Cloning, Expression Analysis, and Detection of the Vitellogenin in the Chinese Black Sleeper Bostrychus sinensis

Abstract

Endocrine disruptors in marine environments represented by estrogens lead to reverse health phenomena. To obtain a more effective way to reflect and detect environmental estrogens pollution, a method was developed to obtain the full-length cDNA coding vitellogenin gene in B. sinensis, induced by 17β-estradiol (E2) solution. We have downloaded 16 fish gene sequences from the NCBI database and designed PCR primers accordingly. Based on the quantitative real-time PCR method (qRT-PCR), we analyze the differences in gene expression under the conditions of different E2 exposure times in the low, middle, and high-dose groups. The full-length cDNA consists of 4738 nucleotides with a reading frame encoding 1540 amino acid residues. In vitro recombinant plasmids were constructed and transferred to E. coli BL21 for vitellogenin expression. Efficient fusion expression was obtained by IPTG at 16°C, and the expressed target protein (680 amino acids, 75 kDa) existed in a soluble state, accounting for more than 25% of the total soluble protein. We prepared monoclonal antibodies using established immunohistochemistry to detect vitellogenin expression sites in sexually mature female fish. Our study shows that the expression sites of Vg in sexually mature female fish are mainly distributed in the fishtail, hepatopancreas, intestine, muscle, ovary, and pronephric kidney. In conclusion, the vitellogenin from B. sinensis could be used as a biomarker of environmental estrogens to achieve rapid detection in the marine environment and the subsequent experiments of development in colloidal gold strips after this research would be established to provide a highly efficient and convenient detection method for environment pollution.