【作者】 管蕊； 沈中元； 唐旭东； 徐莉； 朱峰； 陶恒平； 陈大睿；

【Author】 GUAN Rui 1 SHEN Zhong-Yuan 2,* TANG Xu-Dong 2 ZHU Feng 1 CHEN Da-rui 1(1Jiangsu University of Science and Technology,Zhenjiang 212018,China,2Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang 212018,China)

【机构】 中国农业科学院蚕业研究所； 江苏科技大学生物与应用化学学院；

【摘要】 为了探索家蚕核型多角体病毒(BmNPV)蛋白间的相互作用,采用CLONTECH SMART(switching mechanism at 5′end of RNA transcript)技术,在酵母菌株Y187中构建了感染BmNPV家蚕中肠组织的cDNA文库。家蚕五龄第三天添食BmNPV,提取感染后12小时与96小时家蚕中肠组织的总RNA并混合均匀。利用经修饰的CDSⅢ引物合成cDNA第一链,在反应体系中再加入SMARTIII-modified Oligo作为cDNA第二链合成的引物,进行长距离LD-PCR合成双链cDNA,后者经CLONTECH CHROMA SPINTE-400柱纯化后,与线性载体质粒pGADT7-Rec共转化入感受态酵母菌Y187中,二者利用酵母细胞内较高的同源重组酶活性,进行同源重组产生环形有复制活性的文库质粒,然后在缺亮氨酸(LEU)培养基板上筛选出所有克隆,即为感染BmNPV家蚕中肠组织的cDNA文库。实验结果表明:提取RNA的A260/A280=1.82,纯度较高;双链cDNA文库大于0.2kb,且弥散较长,成瀑布条带状;该文库具有基因多样性和足够大的库容量,共获得6.8×108转化子,滴度为4.4×107cfu/ml,文库扩增后,插入cDNA的平均长度为750bp,文库的重组率为95%。该文库的构建为筛选相互作用蛋白并进一步阐明其生理意义提供了基础。更多还原

【Abstract】 In order to explore the protein interaction between Bombyx mori nuclear polyhedrosis virus (BmNPV) and its host,cDNA library from the BmNPV infected silkworm was constructed by CLONTECH SMART technology.Methods:silkworms larvae on the third day of the fifth instar were challenged with 109 BmNPV polyhedron.Total RNA of midguts at 12 and 96h p.i.was extracted respectively and mixed well.The first chain of cDNA was transcripted with a modified CDSⅢ primer and its terminal was automatically extended with CCC.The second chain was synthesized with the SMARTⅢ-modified Oligo primer by Long Distance-PCR.The resulted PCR products were purified by CLONTECH CHROMA SPINTE-400 column.Finally the purified PCR products were co-transformed with the linearized plasmid pGADT7-Rec into the competent yeast Y187.They were recombinated at the presence of yeast homologous recombinase in the yeast cells and became the reproductive cyclic plasmid.The transformed yeasts grew in the SD/-Leu plates.All the growing clones were harvested and then constituted the cDNA 1ibrary.Results:the library conversion rate was 6.8×10 8 transfers.The titer of the library was 4.4×10 7 cfu/ml,and the reorganization rate was about 95%.The average size of inserted fragment was 750bp.Conclution:The yeast two hybrid cDNA library was successfully constructed and could be used for screening proteins related to BmNPV infection.更多还原