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Cloning of the full-length cDNA of the gene encoding complement C5 from grass carp (Ctenopharyngodon idella) and its expression in different tissues by following grass carp reovirus infection

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Abstract

Grass carp (Ctenopharyngodon idella) is the most widely produced freshwater aquaculture product worldwide. However, its susceptibility to microbial infections during its cultivation restricts its development. The complement system plays an important role in immune defence. To investigate the transcription of the complement factor C5 and production of its cleavage product C5a in grass carp infected with grass carp reovirus (GCRV), the full-length C5 gene sequence was cloned and sequenced using the rapid amplification of cDNA ends method, and C5 transcription and C5a production in different grass carp tissues at different GCRV infection stages were determined by RT-qPCR and western blotting, respectively. Our results showed that the full-length cDNA of the C5 gene was 5365-bp long, encoding 1687 amino acids. Signal peptide prediction showed that C5 had a secretory signal peptide. After artificial infection with GCRV, C5 transcription in grass carp liver increased significantly before the appearance of symptoms. C5a generation in the liver increased significantly from the onset of disease symptoms, and continued to increase until recovery. GCRV induced C5 cleavage in grass carp, with C5a production.

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Data availability

The spliced full-length cDNA sequence of the C5 gene of grass carp was submitted to the NCBI GenBank database (access ID: MK256357).

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Acknowledgements

We would like to thank professor XiangJiang Yang at Yuanjiang Institute of Fisheries Sciences for presenting the GCRV-AH528 virus, and anonymous technicians at Guangdong Meilikang Bio-Science Ltd., China for assistance with data analysis.

Funding

This work was supported by the National Natural Science Fund of China (No. 31702335) and the Hunan Natural Science Fund (No. 2019JJ50265).

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Author notes

  1. Baohong Xu and Ligang Lv contributed equally to this work.

Authors and Affiliations

  1. Hunan Engineering Technology Research Center of Featured Aquatic Resources Utilization, Hunan Agricultural University, Changsha, 410128, Hunan, China

    Baohong Xu, Ligang Lv, Tiaoyi Xiao, Qiaolin Liu, Hang Su & Yi Liu

  2. Dongguan Key Laboratory of Medical Bioactive Molecular Developmental and Translational Research, Guangdong Medical University, Dongguan, 523808, China

    Jiajia Ni

  3. Research and Development Center, Guangdong Meilikang Bio-Sciences Ltd., Dongguan, 523808, China

    Jiajia Ni

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  1. Baohong Xu
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Contributions

B. Xu, Q. Liu, and T. Xiao designed the study. B. Xu, L. Lv, Q. Liu, H. Su, Y. Liu, and J. Ni conducted the experiments and collected the data. B. Xu, L. Lv, Q. Liu, H. Su, and J. Ni analysed the data. B. Xu, Q. Liu, and T. Xiao wrote the main manuscript text. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Tiaoyi Xiao or Qiaolin Liu.

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Ethics approval

The experiment was carried out in accordance with the ethical guidelines of Hunan Agricultural University for the care and use of laboratory animals and the Regulations on the Administration of Laboratory Animals of China. All experiments were approved by the Animal Ethics Committee of Hunan Agricultural University (approval number: 2019012).

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Conflict of interest

Jiajia Ni is a technician of Guangdong Meilikang Bio-Science Ltd., China.

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Supplementary information

Figure S1

Phylogenetic tree of fishes based on C5 amino acid sequences. The amino acid sequences of other organisms were retrieved from the NCBI database. The codes in the extensions are the amino acid sequence retrieval numbers in the NCBI database. The evolutionary analyses of the amino acid sequences using the maximum likelihood method were conducted in MEGA7. (PNG 1033 kb)

High resolution image (TIF 1597 kb)

Figure S2

Comparison of C5 multiple amino acid sequences. The images were generated using DNAMAN version 7.0.2.176 (https://www.lynnon.com/dnaman.html) and separated into two parts using Adobe Photoshop version 8.0.1 (https://www.adobe.com/products/photoshop.html). (PNG 11327 kb)

High resolution image (TIF 16737 kb)

Figure S3

Original western blotting gels of the pictures showed in Figure 3. A3 - F3 indicate initial infection, incubation period, onset period, death period, recovering period, and restored period, respectively. The samples used for the analysis of C5 expression at the initial infection period and the incubation period were obtained immediately after the injection of the virus and after 12 h of GCRV infection (before the appearance of GCHD symptoms), respectively. The onset period was the stage at which the symptoms of GCHD emerged, which was 24-30 hours after the injection of the virus. The death period was the stage at which grass carp began to die, which was 48-72 hours after the injection of the virus. The recovering period was the stage at which the grass carp began to recover after death, which was 144 hours after the injection of the virus. The restored period was the stage at which the grass carps recovered completely and the symptoms of GCHD disappeared, which was 216 h after the injection with the virus. Three grass carp samples from each group were collected and analysed repeatedly at each stage. (PNG 5112 kb)

High resolution image (TIF 15852 kb)

Table S1

Protein sequences of functional conserved domains. (DOCX 15 kb)

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Xu, B., Lv, L., Xiao, T. et al. Cloning of the full-length cDNA of the gene encoding complement C5 from grass carp (Ctenopharyngodon idella) and its expression in different tissues by following grass carp reovirus infection. Aquacult Int 29, 2035–2048 (2021). https://doi.org/10.1007/s10499-021-00733-2

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