Amplification of full-length cDNA sequence of Anopheles dirus vitellogenin gene and expression and purification of its functional domain

Abstract

Objective To amplify and analyze the full-length cDNA of Anopheles dirus vitellogenin (AdVg) gene, and express and purify its functional domain. Methods Based on the cDNA fragments of AdVg gene cloned earlier, the full-length sequence was obtained through rapid amplification of cDNA ends (RACE). The sequences of AdVg cDNA fragment, 5' end and 3' end were corrected and assembled to obtain a complete AdVg cDNA full-length sequence by Vector NTI software. Then the full-length sequence of AdVg was used to predict the functional domain. Finally, recombinant plasmid pet28A (+) was used to express and purify the AdVg functional domain. Results The full-length cDNA sequence of the AdVg gene was successfully obtained, including a 117 bp 5' UTR sequence, a 192 bp 3'UTR sequence, and a 6 186 bp open reading frame (ORF) sequence. Meanwhile, the analysis showed that the AdVg protein mainly contained 3 structural domains, including Vit N, DUF1943 and vWD. Then we successfully expressed and purified the functional domain AdVg Vit N by constructing a recombinant plasmid. Conclusion The full-length cDNA of AdVg is successfully cloned, and the recombinant protein of its functional domain Vit N is obtained in this study.

Keywords

• anopheles dirus
• vitellogenin
• race
• protein expression and purification