家蝇酵母双杂交cDNA文库构建和MD14-3-3互作蛋白筛选

Construction of yeast two-hybrid cDNA library of Musca domestica (Diptera: Muscidae) and screening of MD14-3-3 interacting proteins

【目的】构建家蝇Musca domestica酵母双杂交cDNA文库,筛选MD14-3-3互作蛋白。【方法】以家蝇为研究对象,经mRNA纯化、cDNA初级文库构建、重组pGADT7-DEST构建次级文库,构建酵母双杂交筛选系统,构建pGBKT7-MD14-3-3质粒作为诱饵筛选与MD14-3-3相互作用的蛋白,并进行回转验证。【结果】成功构建文库容量为1.6×10^(7) CFU,重组率为100%的家蝇酵母双杂交cDNA文库。酵母双杂交筛选获得2个靶蛋白,回转试验表明mucin-like protein HKR1与抗菌肽ctenidin-1为MD14-3-3的互作蛋白。【结论】从成功构建的家蝇酵母双杂交cDNA文库中筛选出MD14-3-3蛋白的2个互作蛋白,为进一步探究家蝇免疫应答机制奠定了基础。

【Aim】To construct a yeast two-hybrid(Y2H)cDNA library of Musca domestica and investigate the proteins that could interact with the MD14-3-3.【Methods】Taking M.domestica as the research object,the mRNA was purified,the primary cDNA library was constructed,the secondary library was constructed by the recombinant pGADT7-DEST vector,so as to construct the Y2H.Then,the plasmid pGBKT7-MD14-3-3 were constructed as bait and used for screening the proteins interacting with MD14-3-3.Finally,the transformation and validation were verified.【Results】A Y2H cDNA library of M.domestica was successfully constructed,with the library storage capacity of 1.6×10^(7) CFU and the recombination rate of 100%.Two target proteins were obtained by Y2H screening.The results of transformation and validation showed that mucin-like protein HKR1 and antibacterial peptide ctenidin-1 interacted with MD14-3-3.【Conclusion】Two proteins that interact with MD14-3-3 were identified from the successfully constructed Y2H cDNA library of M.domestica.Our results provide a basis for investigating the mechanism of immune response of M.domestica.