Chicken defensin 9 gene expression vector and preparation method and use thereof

A chicken defensin and gene expression technology, applied in the field of genetic engineering, can solve the problems of lack of drugs and high cost of chemically synthesized antimicrobial peptides

Inactive Publication Date: 2013-01-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a chicken defensin 9 gene expression vector according to the defects of high cost of chemically synthesized antimicrobial peptides and lack of drugs prepared by biological methods in the prior art for the treatment of oviduct disease in breeders

Method used

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  • Chicken defensin 9 gene expression vector and preparation method and use thereof
  • Chicken defensin 9 gene expression vector and preparation method and use thereof
  • Chicken defensin 9 gene expression vector and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Cloning of chicken defensin 9 gene

[0043] Use primer sequence SEQ ID NO:1~4 to carry out PCR according to the following procedures: primers (20μmol / L) each 1μL dNTPs (10mmol / L) 1μL, 10×buffer(Mg 2+ ) 5μL, rTaq DNA polymerase (2.5U / μL) 1μL, ddH 2 Add O to 50 μL. Use the obtained PCR product as a template, and use SEQ ID NO:1 and SEQ ID NO:5 as primers for the second round of PCR amplification. The composition of the PCR reaction system is as follows: 10×buffer 5 μL, dNTPs 2 μL, primers (20 μmol / L) each 2 μL, rTaq DNA polymerase (2.5U / μL) 0.5 μL, template 1 μL, ddH 2 O make up to 50μL. 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 after extension for 5min.

[0044] When PCR products are detected by 1.5% agarose gel electrophoresis, specific bands ranging from 100bp to 250bp can be observed, see Figure 4 .

Embodiment 2

[0045] Example 2 Amplification of ov sequence

[0046] The total DNA of the 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) each 1 μL, rTaq DNA polymerase (2.5U / μL) 0.5 μL, template 0.5 μL, ddH 2 O make up to 25μL. PCR reaction program: 94°C pre-change 5min, 1 cycle; 94°C 40s, 55°C 40s, 72°C 80s, 72°C, 30 cycles; 72°C extension 10min. Using the above product as a template, perform another PCR, double the amount of each item added to the system, and the total volume is 50 μL.

[0047] When the PCR product is detected by 1.5% agarose gel electrophoresis, a specific band of about 1000 bp 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 Carry out restriction digestion, and act at 37°C for 3 hours. The reaction system is as follows: 10×buffer M 5μL, Nhe I 1μL, EcoR I 1μL, PCR product 25μL, ddH 2 Make up O to 50μL, and use the same endonuclease for pVAX1 plasmid (invitrogen) Nhe I and EcoR I for enzyme digestion. After 3h at 37℃, the reaction system is as follows: 10×buffer M 5μL, Nhe I 1μL, EcoR I 1μL, PCR product 20μL, ddH 2 Make up to 50μL of O and dephosphorylate with CIAP (alkaline phosphatase). All the digested reaction solution was electrophoresed with 1.5% agarose, and the band of the target fragment was cut under ultraviolet light, and the target DNA fragment after digestion was recovered with a gel recovery kit. Under the action of T4 ligase, connect the digested PCR product of AvBD9 gene and digested pVAX1 plasmid, ligate overnight at 4°C, ...

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Abstract

The invention discloses a chicken defensin 9 gene expression vector and a preparation method and use thereof in prevention and treatment of uterotubal diseases in breeding chickens, and belongs to the technical field of genetic engineering. The expression vector is constructed by using a eukaryotic vector Pvax1 as a starting vector, substituting chicken ovalbumin coding sequence for a CMV promoter in pVAX1 and inserting a chicken defensin 9 gene in the downstream of the chicken ovalbumin coding sequence. The preparation method of the expression vector comprises: connecting the chicken defensin 9 gene to the vector pVAX1; connecting a chicken ovalbumin gene to a vector Pmd18-T; subjecting the connected vectors to double enzyme digestion; recombining; and constructing a pVAX1 recombinant vector containing the chicken ovalbumin gene and the chicken defensin 9 gene, namely the chicken defensin 9 gene expression vector. The expression vector disclosed by the invention can be safely used inthe preparation of medicines for treating uterotubal diseases in chickens with obvious treatment effect but not toxin, and therefore has a bright application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a chicken defensin 9 gene expression vector, a preparation method thereof and an application in preventing and treating oviduct diseases of breeder chickens. Background technique [0002] Egg-borne diseases are diseases caused by pathogens passed from infected or infected hens to offspring. Due to the spread of egg-borne diseases, the result is that the commercial generation chicks are contaminated during the hatching process, the hatched chicks often die, and the remaining ones also become recessive carrier chickens. Because chicken breeding enterprises generally use regular feeding of effective antibacterial drugs to control the occurrence of diseases, but the abuse of drugs has led to a rapid increase in drug-resistant strains. People focus on antibacterial peptides. Antimicrobial polypeptides are rarely expressed in the natural environment, and the cost of chem...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N15/12C12N15/66A61K48/00A61P15/00A61P31/04
Inventor 谢青梅赵亚华林崇韫毕英佐高向阳马静云陈峰
Owner SOUTH CHINA AGRI UNIV
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