Pathogens that invade vertebrates are first identified by the innate immune system through various pattern recognition receptors (PRRs). TLRs (Toll-like receptors) are transmembrane proteins expressed by cells of the innate immune system (Lundber et al., 2013), which recognize invading microbes and activate signaling pathways that launch immune and inflammatory responses to destroy the invaders. In mammals, TLR receptors other than TLR3 are involved in MyD88-dependent pathway signaling. MyD88, as a key adaptor protein, participates in the signaling process of MyD88-dependent pathways. MyD88 was originally discovered during the differentiation of myeloid cells. MyD88 contains an N-terminal death domain (DD) that interacts with IL-1R-related kinase 4 (IRAK-4) through its own DD structure (Suzuki et al., 2002). IRAK4 phosphorylates IRAK1 and IRAK2 (Kawagoe et al., 2008); they in turn activate TNF receptor-associated factor 6 (TRAF6), which is inhibited by another IRAK family member IRAK-M (Kobayashi et al., 2002). TRAF6 binds to UBC13 and UEV1A and forms multiple ubiquitin chains on the necessary regulator of NF-κB (NEMO or IKKγ) (Siggs et al., 2010). TRAF6 also activates MAPKKK member TGF-β activated kinase 1 (TAK1), which is associated with two adaptor proteins: TAK1 binding protein 1 (TAB1) and TAB2. TAB2 acts as a receptor for the polyubiquitin chain on TRAF6 and NEMO and binds these proteins together. TAK1 phosphorylates IKKβ and also activates a series of mitogen-activated kinases (MAPK), starting with MAPKK3 and MAPKK6, leading to phosphorylation of Jun kinase (JNKs), p38, and ultimately CREB. IKK protein phosphorylates IkB, leading to its degradation and nuclear translocation of NF-κB. Eventually, the activation of NF-κB and AP-1 transcription factors will induce the transcription of genes such as proinflammatory cytokines (such as TNF and IL-12) (Blasius and Beutler, 2010).

When the body is externally stimulated, it first triggers innate immunity and induces TLR4 expression, resulting in an adaptive immune response. It then activates NF-κB through a signal transduction pathway dependent on MyD88. Some endogenous ligands activate NF-κB by activating TLR4 receptors, leading to the release of pro-inflammatory factors, such as TNF-α, IL-1β, etc. (O’Neill and Bowie, 2007). The absence of TRAF6 will inactivate the TLR signaling pathway, thereby inhibiting the activation of NF-κB and reducing the production of downstream cytokines (Lutgens, 2012). Therefore, the two linking molecules MyD88 and TRAF6 in the TLR signaling pathway play a very important role in the innate immune response.

Zebrafish (Danio rerio) is a small cyprinid fish, named after its side pattern is similar to zebra stripes (Sun et al., 2006). As early as the 1930s, zebrafish were used by scientists in scientific experiments, and now it has become a widely recognized and popular model organism. There are many advantages as a model organism. Firstly, the zebrafish is fertilized in vitro and the embryo is transparent, which is easy to observe and manipulate the embryo; secondly, the zebrafish embryo develops very quickly and the growth cycle is short, only about 3 months. Mature; more importantly, its embryos only have a natural immune response within 3 weeks after fertilization. As an in vivo experimental model for assessing gene function, zebrafish is increasingly used to simulate human diseases, including cancer (Joerger and Fersht, 2016), so it has a high level of research in development, disease and immunity. Research value (Danilova et al., 2004).

Zebrafish has unique advantages as a model organism for studying innate immune responses. Nowadays, a variety of pathogen infection models have been established. Fluorescently labeled pathogens and neutrophils or macrophages can be used to visualize dynamic processes (Mazon-Moya et al., 2017). However, due to the lack of antibodies and cell lines, there are still bottlenecks in the study of specific molecular mechanisms. To solve this problem, we constructed eukaryotic expression vectors for zebrafish myd88 and traf6. Phylogenetic tree analysis found that the zebrafish myd88 and traf6 genes are highly homologous and structurally conservative with the corresponding genes of other species. In all species, they are very close to bony fish. They will be constructed the good zebrafish pCMV-myd88 and pCMV-traf6 were transfected into HEK293T cells. The dual luciferase reporter gene system was used to detect the activity of the zebrafish NF-κB family nfκb1 promoter. It was found that overexpression of myd88 and traf6 can significantly promote nfκb1 promoter transcription and expression. This study provides a reliable experimental materials to explore the innate immune function of important molecules in zebrafish.

1 Results

1.1 Amplification of the full-length CDS fragment of zebrafish myd88 and traf6 gene

Using 1dpf-sized wild-type zebrafish cDNA as a template, the primer sequences designed in Table 1 were amplified by PCR. The amplified fragments were sequences containing the upstream 5'UTR and the downstream 3'UTR of the coding region. After reaching the pMD19-T vector, PCR amplification was performed using the double restriction site primers designed in Table 2 to obtain the full-length CDS fragment. The zebrafish myd88 gene full-length CDS was 855 bp, and the traf6 gene CDS was 1 629 bp (Figure 1).

1.2 Bioinformatics analysis of Myd88 and TRAF6 amino acid sequences

The zebrafish MyD88 and TRAF6 domains were analyzed online by SMART. The zebrafish MyD88 and TRAF6 amino acid sequences were compared with those of Homo sapiens, Gallus gallus, Larimichthys crocea, and Eudontomyzon morii MyD88 and TRAF6 by DNAMAN.

The results showed that the zebrafish MyD88 encodes 284 amino acids and consists of three parts. The 22~101 position is the DD death domain, the 102~147 position is the middle region, and the 148~284 position is the TIR domain. The highest amino acid identity of zebrafish and Larimichthys crocea MyD88 is 66.55%, and that of Homo sapiens, Gallus gallus and Eudontomyzon morii are 58.22%, 56.52% and 46.88% respectively. The homology of TIR domain is higher than that of DD death domain, indicating that the TIR domain is more conservative (Figure 2A).

Zebrafish TRAF6 encodes 542 amino acids and contains four domains: N-terminal RING domain (71-109aa), two zinc finger structures (129-178 aa, 205-262 aa), and C-terminal loop-loop (coiled-coil) α helix domain (311-374 aa) and TRAF homology domain-MATH (379-503 aa). The highest amino acid identity of TRAF6 between zebrafish and Larimichthys crocea is 62.26%, and 55.27%, 56.60%, and 40.17% with Homo sapiens, Gallus gallus, and Eudontomyzon morii, respectively (Figure 2B).

The phylogenetic tree constructed by the NJ method showed that the myd88 and traf6 of the zebrafish and osteichthyes are grouped together, while the mammal and the bird family are each grouped into another branch, as well as invertebrates, amphibians and Cyclostomata lamprey and other relatives are relatively low and form their own branches (Figure 3).

1.3 Construction of zebrafish pCMV-myd88 and pCMV-traf6 recombinant plasmids and identification by double enzyme digestion

Double digestion of pCMV-Tag2B vector, full-length CDS fragments of myd88 and traf6 genes, purification and ligation, transformation, overnight culture plate, 12-16h picking of monoclonal bacterial solution PCR, and positive samples after agarose gel electrophoresis Clones No.3 and No.6 were sent to Shanghai Biotech Co., Ltd. for sequencing. The similarity between the sequencing results and the reference sequence alignment was more than 99% (Figure 4).

Add the positive monoclonal corresponding to the preserved bacterial solution to 25 mL of Kana-resistant LB liquid medium to expand the bacterial solution for 12~16 h. Use EndoFree PlasmidezFlow MiniPrep Kit II (BIOMIGA, PD1222-01) endotoxin-free kit to extract the recombinant plasmid after double digestion, the band position was detected by agarose gel electrophoresis. The results showed that there were two bands (Figure 5). The target fragment, the full-length CDS of myd88 gene is 855 bp, and the full-length CDS of traf6 gene is 1 629 bp.

1.4 Effect of overexpression of zebrafish myd88 or traf6 gene on the activity of NF-κB family gene nfκb1 promoter

Co-transform the constructed recombinant plasmid pCMV-myd88 or pCMV-traf6 with the NF-κB family nfκb1 promoter reporter gene to HEK293T cells, ie overexpress zebrafish myd88 or traf6 gene in HEK293T cells. The transcriptional activity of the NF-κB family nfκb1 promoter reporter gene was detected in cells by dual-luciferase reporter assay system. After overexpressing the zebrafish myd88 gene, the transcriptional activity of the nfκb1 promoter increased significantly, about 2.5 times that of the unloaded control group; after overexpression of the zebrafish traf6 gene, the transcriptional activity of nfκb1 increased significantly, which was about the empty control group 8 times the activity (Figure 6).

2 Discussion

MyD88 and TRAF6 molecules are important linker molecules of TLR signaling pathway in innate immune response and have very important functions. This paper successfully constructed the eukaryotic expression vectors of zebrafish myd88 and traf6, and can significantly activate the activity of NF-κB reporter gene. Our research results show that zebrafish has high amino acid sequence identity with Homo sapiens, Gallus gallus, Larimichthys crocea, and Eudontomyzon morii, ranging from 40.17% to 66.55%. This is related to the structural studies of MyD88 in lamprey (Zhao et al., 2018) and grouper (Li et al., 2015). And in mammals (Park et al., 2015), lamprey (Ding et al., 2019), grass carp (Kongchum et al., 2011) are similar to TRAF6 in fish. It shows that MyD88 and TRAF6 molecules are very conservative in evolution, and their structures and functions are highly similar between different species.

A large number of in vitro studies have confirmed that MyD88 and TRAF6 are widely involved in the inflammatory process. Multiple studies have shown that the main cause of myocardial tissue damage is due to TLR4 / MyD88 / NF-κB signaling that controls the production of pro-inflammatory factors and induces inflammatory responses in myocardial tissue (Ma and Xie, 2017; Zhang et al., 2017). In addition, TLR4 / MyD88 / NF-κB signaling also promotes the inflammatory cascade by activating nod-like receptor protein 3 (NLRP3), which aggravates myocardial damage (Yu et al., 2016). Yang et al. (2008) found that TLR4 / MyD88 / TRAF6 signaling as a common pathway for cell-mediated inflammation can cause LPS-induced cell activation leading to the production of TNF-α and IL-13. A study in 2018 used fish rhabdovirus to infect zebrafish and found that MyD88 / NF-κB inflammation signaling pathway is involved in the regulation process of antiviral response (Song Yajiao et al., 2018). Transcriptome data analysis of knocked out traf6 gene in Salmonella infection showed that the inflammation response during infection was highly dependent on traf6 gene (Stockhammer et al., 2010).

Zebrafish has unique advantages as a model organism for studying innate immune response. In order to study the functional mechanism of immune molecules in detail and whether to use MyD88 and TRAF6 as adaptor molecules to play a regulatory role in the immune response, we constructed the zebrafish myd88 and traf6 genes Eukaryotic expression vector. After sequencing and comparison with the reference sequence, the similarity was higher than 99%. After double-digestion of the recombinant plasmid, the linearized pCMV-Tag2B plasmid vector band and the target fragment were found by electrophoresis detection Bands. After overexpressing the zebrafish myd88 gene and traf6 gene, the transcriptional activity of the nfκb1 promoter in the NF-B family increased significantly, which was about 2.5 times and 8 times that of the unloaded control group, respectively. The above results prove that the zebrafish pCMV-myd88 and pCMV-traf6 eukaryotic expression vectors were successfully cloned, and the overexpression of the adapter molecules MyD88 and TRAF6 in the TLR signaling pathway can significantly activate the transcription and expression of the NF-B signaling pathway, thereby regulating inflammation Reaction and other processes. Our experimental results have laid the foundation for the follow-up study of the mechanism of MyD88 and TRAF6 in innate immune regulation, providing a valuable research tool.

3 Materials and Methods

3.1 Materials

The experimental wild-type (WT) adult zebrafish (strain AB) were obtained from the zebrafish platform of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Feeding and breeding were conducted by our laboratory with reference to WESTERFIELD (Loosli et al., 2003). Research methods were approved by the Shanghai Ocean University Experimentation Ethics Review Committee (SHOU-DW-2016-002). The pGL3-Enhancer Vector and plasmid phRL-TK used in Dual-Luciferase Reporter Assay System and the human embryo kidney cell line (HEK293T) were all donated by professor Cao Xuetao from the state key laboratory of medical immunology, Naval Medical University of China. They were preserved and cultured by our laboratory according to the methods provided by ATCC database. And the nfκb1 gene promoter was constructed by our laboratory.

DNA polymerase, DNA Marker, pMD™19-T Vector Cloning Kit (TAKARA, 6013) and PrimeScript™ RT reagent Kit with gDNA Eraser were purchased from Takara Biomedical Technology (Beijing) Co., Ltd. Trizol Reagent was purchased from Invitrogen under the Thermo Fisher Scientific corporation. DNA Clean & Contentrator-5, FastPure Gel DNA Extraction Mini Kit (Vazyme, DC301-01), TIANprep Rapid Mini Plasmid Kit and DH5α Competent Cell (TIANGEN, CB101-03) were purchased from Tiangen Biotech (Beijing) Co., Ltd. EcoR Ⅰ -HF, Xho Ⅰ and Apa Ⅰ were purchased from New England Biolabs. LB Broth agar, glycerin, ethyl alcohol and other necessary chemical products were purchased from Sangon Biotech (Shanghai) Co., Ltd. Dual-Luciferase® Reporter Assay System and FuGENE Hdjun were purchased from Promega Corporation. Primer synthesis and DNA sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd.

3.2 Methods

3.2.1 Primer design

Firstly, the CDS sequences of zebrafish myd88 and traf6 genes and their 5’UTR and 3’UTR sequences were downloaded from NCBI. The upstream primer F and the downstream primer R were designed at the 5 'UTR and 3' UTR sequences respectively (Table 1). According to the CDS sequences and the sequence of pCMV-Tag2B vector (Figure 7), EcoR Ⅰ -HF and Xho Ⅰ were chosen as restriction enzyme digest sites of myd88 gene, and EcoR Ⅰ -HF and Apa Ⅰ were chosen as restriction enzyme digest sites of traf6 gene (Table 2).

3.2.2 Total RNA extraction and reverse transcription

Trizol method was used to extract RNA from the whole embryo of 20 wild-type zebrafish with the size of 1dpf. And 1 μg RNA was used to make cDNA products according to the instruction of reverse transcription kit.

The cDNA of 1dpf wild-type zebrafish was used as the template. And the designed primer sequences were amplified at different temperatures to optimize annealing temperature (Table 1). And the PCR products were detected by agarose gel electrophoresis. The bright and consistent target band was selected, and the temperature it represented was regarded as the optimal annealing temperature. And the designed primer sequences were amplified again at the annealing temperature. PCR conditions: predenaturation for 2 SEC at 98℃; cycling procedures (a total of 34 cycles): denaturation for 10 SEC at 98℃, annealing for 30 SEC at their own annealing temperatures and extensions for 1min at 72℃; and then further extension for 5 min at 72°C and storage at 4℃.

3.2.3 Construction of the eukaryotic expression vectors of zebrafish myd88 and traf6 genes

The purified PCR products were ligated into pMD19-Tt vectors, and then transformed into E·coli competent cells DH5α. After that, 500 μL non-resistant LB liquid medium was added and  they were placed in the constant temperature shaker for 1 h. 200 μL of the solution above was coated on LB agar plate and cultured at 37℃ for 12~16 h. The monoclonal colonies were selected and cultured in 200 μL LB liquid medium for 4h. And after its PCR amplification, the products of agarose gel electrophoresis whose bands appeared to be bright and consistent or fresh were selected. And the bacteria liquid they represented were sent to Sangon Biotech Co., Ltd for DNA sequencing.

The similarity between the tested sequences and the reference sequences were above 99%. According to the sequences of CDS regions and the pCMV-Tag2B expression vector map (Figure 7), EcoR Ⅰ -HF and Xho Ⅰ were selected as restriction enzyme digest sites of myd88 gene, and EcoR Ⅰ -HF and Apa Ⅰ were selected as restriction enzyme digest sites of traf6 gene. The designed primers with restriction enzyme digest sites were amplified with reference to Table 2, and the PCR conditions were the same as above. After agarose gel electrophoresis, PCR products were purified and recovered. Double digestion was performed on both PCR purified products and pCMV-Tag2B control vector. At 37℃, double digestion was performed for 12 h on both myd88 target sequence and pCMV-Tag2B vector, with EcoR Ⅰ -HF and Xho Ⅰ; at 37℃, double digestion was performed on both traf6 target sequence and pCMV-Tag2B vector with EcoR Ⅰ -HF, and 6 hours later, Apa Ⅰ was added and the double digestion was performed for 12 h at 25℃. Based on the volume ratio target genes: plasmid vectors = 1:5, we placed them at 16℃ for the night (> 12 h). Then, the ligated products were transformed into competent cells DH5α, and the rest of the experiment were the same as above. The primers used in PCR amplification of bacteria solution are universal primers of pCMV-Tag2B vectors. After agarose gel electrophoresis, the products represented by the bright and consistent band or fresh bacteria solution were sent to Sangon Biotech Co., Ltd for DNA sequencing.

3.2.4 Bioinformatic analysis of MyD88 and TRAF6 gene sequences

The gene sequences of MyD88 and TRAF6 in different species were obtained form NCBI. Neighbor-joining in MEGA 7.0 was used to construct a phylogenetic tree. And SMART was applied to predict and analyze structural domains and DNAMAN was used to compare amino acid consistency between different species.

3.2.5 Transcriptional activity detection of pGL3-nfκb1-Enhancer after overexpression of zebrafish myd88 or traf6 gene by Dual-Luciferase® Reporter Assay System

HEK293T cells were cultured in 10% FBS-DMEM culture solution and placed in 5 % CO₂ incubator at 37℃, and every passage took 48 hours. HEK293T cells were seeded in 24-well plates 24 h before transfection with the stock density of 5×10⁴ cells per well, and then transfected at a cell fusion rate of 80%. The exact procedures were as follows: HEK293T cells were cultured in the culture dish with the diameter of 10 cm. After the cell fusion rate reached 89%-90%, the old culture medium was sucked out, and 2 mL DPBS buffer was added along the wall. After slight shaking, DPBS was sucked out, and the cleaning was repeated twice. 500 μL trypsin was added and they were placed in an incubator at 37℃ for 1min digestion. The adherent cells were completely detached from the wall by gently tapping the bottom of the culture dish, and the digestion was terminated by adding DMEM complete medium (containing 10% FBS). And then they were transferred to a 15 mL centrifuge tube for centrifugation for 3 min (800 rpm). The supernatant was sucked out and the proper amount of DEME was added to the complete culture medium to resuspend cells. Then, they were calculated by the blood counting chamber. 1×10⁵ cells were placed in each well of the 24-well plate and cultured at 37℃.

The zebrafish eukaryotic expression vectors (pCMV-myd88 or pCMV-traf6), NF-κB gene promoter recombinant plasmid pGL3-nfκb1-promoter-Enhancer and TK internal reference fluorescence reporter gene vector were all transfected into HEK293T cells, and the gene promoter activity detection of pGL3-nfκb1-Enhancer after overexpression of zebrafish myd88 or traf6 gene by Dual-Luciferase® Reporter Assay System was performed. The transfection system was as the following (Table 3).

TAfter the 24 h transfection, FlexStation3 was used to detect the transcription activity according to Dual Luciferase® Reporter Assay System instruction. Each group was repeated for three times and the experiments were repeated for three times. The results were expressed as mean±SEM, and the significant differences was analyzed by Prism 6, and p<0.05 was considered as existing significant difference.

Authors’ contributions

Xu Xing was mainly in charge of the vector construction, the design of double luciferase reporter gene assay experiment, data analysis and paper writing; Liu Heyi mainly participated in the vector construction experiments and part of paper writing; Wang Zirui and Zhang Ying were responsible for bioinformatic analysis; Zhang Qinghua and Li Weiming provided guidance for the experiment design, paper writing and paper revision. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (D-8002-15-0042), Shanghai Municipal Education Commission Key Innovation Project (13ZZ127) and China-US Ocean Research Center Fund (A1-3201-19-3013).

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