Cloning and sequence analysis of leptin receptor overlapping transcript-like 1 gene from Dermatophagoides farinae

Abstract

Objective To obtain the leptin receptor overlapping transcript-like 1 encoding gene ( LepROTL1 gene) from Dermatophagoides farina, investigate the molecular characteristics of the gene and construct a prokaryotic expression vector to express this gene.

Methods The LepROTL1 gene-encoding sequence fragments were captured based on the transcriptome sequencing results, and the full-length gene fragments were amplified from total RNA of D. farinae using a RT-PCR assay, and used to construct the expression plasmid pET28a(+)-LepROTL1, followed by sequencing. The plasmid was transformed into E. coli BL21 (DE3) T1R for the induction of IPTG expression. The expression product was characterized by SDS-PAGE and Western blotting. Bioinformatics analyses were performed to analyze the sequence and the molecular characteristics of its encoded protein.

Results The amplification products of the RT-PCR assay showed a clear band on agarose gel electrophoresis, and sequencing analysis of the pET28a(+)-LepROTL1 plasmid showed 417 bp in length of the coding gene from the start codon ATG to the termination codon TAA. Following the plasmid transformation into E. coli and induction with IPTG, a specific band was seen on SDS-PAGE, indicating successful expression. Bioinformatics analysis showed that the LepROTL1 gene-encoded protein was composed of 134 amino acids, and had a relative molecular weight of 14 378.13 Da, a hydrophilicity index of 1.149, and certain hydrophobicity. The secondary structure was composed of alpha-helix (19 aa, 14.18%), extended strand (48 aa, 35.82%) and random coil (67 aa, 50.00%). The deduced amino acid sequence was used to obtain homologous genes by BLAST, and the phylogenetic tree showed that D. farinae was clustered with D. pteronyssinus.

Conclusion The full-length sequences and expression plasmid of the LepROTL1 gene are obtained, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene.

Abstract

［摘要］ 目的 获得粉尘螨瘦素受体叠加转录蛋白样 1 (Leptin receptor overlapping transcript-like 1, LepROTL1) 的编码基因及其分子特征, 并构建表达质粒。 方法 根据转录组测序结果, 获得粉尘螨 LepROTL1 编码基因序列片段。据此设计引物, 应用实时PCR技术从粉尘螨总RNA中扩增全长基因片段, 构建原核表达质粒pET28a(+)-LepROTL1后测序。将该质粒转化至大肠埃希菌 BL21 (DE3) T1R, 异丙基-b-D-硫代半乳糖苷 (IPTG) 诱导表达, 采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 和免疫印迹 (Western blotting) 鉴定表达产物。采用生物信息学软件分析该序列及其编码的蛋白质分子特征。 结果 PCR扩增产物经琼脂糖凝胶电泳显示 1 个清晰条带; 构建的质粒pET28a(+)-LepROTL1经测序显示, 该编码基因从起始密码子ATG至终止密码子TAA共 417 bp, 该质粒转化大肠埃希菌后经IPTG诱导, SDS-PAGE见特异性条带, 提示有表达。生物信息学分析显示, 该基因编码的蛋白质由134 个氨基酸组成, 相对分子质量为 14 378.13 Da, 其亲水性指数为 1.149, 有一定疏水性; 二级结构包括α-螺旋 (19 aa, 14.18%)、延伸主链 (48 aa, 35.82%) 和无规卷曲 (67 aa, 50.00%)。将该基因推导出的氨基酸序列进行BLAST获得同源基因, 构建的系统进化树中粉尘螨与屋尘螨聚成一簇。 结论 本研究获得了粉尘螨 LepROTL1 编码基因全长及其表达质粒, 通过生物信息学分析获得其分子特征, 为进一步研究该基因奠定了基础。