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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[0042] Example 1 Cloning of chicken defensin 9 gene
[0043] PCR was performed with primer sequences SEQ ID NOs: 1~4 according to the following procedures: primers (20 μmol / L), 1 μL dNTPs (10 mmol / L), 1 μL, 10×buffer (Mg 2＋ ) 5μL, rTaq DNA polymerase (2.5U / μL) 1μL, ddH 2 O was added to 50 μL. Using the obtained PCR product as a template, a second round of PCR amplification was performed 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 supplemented to 50 μL. PCR reaction program: pre-denaturation at 94°C for 2 min, 1 cycle; 94°C for 30s, 52°C for 30s, 72°C for 40s, 30 cycles; extension at 72°C for 5min.
[0044] When PCR products are detected by 1.5% agarose gel electrophoresis, specific bands of 100bp~250bp can be observed, see Figure 4 .
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[0045] Example 2 Amplification of ov sequences
[0046] Taking chicken oviduct total DNA as template, specific primers SEQ ID NO: 6~7 were used to amplify the ov sequence fragment. 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 (2.5 U / μL) 0.5 μL, template 0.5 μL, ddH 2 O supplement to 25 μL. PCR reaction program: 94°C for 5 min, 1 cycle; 94°C for 40s, 55°C for 40s, 72°C for 80s, 30 cycles; 72°C for 10min extension. Using the above product as a template, PCR was performed again, and the amount of each addition in the system was doubled, and the total volume was 50 μL.
[0047] When PCR products are detected by 1.5% agarose gel electrophoresis, a specific band of about 1000bp can be observed, see Figure 5 .
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[0048] Example 3 Construction of recombinant eukaryotic expression vector pVAX1-AvBD9
[0049] Restriction endonuclease for AvBD9 gene Nhe I and EcoR I was digested with enzyme digestion at 37°C for 3h. The reaction system was as follows: 10×buffer M 5μL, Nhe I 1 μL, EcoR I 1 μL, PCR product 25 μL, ddH 2 O supplemented to 50 μL, and the pVAX1 plasmid (invitrogen) was also treated with endonuclease. Nhe I and EcoR I for enzymatic digestion. 37℃ for 3h, 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 supplemented to 50 μL while dephosphorylating with CIAP (alkaline phosphatase). The reaction solution after digestion was all electrophoresed with 1.5% agarose, and the band of the target fragment was cut out under an ultraviolet lamp, and the target DNA fragment after digestion was recovered by a gel recovery kit. The digested PCR product of AvBD9 gene and the digested pVAX1 plasmid were ligated u...
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