Universal Primers for Cloning Avian α-Interferon and Its Application
A degenerate primer and poultry technology, applied in the biological field, can solve problems such as difficulty in starting α-interferon cloning
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Embodiment 1
[0031] Example 1 Primer Design
[0032] According to the sequences of 10 known poultry IFN-α genes, the present invention analyzes three relatively conservative nucleotide sequences (such as figure 1shown), based on the three sequences, three degenerate primers were designed and synthesized for cloning the avian IFN-α gene. The nucleotide sequence of one of the upstream degenerate primers AIF-TY1a is: 5'-CASCRSYACRBCCASCASCTCSAGC-3' (shown in SEQ ID NO.1), and the nucleotide sequence of the other upstream degenerate primer POAU is: 5'-CYRSYSCTCNYGCTCCTYCT- 3' (shown in SEQ ID NO.2), the nucleotide sequence of its common downstream degenerate primer AIF-TY2b is: 5'-AGGMGGACGRGKTCCCASGCGCAG-3' (shown in SEQ ID NO.3). Where R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, B=C / G / T, N=A / C / G / T.
[0033] Primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. Using the pair of primers, the IFN-α gene was amplified by PCR technique.
[0034] Reference sequence and sequence num...
Embodiment 2
[0036] Example 2 Application of primers AIF-TY1a and AIF-TY2b in amplifying IFN-α gene
[0037] Using this pair of primers, the IFN-α gene was amplified from the genomic DNA of chicken, duck and goose respectively, and the fragment length was 167bp.
[0038] Operating procedures:
[0039] 1. According to the simple and rapid extraction method of poultry blood genome DNA (Wu Huiying, Liu You, Jia Rumin and Liu Yanfen, etc. Research on the simple and rapid extraction method of goose blood genome DNA [J]. Guangdong Animal Husbandry. Veterinary Science and Technology, 2010,4(35) :29-31), Genomic DNA was extracted from the blood of chicken, duck and goose respectively;
[0040] 2. Using the genomic DNA of chicken, duck, and goose as templates, and AIF-TY1a and AIF-TY2b as primers, PCR amplifies the IFN-α gene. The reaction system and reaction conditions are as follows:
[0041] PCR reaction system:
[0042]
[0043]
[0044] Reaction conditions: Pre-denaturation at 94°C fo...
Embodiment 3
[0048] Example 3 Application of Primers AIF-TY1a and AIF-TY2b in Amplifying IFN-α Gene
[0049] In Example 2, species with known interferon genes were selected. The species used in this example are red-crowned crane and peacock, and these two species have not been cloned yet. PCR was amplified according to the operation process in the example, and the sequencing results showed that the amplified products of the two species were highly homologous to IFN-α through AIF-TY1a and AIF-TY2b. The primers are also suitable for unknown IFN-α species. The results of PCR amplification were as figure 2 shown.
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