Molecular cloning and sequencing analysis of the interferon β from Coturnix

One pair of primers was designed according to Gallus and Meleagris gallopavo interferon β (IFN-β) sequences published in GenBank. The primers and RNA extraction from the spleen of Coturnix were used to amplify Coturnix IFN-β cDNA by real-time polymerase chain reaction (RT-PCR). The product was cloned into pEasy-T1 vector. Evaluating recombinant plasmid by PCR and restriction enzyme digestion. Sequence the cloning sequences, comparing the sequencing results by NCBI. We successfully got a Coturnix IFN-β partial sequence. The sequence was subtyped and put to homologous analysis. The results suggested the homology of IFN-β gene of Coturnix and gene of Coturnix and chicken (88.7%), the homology of IFN-β gene of Coturnix and chicken (88.7%), the homology of IFN-β gene of Coturnix and Anas platyrhynchos (72.5%), the homology of IFN-β sequence registered in GenBank. The analysis of the genetic tree showed that the relationship of Coturnix and chicken IFN-β had a high homology. It can be seen that in this study we successfully got a partial sequence of IFN-β of quail.


Introduction
The immune response against viral pathogens includes specific and non specific mechanisms. Cytokines are peptides which can play a role in the non specific immunity [1]. Interferon (IFN) is a kind of highly active multifunctional glycoprotein, and it is an important cytokine with a broad range of biological activities [2]. Interferon is a secretory glycoprotein produced by the biological cells when it is subjected to the influence of the virus or other inducing agent [3]. Interferon has many kinds of bioactivity, such as antiviral activity, immune regulation and so on [4]. Interferon is an important part of the body's defense system. Interferon has broad-spectrum resistance [5]. When interferon acts on the body's organic tissue cells, it can make it obtain the ability to resist a variety of viruses and microbes [6]. Interferon has strict selectivity for a somatic cell, and has a relative species specificity. But the specificity is relative, not absolute [7]. Interferon can be divided into type I and type II [8]. Interferon of type I is a product of many gene families, including 14-20 interferon α genes, 1 kind of interferon β gene; interferon of type α contains only one family member, namely the interferon g [9]. Type I interferons are the most effective antiviral cytokines. Type I interferon genes are located on chromosome 9, and are segregated in a "modern" and an "ancestral" group with distinctive effects on the cells. Interferons α are represented by a large family of structurally related genes while the IFN-β is encoded by a single gene [1,10,11].
The meat and eggs of Coturnix were delicious in China and known as "animal's ginseng". Currently, the number of Coturnix raised is about 200 million in China, which is 1/5 of the world production. Coturnix is an important part of the economic animal production in China. But the occurrence of avian influenza and New-castle diseases posed a serious threat to the production of Coturnix. Establishing the Coturnix immune mechanism, looking for a new and efficient security immune route makes it necessary to breed Coturnix. By contrast, the research on animal interferon lags behind. There/This is still the main stay/subject? in basic research and clinical trials, and most concentrating on a few animals such as pigs, chicken, fish. But a big progress has been made in recent years. There are commercial interferon of pigs, dogs, chicken and recombinant interferon product coming to the market. However, there are very few research reports about the Coturnix interferon.

Sample
Spleen was collected from Coturnix. Coturnix was brought as a live mature Coturnix from a common farm and kept under inspection for 5 days to be sure that it is Molecular cloning and sequencing analysis of the interferon β from Coturnix free from any clinical infection, then the sample was collected and stored at -20°C.

Primer design
We downloaded the complete IFN-β mRNA sequences of the gallus (NM_001024836) and Meleagris gallopavo (U28140) from GenBank. The sequence was aligned using the ClustalW application (Dnastar software) where we designed primers match with the alignment of the sequences.

Amplification of cDNA sequence
Total RNA from Coturnix spleen was extracted by Trizol (TransGen) according to the manufacturer's protocol and was reverse transcribed to cDNA. Reverse transcription is processed with a Prime script real-time (RT) reagent kit (TaKaRa) according to the manufacturer's instructions in 10 μl reaction system. The reagent was blended and put in the polymerase chain reaction (PCR) Amplifier at 37°C for 15 min and inactivated at 85°C for 5 s.
The PCR using the reverse transcription product was performed to amplify the target gene using specific primers. Polymerase chain reaction mixtures contained 50 pmol for each forward and reverse primer, 1 μl template cDNA, 200 μM dNTP mix and 0.2 μl 5 U Ex Taq polymerase (TransGen) in 1× Ex buffer up to 25 μl with ddH 2 O. The PCR was processed in the PCR Amplifier as follows: initial denaturation at 94°C for 5 min, 35 cycles at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, final extension at 72°C for 10 min. The PCR amplicons were verified by 0.8% agarose gel electrophoresis at 120 mA for 30 min and products were visualized by staining with DuRed (Fanbo Biochemicals), we checked the band and got the images with GelDoc™ XR+system (Bio-RAD, USA).

Construction and identification of recombinant plasmid with interferon
The Gel Extraction Kit was used to purify and recycle the PCR products. With reference to the instruction of cloning vector pEasy -T1, in order to join the components in the 10 ml reaction system, the following steps were taken. After gently blending, we put it in the PCR instrument to connect 25°C 10 min for conversion. Getting 5 ml connecting product into fresh preparative DH5a cells, coating converted products on the LB/Amp plate evenly under aseptic conditions. Putting the tablet at 37°C thermostat in 12 h and picking a single colony after culturing, preparing and evaluating plasmid DNA. Picking a single colony from the above overnight culture tablet randomly, vaccinating in 5 ml LB liquid medium containing Amp. Shaking culture overnight in 37°C, preparing plasmid DNA using GenEluteTM High Performance Plasmid Kits. Evaluating recombinant plasmid by PCR and restriction enzyme digestion. In order to further determine the resulting clone, recombinant plasmid identified as positive clones after the above steps was sent to Shanghai Sangon Biological Company for DNA sequencing. Sequencing results and the nucleotide sequence registered in GenBank was subtyped and put to homologous analysis.

Interferon-nucleotide sequence homology comparison and the analysis of the phylogenetic tree
The sequence of the Coturnix IFN-β mRNA was blast in the GenBank using nucleotide blast and the translated amino acids were also blast by protein blast to check whether the new sequence is related to any other cloned gene.

Interferon gene cloning results
Total RNA was extracted from Coturnix spleen cells, then amplified to products by RT-PCR. The PCR products underwent electrophoresis in 0.8% agarose gel. There was a specific band in 400 bp with expectations (Fig. 1).

Interferon sequence analysis
The partial mRNA sequence of Coturnix IFN-β consists of 396 nucleotides. The homology of IFN-β gene of Coturnix and gallus was 88.7%. The consensus cDNA sequence showed 72.5% identity with a. platyrhynchos and 71.5% identity with Meleagris gallopavo as compared with the nucleotide sequence registered in GenBank (Figs. 2, 3)

Interferon β amino acid sequence analysis
The predicted protein encoded by Coturnix IFN-β mRNA sequence is composed of 132 amino acids. The predicted amino acid sequence of Coturnix IFN-β was compared with the IFN-β of gallus and Meleagris gallopavo using DNAMAN (Fig. 4). The homology of IFN-β amino acid of Coturnix and gallus was 78.8%, the homology of IFN-β amino acid of Coturnix and Meleagris gallopavo was 56.8% as compared with the amino acid sequence (Figs. 5, 6). The analysis of the phylogenetic tree   showed that the relationship of Coturnix and gallus IFN-β had a high homology.

Conclusions
Temporarily quail β interferon gene sequence is not published at home and abroad. This is the first study to focus on the sequence of Coturnix IFN-β. We successfully got a Coturnix INF-β partial sequence. The sequence was subtyped and put to homologous analysis. Our results proved that the relationship of Coturnix and gallus IFN-β had a high homology.
The predicted protein encoded by Coturnix IFN-β mRNA sequence has a high homology to gallus. This may have a role in the difference of the immune response against pathogen between Coturnix and gallus. The result can be a reference for further research and practical application.
authors declare no conflict of interest.