Method for preparing chicken genetic engineering immunopotentiator by akirin2 gene sequence

An immune enhancer and genetic engineering technology, which is applied in the field of preparing chicken genetically engineered immune enhancer, can solve the problems of not being able to widely improve the immune effect of vaccines and bacterins, poor enhancement effect, and high preparation costs, and reduce vaccine production. Cost, broad application prospects, low cost effect

Inactive Publication Date: 2011-11-16
HARBIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that the existing traditional vaccine immune enhancer has poor enhancement effect, high preparation cost, large toxicity and side effects, and the existing geneti

Method used

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  • Method for preparing chicken genetic engineering immunopotentiator by akirin2 gene sequence
  • Method for preparing chicken genetic engineering immunopotentiator by akirin2 gene sequence
  • Method for preparing chicken genetic engineering immunopotentiator by akirin2 gene sequence

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specific Embodiment approach 1

[0018] Specific embodiment one: the present embodiment utilizes akirin2 gene sequence to prepare the method for chicken genetic engineering immunopotentiator to carry out according to the following steps:

[0019] 1. Using the EST database of red jungle fowl in NCBI, the coding sequence (CDS) of the human akirin2 gene (GenBank: NM 001007589) was used as the target, and the chicken akirin2 gene coding sequence was obtained by using BLAST software for electronic cloning;

[0020] 2. Using Trizol reagent to extract the total RNA of the skeletal muscle of 3-day-old chicks, and using the two-step RT-PCR technique to obtain the full-length coding sequence of the chicken akirin2 gene, after purification, clone it into the vector pMD18-T and sequence and identify it, and obtain the chicken akirin2-containing gene Gene recombinant pMD18-T vector;

[0021] 3. According to the full-length coding sequence of the chicken akirin2 gene, design primers P1 and P2, and make the 5'-ends of the p...

specific Embodiment approach 2

[0047] Embodiment 2: This embodiment differs from Embodiment 1 in that the 3-day-old chicks in step 2 are commercial Hailan brown chickens. Other steps and parameters are the same as those in Embodiment 1.

[0048] In this embodiment, the 3-day-old commercial Hailan brown chickens, whose average antibody titers to Newcastle disease virus (NDV) before immunization, were all less than 2 Log2, were purchased from the hatchery of Northeast Agricultural University.

[0049]

[0050]

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Abstract

The invention provides a method for preparing a chicken genetic engineering immunopotentiator by an akirin2 gene sequence, relating to a method for preparing a chicken genetic engineering immunopotentiator. The invention solves the problem that the existing genetic engineering immunoenhancement nucleic acid vaccines can not extensively improve the immunity effects of vaccines and bacterins. The method comprises the following steps of: acquiring a chicken akirin2 gene coded sequence; acquiring a recombinant pMD18-T vector containing the chicken akirin2 gene; designing a primer and enabling the 5'- terminals to have HindIII and EcoRI restriction sites, subcloning the chicken akirin2 gene in the recombinant pMD18-T vector into an expression vector to construct a recombinant expression vector; and diluting the recombinant expression vector by a sterile PBS (phosphate buffer solution). The chicken genetic engineering immunopotentiator provided by the invention can improve the expression levels of multiple immunity related factors such as IL-1, IL-6, toll-like receptors and the like, and has the characteristics of wide applicable range and the like for various vaccines and bacterins.

Description

technical field [0001] The invention relates to a method for preparing chicken genetic engineering immune enhancer. Background technique [0002] Akirins, discovered in 2008, are highly conserved nucleoproteins among different species. Akirin is a protein encoding 201 amino acids, showing two conserved structural domains at the N-terminus and C-terminus respectively, and there is a nuclear localization signal between 24-29 amino acid residues at the N-terminus. There are two homologous genes (akirin1 and akirin2) in zebrafish, Xenopus laevis, human and mouse, and only one copy (akirin2) in insects (honey bee, mealworm, anopheles gambiae and fruit fly) and chicken, but The gene is not found in plants, yeast and bacteria. Akirin2 functions as a nuclear factor required for innate immune response signaling pathways in immune and inflammatory responses in Drosophila and mice. [0003] Genetic engineering immune enhancer is to clone one or more cytokine genes (currently commonl...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K39/00A61K48/00C12N15/85
Inventor 满朝来
Owner HARBIN NORMAL UNIVERSITY
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