制提供理论依据。【方法】根据已知昆虫的USP基因序列设计简并引物，获得ALUSP的保守序列；再通过设计特异性引物，利用RACE方法，分别克隆ALUSP 5′及3′段序列，然后通过拼接获得ALUSP全长序列；应用生物信息学方法，对ALUSP基因序列特征及其所编码蛋白的特性进行分析，建立系统进化树；将含有ALUSP的T载体经EcoR I及Xho I双酶切，构建ALUSP原核表达载体pEGX6P1-ALUSP，将该表达载体分别在15、25、30和37℃条件下进行诱导表达，再通过GST琼脂糖亲和层析和分子筛层析后获得ALUSP功能区纯化蛋白。【结果】ALUSP开放阅读框长1 005 bp，编码334个氨基酸，预测分子量56.63 kD，理论等电点8.42；序列特征分析表明，该基因翻译后的蛋白质具有超气门蛋白的典型特征，由A/B域（249 bp）、C域（198 bp）、D域（69 bp）和E域（489 bp）组成，其中C域高度保守，包含2个锌脂结构并含有1个P-盒和1个D-盒，且含有由8个氨基酸构成的核定位信号，D域含有1个识别DNA元件的T-盒，E域含有1个由8个α螺旋和1个β折叠形成袋状结构；氨基酸比对结果表明，ALUSP的氨基酸序列与稻绿蝽USP序列一致性最高（57.82%），与其他昆虫的USP序列一致性较低，如膜翅目的Melipona scutellaris（46.05%）、Scaptotrigona depilis（45.94%）；系统进化树分析结果显示，半翅目与膜翅目、直翅目等昆虫的USP较为分化，位于不同分支，ALUSP与稻绿蝽USP进化关系最近，可能来源于共同的祖先。EcoR I和Xho I双酶切的重组克隆载体可成功亚克隆到pEGX-6P-1载体上，命名为pEGX6P1-ALUSP；经37℃、1.0 mmol•L-1的IPTG诱导的pEGX6P1-ALUSP重组质粒可特异性表达1个约65 kD的蛋白，并且该重组ALUSP蛋白主要以包涵体形式存在；收集含有目的基因的菌株进行GST琼脂糖亲和层析和分子筛层析，最后进行复性和纯化后在65 kD附近仅出现1条清晰的特异性条带，表明已得到纯化的靶蛋白。【结论】克隆了ALUSP全长，其具有昆虫超气门蛋白的典型特征，并获得了原核表达的重组蛋白。

关键词: 绿盲蝽 , 超气门蛋白 , 基因克隆 , 原核表达

Abstract: 【Objective】 The objectives of this study are to clone the ultraspiracle protein gene of Apolygus lucorum (ALUSP) and obtain the recombinant protein of prokaryotic expression, and to reveal the function of ALUSP and offer a basis for research of the ecdysis and metamorphosis of A. lucorum. 【Method】 The conserved sequence of A. lucorum was obtained by using the degenerate primers from the known USP genes of the other insects. A new set of gene-specific primers matching the primers in the 3′- and 5′-full RACE kit were designed and the original sequence files were assembled to obtain the complete cDNA sequence of the ALUSP. The characteristics of ALUSP gene sequences and which coding proteins were analyzed by using the bioinformatics methods, and the phylogenetic tree was also constructed. To construct its prokaryotic expression vector, T-vector containing ALUSP was dual-enzyme digested by EcoR I and Xho I. The expression vector was induced to express at 15, 25, 30 and 37℃. ALUSP purified protein gene in functional areas was gotten by the GST agarose affinity chromatography and molecular sieve chromatography.【Result】The open reading frame of ALUSP was 1 005 bp in length, encoding a 334 amino acid polypeptide with a predicted molecular weight of 56.63 kD and the theoretical isoelectric point of 8.42. Protein signature analysis revealed that the protein encoded by ALUSP shared typical structural features of ultraspiracle proteins with other insects, including A/B domain (249 bp), C domain (198 bp), D domain (69 bp) and E domain (489 bp), respectively. C domain was highly conserved, including two zinc fingers, a P-box, a D-box and a nuclear location signal that was constituted of eight amino acids. D domain included a T-box which could identify the DNA components and E domain, including a bag structure, was constituted of eight α-helix and β-strand. The blast results showed that the amino acids sequence of ALUSP was the highest in similarity (57.82%) with Nezada viridula, and low in similarity with other insects, such as Melipona scutellaris (46.05%), Scaptotrigona depilis (45.94%), respectively. Phylogenetic tree analysis results showed that the USP genes from Hemiptera were more differentiation between Hymenoptera, which were located in different branches, ALUSP and N. viridula USP was the nearest in genetic evolution, which may be derived from common ancestors. The recombinant plasmid pEGX6P1-ALUSP was high-efficient expression in Rosetta gami B when induced by 37℃ and 1.0 mmol•L-1 IPTG. The 65 kD target protein from the strain containing ALUSP was obtained by the GST agarose affinity chromatography and the molecular sieve chromatography.【Conclusion】The full-length cDNA of ALUSP from A. lucorum was obtained and also got the recombinant protein. The protein had typical characteristics of insect ultraspiracle protein.

Key words: Apolygus lucorum , ultraspiracle protein , gene cloning , prokaryotic expression

谭永安, 肖留斌, 孙洋, 赵静, 柏立新. 绿盲蝽超气门蛋白ALUSP的克隆、原核表达及纯化[J]. 中国农业科学, 2014, 47(11): 2162-2172.

TAN Yong-An, XIAO Liu-Bin, SUN Yang, ZHAO Jing, BAI Li-Xin. Cloning, Expression and Purification of Ultraspiracle Protein Gene from Apolygus lucorum[J]. Scientia Agricultura Sinica, 2014, 47(11): 2162-2172.

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