雞胚成纖維細胞cDNA文庫構建及其與禽呼腸孤病毒σA相互作用宿主蛋白的篩選

葉麗娜 謝芝勛 謝麗基 王盛 鄧顯文 謝志勤 范晴 黃嬌玲 曾婷婷 張艷芳 羅思思

摘要：【目的】構建雞胚成纖維細胞（CEF）的cDNA文庫并篩選與禽呼腸孤病毒（ARV）σA蛋白相互作用的宿主蛋白，為揭示互作蛋白對ARV復制及凋亡的分子調控機制打下基礎。【方法】采用TRIzol法提取CEF細胞總RNA，以SMART技術和LD-PCR合成雙鏈cDNA（ds cDNA），ds cDNA和pGADT7-Rec共轉化Y187酵母感受態細胞，構建cDNA文庫;將pGBKT7-σA誘餌質粒轉至Y2HGold酵母感受態細胞中，并與構建的cDNA文庫進行酵母雙雜交篩選，篩選出的候選文庫菌經質粒提取后與pGBKT7-σA誘餌質粒再共轉化Y2HGold酵母菌，篩選出能在SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba固體培養基上生長且明顯變藍的菌落，最后提取藍色酵母菌質粒進行測序分析。【結果】構建的CEF細胞cDNA文庫庫容為1.6×106 CFU，文庫滴度為3.1×108 CFU/mL，插入片段大小集中在300～2000 bp，重組率為91.67%。以含有pGBKT7-σA的Y2HGold酵母菌與cDNA文庫酵母菌進行雙雜交，陽性克隆能在SD-/Trp/-Leu/-Ade/-His/X-α-Gal/Aba（SD/-4/X/A）固體培養基上長出菌落且明顯變藍，測序結果顯示共篩選出4個能與σA蛋白相互作用的宿主蛋白，分別是初期多肽相關復合體α亞基（NACA）、核苷二磷酸激酶2（NME2）、假定蛋白及線粒體核糖體蛋白S9（MRPS9）。【結論】通過SMART技術構建的CEF細胞cDNA文庫具有較好的庫容和滴度，且從cDNA文庫中篩選出4種與ARV σA蛋白相互作用的宿主蛋白，為后續研究互作蛋白對ARV復制及凋亡的分子調控機制提供了可靠數據。

關鍵詞： 禽呼腸孤病毒;σA蛋白;cDNA文庫;互作蛋白;酵母雙雜交

中圖分類號： S852.657? ? ? ? ? ? ? ? ? ? ? ? ?文獻標志碼： A 文章編號：2095-1191（2019）06-1362-07

Abstract：【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 cDNA library was established. The bait plasmid pGBKT7-σA was transformed into Y2HGold 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 Y2HGold 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 Y2HGold yeast strain containing pGBKT7-σA and cDNA library yeast strain， and the po-sitive 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 cDNA library for further study of the molecular mechanisms interacting with ARV replication and apoptosis.

Key words： avian reovirus; σA protein; cDNA library; interacting proteion; yeast two-hybrid

收稿日期：2018-06-29

作者簡介：*為通訊作者，謝芝勛（1963-），研究員，主要從事動物傳染病防控技術研究工作，E-mail：xiezhixun@126.com。葉麗娜（1989-），主要從事……