Recombinant DNA encoding the major allergen of plantago lanceolata pollen, PLA L 1, and applications thereof

A major allergen, plantain technology, applied in recombinant DNA technology, applications, allergic diseases, etc., can solve problems such as uncharacterized plantain allergens

Inactive Publication Date: 2005-03-16
ALK ABELLO SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Despite the reports mentioned above, no data characterizing the major allergens of plantain have been re...

Method used

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  • Recombinant DNA encoding the major allergen of plantago lanceolata pollen, PLA L 1, and applications thereof
  • Recombinant DNA encoding the major allergen of plantago lanceolata pollen, PLA L 1, and applications thereof
  • Recombinant DNA encoding the major allergen of plantago lanceolata pollen, PLA L 1, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] This example is used to describe the isolation of RNA from P. lanceolata pollen, the synthesis of first-strand cDNA, and the amplification of the 3' fragment of the Pla l 1-specific cDNA.

[0102]Total RNA was extracted from staghorn plantain pollen by prolonged sonication of pollen grains in a denaturing solution, followed by phenol-chloroform extraction of the supernatant. Pollen (0.6 g) was suspended in 7 mL of denaturing solution (4M guanidinium thiocyanate, 25 mM sodium citrate pH 7.0, 0.5% (w / v) sarcosinate lauryl, 100 mM β-mercaptoethanol) (25) , and then the suspension was sonicated in a Vibracell sonicator VC300 for 30 min, with a working cycle of 50% ultrasonic exposure, at setting 9. Then 0.7mL of 2mM sodium acetate solution with pH4.0, 7mL of water-saturated phenol and 1.4mL of chloroform: isoamyl alcohol solution (49:1, v / v) were added to the sonicated suspension, and warmed at 4°C. Incubate for 15 minutes, shaking occasionally. The two phases were separa...

Embodiment 2

[0111] This example describes the cloning and sequencing of the Pla 1 1-cDNA 3' fragment.

[0112] The Pla 1 1-cDNA 3' fragment amplified by 3'-RACE PCR was purified from agarose gel, and then it was cloned into the pGEM Easy vector carrying a T nucleoside hanging end (Promega Company)[ 26], the plasmid carries an ampicillin resistance gene that can be used to screen transformants and multiple restriction enzyme sites on both sides of the cloning site. Under the action of T4 DNA ligase, incubate at 4°C for 16 hours to carry out the ligation reaction. The ligation product was then transformed into E. coli DH5α. Plasmid DNA was prepared from recombinant transformants by using the method of Wizard Plus Minipreps (Promega). In order to confirm that the screened transformant contains the desired insertion sequence, the plasmid DNA sample was digested with EcoR I (Boehringer), and then analyzed by agarose gel electrophoresis. The complete double-stranded nucleotide sequences of s...

Embodiment 3

[0115] This example describes the synthesis, amplification, cloning and sequencing of the 5' fragment of Pla 11, which includes the nucleic acid sequence encoding the leader peptide and the N-terminal fragment of the protein.

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PUM

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Abstract

A nucleic acid molecule encoding a peptide or protein comprising at least one epitope of the major allergen of Plantago lanceolata, Pla I 1, wherein the nucleic acid molecule a) has the sequence of SEQ ID NOS.: 5-7, b) is a fragment of the sequence SEQ ID NOS.: 5-7, c) has a sequence encoding the amino acid sequence of SEQ ID NO.: 8 or a fragment thereof, d) has a sequence hybridising to SEQ ID NOS.: 5-7 under stringent conditions, e) has a sequence derivable by degeneration of SEQ ID NOS.: 5-7, or f) a complementary strand of any of the sequences a)-e).

Description

field of invention [0001] The present invention relates to nucleic acid molecules containing at least one antigenic epitope of Pla l 1, the major allergen of Plantago lanceolata. Background of the invention [0002] In recent years, type I allergens affect tens of thousands of people worldwide, and its incidence has been on the rise in developed countries, leading to increased deaths and economic losses (1). Pollen allergens are proteins or glycoproteins that can cause IgE-mediated allergic diseases such as hay fever and asthma, and approximately 17% of the population is genetically predisposed to allergic diseases. [0003] The current methods of treating these diseases mainly focus on alleviating symptoms. Patients often take some antihistamines and steroids to relieve symptoms, but these drugs do not inhibit the formation of IgE antibodies and often cause some side effects. As stated in the WHO Position Paper (2), immunotherapy is the only treatment that affects the nat...

Claims

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Application Information

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IPC IPC(8): G01N33/53A61K38/00A61K39/00A61P37/08C07K14/415C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/29C12P21/02
CPCC07K14/415A61K38/00A61K39/00A61P37/08
Inventor B·C·弗赖尔A·D·佩拉莱斯D·B·赫尔南德兹G·S·杜兰F·P·克拉莱斯
Owner ALK ABELLO SA
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