关键词:  , 绿盲蝽, 表皮蛋白, RACE, 原核表达, 多克隆抗体

Abstract: 【Aim】This study aims to lay the foundation for the functional study of cuticular protein (CP) 17 of Apolygus lucorum (AlCP17) and to provide a preliminary basis for further analyzing the signal pathway map of cuticular development of A. lucorum by cloning its gene AlCP17, preparing its polyclonal antibody and analyzing its expression profiles.【Methods】Based on the previous transcriptome data, the full-length cDNA sequence of AlCP17 of A. lucorum was cloned and subjected to bioinformatics analysis. The prokaryotic expression vector pCZN1-AlCP17 was constructed and transformed to Escherichia coli to conduct in vitro expression. The purified recombinant protein was used to prepare polyclonal antibody of AlCP17. qRT-PCR was used to analyze the expression profiles of AlCP17 in different developmental stages (1st-5th instar nymphs and adult) and different tissues (head, thorax, leg, midgut, fat body and cuticle) of the early 3rd instar nymphs of A. lucorum.【Results】The full-length cDNA sequence of AlCP17 (GenBank accession no.: OM302231) was cloned from A. lucorum with an open reading frame of 594 bp in length, encoding a putative protein of 197 amino acids with the predicted molecular weight of 21.69 kD and the theoretical isoelectric point of 6.11. AlCP17 has the typical structure characteristics of cuticular protein, containing the conserved chitin binding domain (non-cysCBD) of arthropod cuticular protein, namely Chitin_bind_4 domain. Amino acid sequence alignment result showed that AlCP17 shows the highest amino acid sequence identity (76.29%) with the CPA2B-like of Cimex lectularius of Cimicidae of Hemiptera. Phylogenetic tree showed that CP is a highly conserved protein with high similarity to CPA2B-like of C. lectularius and CP7-like of Halyomorpha halys. The prokaryotically expressed recombinant AlCP17 was obtained by IPTG induction. After purification, the polyclonal antibody of AlCP17 was obtained and had good purity for subsequent experiments. AlCP17 was expressed in different developmental stages and different tissues of the early 3rd instar nymphs of A. lucorum, and its expression level reached the peak in the newly-hatched 1st instar nymphs and increased significantly at the end of each instar. In addition, the expression level of AlCP17 in the midgut of the early 3rd instar nymphs was the highest.【Conclusion】The full-length cDNA sequence of AlCP17 gene of A. lucorum was cloned. AlCP17 has the typical characteristics of insect cuticular protein. The expression of AlCP17 in A. lucorum shows developmental stage specificity and tissue specificity. These results lay the foundation for the future research on the physiological function of this protein in A. lucorum development.

Key words: Apolygus lucorum, cuticular protein, RACE, prokaryotic expression, polyclonal antibody