Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus
Construction and identification of a cDNA library for use in the yeast two-hybrid system from duck embryonic fibroblast cells post-infected with duck enteritis virus
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DOI：
10.1007/s11033-013-2881-z
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发表时间：
2014-01-01
影响因子：
2.8
通讯作者：
Cheng, Anchun
中科院分区：
文献类型：
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作者：
Gao, Xinghong;Jia, Renyong;Cheng, Anchun
为了探索和分离鸭肠炎病毒（DEV）感染后鸭胚胎成纤维细胞（DEF）相关基因，采用SMART（Switching Mechanism At 5' end of the RNA Transcript）技术结合同源重组方法建立了cDNA文库。感染后48小时收获细胞并提取总RNA。然后纯化 mRNA 并使用寡 (dT) 引物 (CDS III) 反转录为第一链 cDNA。随后，进行长距离PCR，纯化双链cDNA，并依次进行转化测定。最终，根据质量评价，成功建立了高质量的文库。转化效率约为2.33×10(6)个转化体/4.34μg pGADT7-Rec(>1.0×10(6))。文库的细胞密度为 1.75 x 10(9) 细胞/mL（> 2x10(7) 细胞/mL）。初级cDNA文库和扩增cDNA文库的效价分别为6.75×10(5)和2.33×10(7)CFU/mL。初级cDNA文库和扩增cDNA文库的数量分别为1.01×10(7)和1.14×10(9)，重组率为97.14%。随机挑选的 27 个独立克隆的测序结果显示，插入片段的范围为 0.323 至 2.017 kb，平均插入片段大小为 0.807 kb。通过非冗余核酸或蛋白质数据库的序列相似性分析获得DEV-CHv LORF3、UL26和UL35基因的全长转录本。在 DEV-CHv 基因组中鉴定出五个多聚腺苷酸位点。此外，在DEV-CHv基因组的IRS基因和US1基因之间发现了一个668 bp的新转录本。因此，我们得出的结论是，构建的cDNA文库将成为DEV与宿主DEF之间相互作用的蛋白质组学分析、发现DEV机制的生物标志物研究以及随后开发原始疫苗和抗病毒药物来预防或治疗疾病的有用工具。
To explore and isolate genes related to duck embryonic fibroblast cells (DEFs) post-infected with duck enteritis virus (DEV), a cDNA library was established using SMART (Switching Mechanism At 5' end of the RNA Transcript) technique coupling with a homologous recombination method. The cells were harvested and total RNA was extracted at 48 h post infection. Then the mRNAs were purified and reverse transcribed to first-strand cDNAs using oligo (dT) primers (CDS III). Subsequently, long distance-PCR was performed, the double-stranded cDNAs were purified, and a transformation assay was carried out in that order. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 2.33 x 10(6) transformants/4.34 mu g pGADT7-Rec (> 1.0 x 10(6)). The cell density of the library was 1.75 x 10(9) cells/mL (> 2x10(7) cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 x 10(5) and 2.33 x 10(7) CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 x 10(7) and 1.14 x 10(9), respectively, and the recombinant rate was 97.14 %. The sequence results of 27 randomly picked independent clones revealed the insert ranged from 0.323 to 2.017 kb with an average insert size of 0.807 kb. Full-length transcripts of DEV-CHv LORF3, UL26 and UL35 genes were acquired through sequence similarity analysis from the non-redundant nucleic acid or protein database. Five polyA sites were identified in the DEV-CHv genome. Also, a new transcript of 668 bp was found between the IRS gene and US1 gene of the DEV-CHv genome. Thus, we concluded that the constructed cDNA library will be a useful tool in proteomic analysis of interactions between the DEV and host DEFs, and discovery of biomarkers studies on the mechanism of DEV and subsequently exploitation original vaccines and antiviral drugs to prevent or cure diseases.