CDS Cloning, Sequencing and Bioinformatic Analysis of Vasoactive Intestinal Peptide in Wahui Pigeon

Fei Wang1, Xiaofen Hu1, Feng Wen2, Xiaoen Tang3, Shanshan Yang1, Shengwei Zhong1, Zuohong Zhou1, Xu Yuan1 and Yong Li1,* 1College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China 2College of Life Science and Engineering, Foshan University, Foshan 528231, Guangdong, China 3Fuzhou Husbandry Breeding Farm, Linchuan 344000, Jiangxi, China Article Information Received 01 August 2021 Revised 03 October 2021 Accepted 12 October 2021 Available online 21 January 2022 (early access)


INTRODUCTION
V asoactive intestinal peptide (VIP) was initially isolated and characterized from porcine duodenum in 1970 (Said and Mutt, 1970). In vivo, VIP widely distributes in various tissues as gastrointestinal hormone and neuropeptide. VIP is considered as the paradigm of an endogenous neuroendocrine-immune mediator with therapeutic potential for a variety of inflammatory disorders such as inflammatory bowel disease (Abad et al., 2012;Jönsson et al., 2012;Wu et al., 2015;Iwasaki et al., 2019). VIP can inhibit the neurodegeneration due to loss of neurons, which may be mediated by glial cells through the production of neurotrophic factors and the inhibition of proinflammatory mediators (Deng and Jin, 2017). In addition, VIP acts as an antimicrobial peptide in the pathological process of rheumatoid arthritis, reduces the expression of pattern recognition receptor and its inflammatory signal, down-regulates the production of proinflammatory cytokines and chemokines, and counterbalances Th subsets decreasing pathogenic Th17 cells and their capacity to shift to Th1 profile (Villanueva-Romero et al., 2018). In the recent study, the signal pathway between nervous system and immune system mediated by VIP opens up a new way for the treatment of allergy and plays an important role in the prevention of allergic diseases (Verma et al., 2017). Another study demonstrates an increased transepithelial passage of live commensal and pathogenic bacteria in the colon of irritable bowel syndrome subjects, the up-regulation of VIP is important in the regulation of this bacterial translocation through the mucosa (Bednarska et al., 2017). So far, VIP has continually been investigated as a known neuropeptide on its aspects, the structural characteristics and biological function of VIP have been abundantly acquired as well.
Pigeon is a kind of common poultry with tender meat, high protein, and low fat in China. In recent years, the market demand for pigeons has gradually increased. However, some bad managements, for example, wet litter, bad ventilation, high density, improper coccidiostat O n l i n e

F i r s t A r t i c l e
consumption and contaminated feeders, exaggerate intestinal parasite problems (Ruff, 1993). The parasitism often brings about severe effects on birds including malnutrition, retarded growth, low egg production, susceptibility to other infections, and death in young birds (Radfar et al., 2012), these similar intestinal diseases account for a large part of pigeon's disease, so as to seriously threaten pigeon's normal breeding. As an effective antiinflammatory factor, VIP has the effect of inhibiting T cell proliferation (Gonzalez-Rey et al., 2007). In addition, VIP inhibits the production of inflammatory cytokines and chemokines by macrophages, microglia and dendritic cells via its receptors VPAC1 and VPAC2 (Gonzalez-Rey et al., 2007). VIP is also a signaling molecule between the nervous and immune system, and plays a role in immunity, especially in local mucosal immunity (Delgado and Ganea, 2013). However, there have been few reports on molecular bioinformatics of VIP such as the related functional structures. Here, Wahui pigeons' intestines were used to obtain the coding region sequences of VIP by cloning and sequencing, and its sequence was analyzed and functionally predicted by bioinformatics software. The present results provide more detailed information for further exploration on the biological function of VIP in pigeon.

Experimental animals
Ten healthy Wahui pigeons with half male and half female (26 days old, weighing 500±5 g) were purchased from Xixia Pigeon Farm (Nanchang, China) and provided with pigeon-starter feed and water. Raised in Animal Anatomy Laboratory for one day of fasting, pigeons were dissected after anesthetization on the next day. The intestines were quickly collected to put in liquid nitrogen, and then stored in refrigerator at -80 o C. All of the procedures were performed in accordance with the Ethics Committee and Guidelines of Animal Experiments of our institute.

RNA extraction and cDNA synthesis
RNA was extracted from ileum according to Trizol method. Briefly, the frozen specimens were powdered in liquid nitrogen and homogenized in RNAiso Plus (TAKARA Bio Inc., Japan), from which 500 μl was transferred to EP tube. 200 μl chloroform was added to each tube, violently shaken for 15 s and centrifuged for 15 min (4 o C, 12000 r/min). The sample was divided into three layers: the yellow organic phase, the middle layer and the upper colorless water phase. The upper water phase was transferred to a new 1.5 ml EP tube, and then 500 μl isopropanol was added in this tube and mixed gently. The mixture was kept at room temperature for 10 min, was centrifuged for 10 min (4 o C, 12000 r/min). After the supernatant was discarded, total RNA was washed with 1 ml 95% ethanol prepared by RNase-Free ddH 2 O. Then 30 μl DEPC water (TAKARA Bio Corp., Japan) was added to fully dissolve and precipitate in refrigerator at -80 o C (Thermo Scientific, USA). RNA concentration and OD value were detected by ultraviolet spectrophotometer (Beckman Corp., USA). The first strand cDNA was synthesized using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Transgen, China), and then placed in refrigerator at -20 o C.

PCR amplification and sequencing
PCR amplification primers were designed by Primer Premier 5.0 software according to VIP sequence of pigeon from GeneBank (Accession number: XM_021294865.1). CDS region of VIP was amplified by PCR using the above cDNA as a template. The PCR amplification mixture of 25 μl was as follows: 12.5 μl of 2×EasyPfu PCR SuperMix, 1 μl of each of the upstream and downstream primers (Table I), 2 μl of template, 8.5 μl of ddH 2 O. PCR reaction conditions was applied as follows: pre-denaturation at 95 o C for 5 min, denaturation at 94 o C for 30 s, annealing at 50.7 o C for 30 s, extension at 72 o C for 40 s, 35 cycles, extension at 72 o C for 8 min. The electrophoresis of PCR products was performed by using 1% agarose gel, and then the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence detection.

Bioinformatic analysis
The biological information of VIP was obtained by a series of predictive analysis based on CDS above. These items and software are shown as Table II.

CDS region of VIP
After PCR amplification, the target CDS region are 511 bp long as agarose gel electrophoresis (Fig. 1), subsequently, the nucleotide sequence was obtained by sequencing, and the open reading frame is of 399 bp length according to the sequence analysis by NCBI online.

Physicochemical properties and hydrophobicity analysis on VIP
The physicochemical properties of VIP were predicted by ProtParam software. The peptide is encoded by 132 amino acids (Fig. 2), the amino acid composition is shown in Table III, and the content of leucine is the highest with 12.9%. The molecular formula and mass of VIP are O n l i n e According to the hydrophobicity prediction by Protscale program, it can be seen that Valine in the 67 th position has the strongest hydrophobicity due to the hydrophobic value with 2.933, and Lysine in the 77 th position has the strongest hydrophilicity due to hydrophilic value with -3.967. The hydrophilicity contributes to approximately 70% with larger hydrophilic region; accordingly, it should be a soluble peptide.

F i r s t A r t i c l e
The signal peptide, transmembrane structure and subcellular localization of VIP The signal peptide of VIP was predicted using SignalP5.0 software, but no signal peptide was found in VIP of Wahui pigeon, it infers that VIP is not a secreted peptide. Meanwhile, the peptide does not have a transmembrane structure based on the prediction of TMHMM software (Fig. 3).

Other structure prediction of VIP
As to the N-glycosylation site prediction of VIP by using NetNGlyc1.0, it was found that VIP has a potential N-glycosylation site at the 71 st amino acid with a score of 0.7126 (Fig. 4). In the phosphorylation site prediction by using the NetPhos 3.1 server software online, when the threshold of potential phosphorylation site is set at 0.5, there are 17 potential phosphorylation sites in VIP, which consists of 12 Serine, 2 Threonine, and 3 Tyrosine sites (Fig. 5). There are probable six conserved protein kinase binding sites of unsp (including PKC, PKA, CKII, GSK3, and INSR in VIP), and its score has the highest value of 0.991 at 120.  The secondary structure of VIP in Wahui pigeon was predicted by ExPAsY's SOPMA program. The Alpha helix of VIP has 67 amino acids, taking up 50.76%, and the Beta turn has 7 amino acids, taking up 5.30%. The extended strand has 12 amino acids, taking up 9.09%, and the Random coil has 46 amino acids, taking up 34.85%. The tertiary structural model was obtained by homology modeling of VIP with Phyre 2. The peptide is folded, bent and forms into a three-stage structure (Fig. 6). According to the genetic sequence analysis established among ten different animals, the phylogenetic tree can be divided into three large groups, from which Wahui pigeon locates in the same small group with Meleagris gallopavo, Gallus gallus and T. guttata, the phylogenetic evolutionary trend indicates that there are the closest relative relationship between Wahui pigeon and other three kinds (Fig. 7).

DISCUSSION
VIP is a neuropeptide extensively distributed in tissues, it exerts pleiotropic functions in multiple systems, such as the gastrointestinal, cardiovascular, nervous, and immune systems (Villanueva-Romero et al., 2018;Kasacka et al., 2015;Benitez et al., 2018). It has been reported that VIP can participate in regulating immune balance as an immunomodulatory and anti-inflammatory factor (Benitez et al., 2018;Li et al., 2017). Therefore, it has been considered as a potential candidate for treating various autoimmune and inflammatory diseases, and also has been effective in prevention of autoimmune diseases, such as diabetes mellitus, rheumatoid arthritis, EAE, sepsis and inflammatory bowel disease (Villanueva-Romero et al., 2018;Jimeno et al., 2010;Ibrahim et al., 2012;Tan et al., 2015;Shi et al., 2016;. In the treatment of lupus erythematosus, VIP modulates immune homeostasis of Th17/Treg by down-regulating serum levels of autoantibodies and renal levels of IL-17 and IL-6, and up-regulating renal levels of Foxp3 and IL-10, thereby improves renal functional defects, proteinuria and renal damage (Fu et al., 2019). VIP and its receptors are overexpressed in many common tumors, including bladder, breast, colon, liver, lung, pancreatic, prostate, thyroid, and uterine cancer, VIP and PACAP stimulate the growth of several cancer cell lines in vitro (Moody et al., 2016), as indicates VIP may affect tumor growth and differentiation. In addition, VIP widely distributes in the gastrointestinal mucosa, which has various functions such as regulating the physiology, biochemistry, immune function of the gastrointestinal tract, and resisting inflammation, taking on immunity, and relaxing muscles (Montagnese et al., 2015). In studies on live Salmonella typhimurium instead of LPS, VIP can down-regulate inflammatory mediators in human monocytes and murine macrophages (Foster et al., 2006;Askar et al., 2015;Ibrahim et al., 2018), thereby S. typhimurium probably increase its survival rate in humans and mice by VIP. According to study on the immunomodulatory effects of VIP and the increase in survival and growth of S. typhimurium, VIP reduced cytokines over-expressed (IL-1β, IL-6, TNF-α, IFN-γ and IL-10) in sepsis, and confirmed that VIP can be used as an adjuvant therapy for antibiotics in sepsis (Askar et al., 2020). The above studies demonstrate that VIP has enormous potential for the treatment of inflammatory diseases in the immunomodulation of innate immune responses.
According to the bioinformatics analyses in present study, on the one hand, Wahui pigeon is clustered with chicken, turkey, and T. guttata. The close relationship suggests the structure of VIP is evolutionarily conservative among these species. On the other hand, this simple structure and fine solubility account for the whole physiochemical and biological properties of VIP, and also greatly explain its extensive distribution in different tissues. It is indicated that the molecular characteristics are appropriate to play its functions in various systems.

CONCLUSIONS
In conclusion, we obtained CDS region of VIP gene with 511 bp in Wahui pigeon, and analyzed its molecular characteristics and properties, this simple molecular structure and characteristics are appropriate to take various functions in multiple systems.