关键词: 中华蜜蜂, cDNA 文库, 滴度, 酵母双杂交, FullCoV技术, 蛋白质相互作用

Abstract: 【Aim】 This study aims to construct the yeast two-hybrid cDNA library for membrane proteins of Apis cerana cerana larvae by cloning the larval membrane protein cDNA into pPR3-N using FullCoV technique. 【Methods】 The total RNA was extracted from the 2-3 day-old worker larvae of A. cerana cerana. After mRNA was isolated, the first strand cDNA of membrane protein was synthesized by reverse transcriptase, and then the double-stranded cDNA was synthesized. After being added to the 5′ end of the double-stranded cDNA, the linker with the recombinant sequence was ligated with the vector pPR3-N by FullCoV technology. The ligation product was electrotransformed into DH10B competent cells to construct the larval membrane protein cDNA library, and the titer of the library and the size of the inserted cDNA fragment were examined and verified. 【Results】 A yeast two-hybrid cDNA library for membrane proteins of A. cerana cerana larvae was successfully constructed by the FullCoV technique. The library had the capacity of 1.5×107 cfu, the titer of 3×10⁶ cfu/mL, and the recombination rate of 100%. 【Conclusion】 In this study the yeast two-hybrid cDNA library for membrane proteins of A. cerana cerana larvae was successfully constructed using FullCoV technique, which is helpful for the further study on the interaction between pathogen infecting A. cerana cerana and host proteins.

Key words: Apis cerana cerana; cDNA library, titer, yeast two-hybrid, FullCoV technology, protein-protein interaction