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Method for preparing oral avian influenza vaccine from transgenic dunaliella

An avian influenza, oral type technology, applied in the field of microalgae (salina) genetic engineering, can solve the problems of impossibility of frequent raw food, wild species weeding, ecological environment destruction, etc., and achieves low production cost, high output, and large-scale realization The effect of chemical production

Inactive Publication Date: 2011-01-26
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the growth of these plants is limited by seasons, and even the fruits and vegetables that can be eaten directly cannot be eaten raw frequently, and it is inconvenient to eat, so it is impossible to widely promote them among the crowd, especially children.
In addition, plant-derived vaccines also have biological safety issues, such as the possibility of exogenous genes in transgenic plants drifting into the environment and destroying the ecological environment, and whether cross-pollination between related plants will lead to weeding of wild species, carrying antibiotics or Whether transgenic plants with herbicide-like selective marker genes are safe, etc.

Method used

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  • Method for preparing oral avian influenza vaccine from transgenic dunaliella
  • Method for preparing oral avian influenza vaccine from transgenic dunaliella
  • Method for preparing oral avian influenza vaccine from transgenic dunaliella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the construction of avian influenza virus HA gene expression vector in salina

[0030] (1) Cloning and identification of cDNA of nitrate reductase (NR) gene of Salina salina: Take 10 ml of Dunaliella salina cells grown to the logarithmic growth phase, extract total RNA according to the instructions of Trizol, analyze its integrity by electrophoresis, and measure its purity. Quantitative. cDNA was synthesized according to the reverse transcriptase reagent instructions. According to the sequence of Salina NR cDNA registered in GenBank (GenBank accession number: AY312143.1), the primers were designed as follows:

[0031] nr1:5'-atgcccgcactcgccaacaacaca-3';

[0032] nr2:5′-tcagaagctcaccgtgcgctcttt-3′

[0033] Using cDNA as a template, the NR gene of Salina salina was amplified. The PCR reaction system was pre-denaturation at 94 °C for 2.5 min, denaturation at 94 °C for 45 s, annealing at 50 °C for 1 min, extension at 72 °C for 1.5 min, a total of 30 cycl...

Embodiment 2

[0072] Embodiment 2, screening and identification of transformed algal strains

[0073] (1) Screening: The high-efficiency expression vector pMNCH of Salina salina was transformed into a nitrate auxotrophic strain of Salina salina by the gene gun method, and the inoculation loop was used to streak on the solid salina salina medium with nitrate as the only nitrogen source, and observed after 7 days Growth of transformed cells and calculation of transformation rate. Single algae colony cells that can recover growth on the solid medium of Salina salina with nitrate as the only nitrogen source were picked, and were respectively expanded and cultured with liquid salina salina medium for further detection and identification.

[0074] (2) Identification of chlorate toxicity: Salina cells with NR activity died due to their sensitivity to chlorate toxicity. The transformed Salina cells were streaked on a solid medium containing chlorate to observe the effect of chlorate on Toxicity of S...

Embodiment 3

[0080] Embodiment 3, the immunology experiment of salina avian influenza vaccine:

[0081] (1) Antibody production test: 80 35-day-old test chickens were randomly divided into 4 groups, 20 in each group. The amount of transgenic salt algae in group Ⅰ was 10g / KG; the amount of transgenic salt algae in group II was 5g / KG; the amount of transgenic salina in group III was 1g / KG; The amount of algae is 10g / KG. At 7, 14, 21, 28, 35, 42 and 49 days after immunization, blood was collected from the heart to separate the serum, and the serum HI antibody level was measured.

[0082] .Hemagglutination test: use 96-well V-type reaction plate to measure antigen titer

[0083] a. First add 25 μl of physiological saline to each well of the reaction plate with a micropipette.

[0084] b. Use a micropipette to pipette 25 μl of the antigen to be tested into the first well and squeeze 6 times to mix, then suck out 25 μl to the second well, and sequentially double-dilute to the 11th well, and...

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Abstract

The invention discloses a method for preparing an oral avian influenza vaccine from transgenic dunaliella. The method particularly comprises the following steps of: introducing a controlling element of the dunaliella, constructing a double-component expression vector which contains recombination genes of avian influenza viruses, namely, HA and NA, transforming the dunaliella and screening and identifying so as to determine a dunaliella transformation strain of a high-expression recombinant C3d-H5N1 fusion protein; and amplifying a stable expression C3d-H5N1 fusion protein transformation strain, performing large-scale open culturing on the strain, centrifugally collecting frustules, refrigerating, drying and packaging under an aseptic condition so as to prepare oral avian influenza vaccine capsules. The dunaliella transformation strain prepared by the method can be cultured in an open state, so that high yield and low cost are realized. Therefore, mass production of the oral animal avian influenza vaccine prepared from the transgenic dunaliella is realized and market demand is met.

Description

technical field [0001] The present invention relates to microalgae (salina) genetic engineering, in particular to a method for preparing oral avian influenza vaccine from transgenic salina, specifically, introducing regulatory elements of salina itself and constructing a high-efficiency expression vector to establish an efficient avian influenza virus The gene expression system provides a technical platform for the production of oral vaccines. Background technique [0002] Avian influenza (Avian influenza, AI) is a disease caused by influenza virus that mainly attacks the respiratory system. It has been found in Italy in 1878 and has a history of more than 100 years. Spreading within a wide range has brought great harm to human health and livestock and poultry production. China is the largest poultry-raising country with a total of 13 billion poultry. At the same time, my country has a large number of poultry, wide distribution of waterfowl, extensive and open breeding, and...

Claims

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Application Information

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IPC IPC(8): A61K39/145C12N1/13C12N15/63A61P31/16
Inventor 薛乐勋潘卫东李杰吕玉民侯桂琴
Owner ZHENGZHOU UNIV
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