Recombinant adenovirus as well as preparation method and application thereof

A recombinant adenovirus and adenovirus technology, applied in the field of molecular biology, can solve the problems of not being able to become an immunotherapy method, inducing severe reactions, and inconvenient administration, etc., and achieve good application prospects, less frequent administration, and high compliance Effect

Inactive Publication Date: 2014-02-12
马仕坤
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although SIT is currently the only treatment for the cause of allergic diseases, it has the following disadvantages: 1) Long course of treatment: it usually starts to take effect half a year to a year after starting treatment, and the total course of treatment takes 3-5 years. 2) One or two injections per week, a total of hundreds of injections are required; 3) There is a certain risk: serious reactions may be induced during the treatment; 4) Inconvenient administration: traditional SIT It is a subcutaneous injection and needs to be injected in a medical institution every time; 5) The allergens currently used in SIT mainly come from natural extracts, which are composed of a variety of sensitizing and non-sensitizing compounds, and the composition is complex and difficult to standardize , and the extraction process has the risk of introducing new allergens
This method has achieved satisfactory results in animal experiments. A clinical trial involving 165 allergen patients also showed that three injections of allergens in the inguinal lymph nodes within two months were as effective in reducing clinical symptoms as the three-year standard. SIT is equivalent; however, the reliance on ultrasonic positioning equipment, related professionals, and the risk of puncture itself make it still not an ideal means of immunotherapy

Method used

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  • Recombinant adenovirus as well as preparation method and application thereof
  • Recombinant adenovirus as well as preparation method and application thereof
  • Recombinant adenovirus as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Inserting the hIL-10 gene into the shuttle plasmid to construct the recombinant shuttle plasmid pAdTrack-hIL-10

[0044] 1. PCR amplification of hIL-10 gene

[0045] (1) Isolation of peripheral blood mononuclear cells from healthy people

[0046] Take 10 ml of peripheral venous blood from healthy people, anticoagulate with heparin sodium, add an equal volume of 0.9% sodium chloride to dilute the anticoagulated blood. Then take 10 ml of the lymphocyte separation solution stored at 4°C in the dark and add it to the bottom of a sterile centrifuge tube, and warm it up to room temperature. Use a Pasteur pipette to draw 20 ml of the diluted blood sample, and spread it slowly along the tube wall on top of the lymphocyte separation medium, trying to avoid disturbing the interface of the liquid layer. The horizontal rotor was centrifuged at 2000 rpm for 20 minutes at room temperature. After centrifugation, it can be seen that the liquid in the centrifuge tube is ...

Embodiment 2

[0080] Example 2: OVA gene was inserted into the shuttle plasmid to construct the recombinant shuttle plasmid pAdTrack-hIL-10-OVA

[0081] 1. PCR amplification of OVA gene

[0082] Firstly, the total RNA of chicken fallopian tube epithelial cells was extracted by TRIzol reagent, and cDNA was synthesized by reverse transcription with Oligo dT (oligomeric T) as a primer. According to the OVA gene sequence (GenBank accession number AY223553.1), primers were designed using Primer Premier5 software. These include the upstream Xho I and downstream Xba I restriction sites (underlined), and the primer sequences were synthesized by Shanghai Sangong.

[0083] Amplification primers are as follows:

[0084] Upstream primer: 5'TAC CTCGAG ATGGGCTCCATCGGTG3' (SEQ ID No. 8)

[0085] Downstream primer: 5'GCGC TCTAGA TTAAGGGGAAACACA3' (SEQ ID No. 9)

[0086] First, pre-denaturation at 95°C for 5 minutes; followed by thermal cycling, the reaction conditions are: denaturation at 95°C for...

Embodiment 3

[0105] Example 3: Construction of recombinant adenovirus plasmid (homologous recombination method in bacteria)

[0106] The linearized recombinant shuttle plasmid pAdTrack-hIL-10-OVA (kanamycin resistance) and the adenovirus backbone plasmid pAdEasy-1 (ampicillin resistance) were co-transformed into BJ5183 bacteria for homologous recombination, containing kanamycin The medium was screened, identified by agarose electrophoresis restriction enzyme digestion and PCR, and the correct recombinant plasmid was named pAd-hIL-10-OVA.

[0107] 1. Preparation of competent state for BJ5183 transformation

[0108] The steps are the same as the above DH5α competent preparation method, except that the culture medium contains 30 μg / ml streptomycin to inhibit the growth of other bacteria.

[0109] 2. Homologous recombination

[0110] Extract the pAdTrack-hIL-10-OVA plasmid with the Omega Plasmid Extraction Kit, and digest it with the restriction endonuclease Pme Ⅰ to make it linear; purify t...

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Abstract

The invention provides a recombinant adenovirus as well as a preparation method and application thereof. The recombinant adenovirus simultaneously comprises an immunomodulatory factor IL-10 and an allergen gene, and the immunomodulatory factor IL-10 and the allergen gene are subjected to fusion expression. The recombinant adenovirus disclosed by the invention can be utilized as an oral vaccine for preventing and treating anaphylactic diseases caused by protein allergen.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a recombinant adenovirus, in particular to a recombinant adenovirus co-expressing IL-10 and an allergen gene and its preparation and application. Background technique [0002] Allergic diseases such as allergic rhinitis, allergic asthma, and food and drug allergies are common health problems that plague people's lives and work, and anaphylactic shock is a life-threatening killer. The World Allergy Organization (WAO) announced the results of an epidemiological survey of allergic diseases in 30 countries on the world's first allergic disease day: 22% of the total population of these countries suffer from IgE-mediated allergic diseases , including allergic rhinitis, asthma, conjunctivitis, eczema, food allergy, drug allergy and anaphylactic shock. According to the World Health Organization (WHO), about 150 million people worldwide suffer from asthma, of which more than 50% of adult...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N7/01A61K39/235A61K35/76A61P37/08
Inventor 马仕坤赵婧文
Owner 马仕坤
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