A kind of chicken defensin 9 gene expression vector and its preparation method and application
A chicken defensin and gene expression technology, applied in the field of genetic engineering, can solve the problems of high cost of chemically synthesized antimicrobial peptides and lack of drugs
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Embodiment 1
[0042] Example 1 Cloning of chicken defensin 9 gene
[0043] Use the primer sequence SEQ ID NO:1~4 to carry out PCR according to the following procedure respectively: each primer (20μmol / L) 1μL dNTPs (10mmol / L) 1μL, 10×buffer (Mg 2＋ ) 5 μL, rTaq DNA polymerase (2.5U / μL) 1 μL, ddH 2 O was added to 50 μL. The obtained PCR product was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 1 and SEQ ID NO: 5 as primers. The composition of the PCR reaction system is as follows: 10×buffer 5 μL, dNTPs 2 μL, primers (20 μmol / L) 2 μL each, rTaq DNA polymerase (2.5U / μL) 0.5 μL, template 1 μL, ddH 2 O to make up to 50 μL. PCR reaction program: 94°C pre-denaturation for 2min, 1 cycle; 94°C for 30s, 52°C for 30s, 72°C for 40s, 30 cycles; 72°C extension for 5min.
[0044] Specific bands at 100bp~250bp can be observed when PCR products are detected by 1.5% agarose gel electrophoresis, see Figure 4 .
Embodiment 2
[0045] Embodiment 2 Amplification of ov sequence
[0046] The total DNA of chicken oviduct was used as a template, and the ov sequence fragment was amplified with specific primers SEQ ID NO: 6-7. The composition of the PCR reaction system is as follows: 10×buffer 2.5 μL, dNTPs 2 μL, primers (20 μmol / L) 1 μL each, rTaq DNA polymerase (2.5U / μL) 0.5 μL, template 0.5 μL, ddH 2 O to 25 μL. PCR reaction program: 94°C pre-change for 5min, 1 cycle; 94°C for 40s, 55°C for 40s, 72°C for 80s, 30 cycles; 72°C extension for 10min. Using the above product as a template, PCR was performed again, and the addition amount of each item in the system was doubled, and the total volume was 50 μL.
[0047] When the PCR product is detected by 1.5% agarose gel electrophoresis, a specific band of about 1000bp can be observed, see Figure 5 .
Embodiment 3
[0048] Example 3 Construction of recombinant eukaryotic expression vector pVAX1-AvBD9
[0049] Restriction enzymes for AvBD9 gene Nhe I and EcoR I carried out enzyme digestion and digestion, and acted at 37°C for 3 hours. The reaction system was as follows: 10×buffer M 5 μL, Nhe I 1 μL, EcoR I 1 μL, PCR product 25 μL, ddH 2 O was added to 50 μL, and the pVAX1 plasmid (Invitrogen Company) was also treated with endonuclease Nhe I and EcoR I performed enzyme digestion. 37°C for 3 hours, the reaction system is as follows: 10×buffer M 5μL, Nhe I 1 μL, EcoR I 1 μL, PCR product 20 μL, ddH 2 O to 50 μL while dephosphorylated with CIAP (alkaline phosphatase). All the digested reaction solutions were electrophoresed with 1.5% agarose, and the target fragment bands were excised under ultraviolet light, and the digested target DNA fragments were recovered with a gel recovery kit. Under the action of T4 ligase, ligate the digested PCR product of AvBD9 gene and dig...
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