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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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Abstract

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 × 106 transformants/4.34 μg pGADT7-Rec (>1.0 × 106). The cell density of the library was 1.75 × 109 cells/mL (>2×107 cells/mL). The titer of the primary cDNA library and amplified cDNA library was 6.75 × 105 and 2.33 × 107 CFU/mL respectively. The numbers for the primary cDNA library and amplified cDNA library were 1.01 × 107 and 1.14 × 109, 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.

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Acknowledgments

The research was supported by China Agricultural Research System (CARS-43-8), the Ministry of Education Program (20125103110013), Sichuan Province Research Programs (2013HH0042/2013TD0015/11ZA084/12TD005/2011ZO0034/2011JO0040) and China 973 program (2011CB111606).

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Authors and Affiliations

  1. Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China

    Xinghong Gao, Renyong Jia, Mingshu Wang, Dekang Zhu, Shun Chen, Meng Lin & Anchun Cheng

  2. Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, 46 Xinkang Road, Ya’an, 625014, Sichuan, China

    Xinghong Gao, Renyong Jia, Mingshu Wang, Dekang Zhu, Shun Chen, Meng Lin, Zhongqiong Yin, Yin Wang, Xiaoyue Chen & Anchun Cheng

  3. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Chengdu, 611130, Sichuan, China

    Renyong Jia, Mingshu Wang, Dekang Zhu, Shun Chen, Xiaoyue Chen & Anchun Cheng

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  1. Xinghong Gao
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Correspondence to Renyong Jia or Anchun Cheng.

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Xinghong Gao, Renyong Jia and Mingshu Wang contributed equally to the work.

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11033_2013_2881_MOESM1_ESM.jpg

Supporting Information-S1 The examination of integrity of the total RNA from the DEF cells infected with DEV 48 h by the 1.2 % formaldehyde degenerating agarose gel electrophoresis. There are three visible strand from up to down: 28 S, 18 S, and 5 S. Both lanes 1 and 2 were total RNAs from two different arrow-necked bottles. (JPEG 11 kb)

11033_2013_2881_MOESM2_ESM.jpg

Supporting Information-S2 Extent of molecular weight examination of the first-strand cDNA from the Poly A+ RNAs (S2a) and total RNA (S2b) respectively. The S2a and S2b, which both displayed a distributed smear from 0.1 ~ 8 kb, were carried out by the 1 % agarose gel electrophoresis. And in S2a, M: wide range DNA Marker 500 ~ 15,000; the lane 1 was the first-strand cDNA from the Poly A+ RNAs of DEF cells post-infected DEV 48 h. In S2b, M1: wide range DNA Marker 500 ~ 15,000 bp; M2: DL 2,000 bp; the lane 1 was production of reverse transcription with water as a negative control, the lane 2 was test example (the first-strand cDNA from the total RNAs of DEF cells post-infected DEV 48 h) and the lane 3 was uniformed mouse liver Poly A+ RNAs as a positive control. (JPEG 23 kb)

11033_2013_2881_MOESM3_ESM.jpg

Supporting Information-S3 The result of the cDNA fractionation purification with the CHROMA SPIN-400 column. M: wide range DNA Marker 500 ~ 12,000 bp. (JPEG 13 kb)

Supporting Information-S4 The classification of genes by sequencing. (JPEG 26 kb)

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Gao, X., Jia, R., Wang, M. et al. 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. Mol Biol Rep 41, 467–475 (2014). https://doi.org/10.1007/s11033-013-2881-z

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