鸡胚成纤维细胞cDNA文库构建及其与禽呼肠孤病毒σA相互作用宿主蛋白的筛选
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摘要
【目的】构建鸡胚成纤维细胞(CEF)的cDNA文库并筛选与禽呼肠孤病毒(ARV)σA蛋白相互作用的宿主蛋白,为揭示互作蛋白对ARV复制及凋亡的分子调控机制打下基础。【方法】采用TRIzol法提取CEF细胞总RNA,以SMART技术和LD-PCR合成双链cDNA(ds cDNA),ds c DNA和pGADT7-Rec共转化Y187酵母感受态细胞,构建c DNA文库;将pGBKT7-σA诱饵质粒转至Y2HGold酵母感受态细胞中,并与构建的cDNA文库进行酵母双杂交筛选,筛选出的候选文库菌经质粒提取后与pGBKT7-σA诱饵质粒再共转化Y2HGold酵母菌,筛选出能在SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba固体培养基上生长且明显变蓝的菌落,最后提取蓝色酵母菌质粒进行测序分析。【结果】构建的CEF细胞cDNA文库库容为1.6×106CFU,文库滴度为3.1×108CFU/mL,插入片段大小集中在300～2000 bp,重组率为91.67%。以含有pGBKT7-σA的Y2HGold酵母菌与cDNA文库酵母菌进行双杂交,阳性克隆能在SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba(SD/-4/X/A)固体培养基上长出菌落且明显变蓝,测序结果显示共筛选出4个能与σA蛋白相互作用的宿主蛋白,分别是初期多肽相关复合体α亚基(NACA)、核苷二磷酸激酶2(NME2)、假定蛋白及线粒体核糖体蛋白S9(MRPS9)。【结论】通过SMART技术构建的CEF细胞cDNA文库具有较好的库容和滴度,且从cDNA文库中筛选出4种与ARVσA蛋白相互作用的宿主蛋白,为后续研究互作蛋白对ARV复制及凋亡的分子调控机制提供了可靠数据。
【Objective】Construction of a cDNA library of chicken embryo fibroblasts(CEF)and screening for host proteins interacting with avian reovirus σA protein were conducted to lay a foundation for revealing the molecular regulation mechanism of interaction protein on ARV replication and apoptosis.【Method】Extraction of CEF total RNA using TRIzol method was carried out,and double-strands cDNA(ds cDNA)was synthesized by SMART and LD-PCR technique. Co-transformation of ds cDNA with linearized pGADT7-Rec in competent Y187 yeast cells was realized,then c DNA library was established. The bait plasmid p GBKT7-σA was transformed into Y2 HGold yeast competent cells,and yeast two-hybrid screening was conducted with the established cDNA library. The selected candidate library strain was extracted by plasmid,then co-transformed with bait plasmid pGBKT7-σAinto Y2 HGold yeast cells. The colonies that grew well and could turn blue in solid media containing SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba were selected,then blue yeast plasmid was extracted for sequencing analysis.【Result】The capacity of CEF cell cDNA library was 1.6×106 CFU,titer was 3.1×108 CFU/mL,the insert size was between 300 bp and 2000 bp,recombination rate was about 91.67%. Two-hybridization was carried out with Y2 HGold yeast strain containing pGBKT7-σA and cDNA library yeast strain,and the positive clone was able to grow colonieson the SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba(SD/-4/X/A)solid media and appeared blue. The sequencing results showed that four host proteins interacting with σA protein were screened,which were the initial polypeptide-related complex α subunit(NACA),nucleoside diphosphate kinase 2(NME2),putative protein and mitochondrial ribosomal protein S9(MRPS9),respectively.【Conclusion】The constructed CEF cDNA library by SMART technique has a good capacity and titer,and four host proteins interacting with ARV σA protein are screened from the c DNA library for further study of the molecular mechanisms interacting with ARV replication and apoptosis.
【Objective】Construction of a cDNA library of chicken embryo fibroblasts(CEF)and screening for host proteins interacting with avian reovirus σA protein were conducted to lay a foundation for revealing the molecular regulation mechanism of interaction protein on ARV replication and apoptosis.【Method】Extraction of CEF total RNA using TRIzol method was carried out,and double-strands cDNA(ds cDNA)was synthesized by SMART and LD-PCR technique. Co-transformation of ds cDNA with linearized pGADT7-Rec in competent Y187 yeast cells was realized,then c DNA library was established. The bait plasmid p GBKT7-σA was transformed into Y2 HGold yeast competent cells,and yeast two-hybrid screening was conducted with the established cDNA library. The selected candidate library strain was extracted by plasmid,then co-transformed with bait plasmid pGBKT7-σAinto Y2 HGold yeast cells. The colonies that grew well and could turn blue in solid media containing SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba were selected,then blue yeast plasmid was extracted for sequencing analysis.【Result】The capacity of CEF cell cDNA library was 1.6×106 CFU,titer was 3.1×108 CFU/mL,the insert size was between 300 bp and 2000 bp,recombination rate was about 91.67%. Two-hybridization was carried out with Y2 HGold yeast strain containing pGBKT7-σA and cDNA library yeast strain,and the positive clone was able to grow colonieson the SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba(SD/-4/X/A)solid media and appeared blue. The sequencing results showed that four host proteins interacting with σA protein were screened,which were the initial polypeptide-related complex α subunit(NACA),nucleoside diphosphate kinase 2(NME2),putative protein and mitochondrial ribosomal protein S9(MRPS9),respectively.【Conclusion】The constructed CEF cDNA library by SMART technique has a good capacity and titer,and four host proteins interacting with ARV σA protein are screened from the c DNA library for further study of the molecular mechanisms interacting with ARV replication and apoptosis.
引文
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