摘要
【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.
作者
陈明明
焦振龙
赵文静
国果
CHEN Ming-Ming;JIAO Zhen-Long;ZHAO Wen-Jing;GUO Guo(Key Characteristic Laboratory of Modern Pathogen Biology,School of Modern Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China;Translational Medicine Research Center of Guizhou Medical University,Guiyang 550025,China;Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guiyang 550025,China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2023年第11期1451-1458,共8页
Acta Entomologica Sinica
基金
国家自然科学基金项目(82060381,32360702)
贵州省科技计划项目(黔科合基础-ZK[2022]一般345,黔科合基础-ZK[2023]一般329)
贵州省卫生健康委员会基金项目(gzwkj-2023-569)。