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

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 17 ss-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. ECcient fusion expression was obtained by IPTG at 16 degrees 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;sh. Our study shows that the expression sites of Vg in sexually mature female;sh are mainly distributed in the;shtail, 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 eCcient and convenient detection method for environment pollution.

Automated summary

A method was developed to detect environmental estrogen pollution in marine environments more effectively by analyzing the differences in gene expression under different exposure times to 17β-estradiol in B. sinensis. The full-length cDNA coding vitellogenin gene was obtained and expressed in a soluble state. The expression sites of vitellogenin in sexually mature female fish were detected, providing a potential biomarker for rapid detection and a convenient method for environmental pollution.