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Method of using pichia yeast expression system to produce German cockroach allergenic Blagarg protein

A German cockroach and allergen technology, applied in the field of genetic engineering, can solve the problems of high cost of insect expression system, limited application, unsuitable for large-scale fermentation production, etc., and achieve the effect of high specific IgE binding activity

Active Publication Date: 2014-06-04
何韶衡
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of the insect expression system is huge, and it is not suitable for large-scale fermentation production, which limits its further clinical application

Method used

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  • Method of using pichia yeast expression system to produce German cockroach allergenic Blagarg protein
  • Method of using pichia yeast expression system to produce German cockroach allergenic Blagarg protein
  • Method of using pichia yeast expression system to produce German cockroach allergenic Blagarg protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Extraction of total RNA of Blattella germanica.

[0023] Take artificially raised frozen German cockroaches (from Jiangsu Provincial Center for Disease Control), grind with liquid nitrogen, and add Trizol reagent at 100 mg / mL Trizol reagent. Total RNA was extracted from Blattella germanica.

Embodiment 2

[0024] Example 2: The gene of Blagarg, the allergen of the German cockroach, was amplified by PCR and cloned.

[0025] The extracted total RNA was reverse transcribed into cDNA. Primers 5'-ATGGTGGATGCCGCAGTTCT-3' and 5'-CAGGGAGCTCTCAATCTTGAT-3' were designed according to the gene sequence of the German cockroach allergen Blagarg registered in genbank, and used PCR method (94°C for 10 minutes, 94°C for 30 seconds, 50°C for 45 seconds , 72° C. for 1 minute, 35 cycles in total), the Blagarg gene was amplified, cloned into the pMD18-T vector, and transformed into Escherichia coli JM109 competent cells. Plate overnight. Positive bacteria were screened, plasmids were extracted and identified by PCR and sequencing. The allergen Blagarg gene of the German cockroach was obtained, the nucleotide sequence of which is shown in SEQ ID No:1.

Embodiment 3

[0026] Example 3: Construction of the Blagarg expression plasmid for the allergen Blagarg of the German cockroach.

[0027] The obtained Blagarg gene was cloned into plasmid pGAPaA through XhoI and NotI restriction sites, and transformed into competent JM109. Positive clones were screened out.

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Abstract

The invention discloses a method of using a pichia yeast expression system to produce German cockroach allergenic Blagarg protein, which includes: extracting total RNA (ribonucleic acid) from German cockroach tissues, reversely transcribing the total RNA into cDNA (complementary deoxyribonucleic acid), and obtaining coded genes of the German cockroach allergenic Blagarg by the PCR (polymerase chain reaction) method; cloning the coded genes to pGAPaA plasmids, and converting a yeast expression strain GS115 for expression; and purifying by affinity chromatography to obtain the German cockroach allergenic Blagarg protein. By the method using affinity chromatography, the problems in Blagarg allergen production are solved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for producing Blagarg protein, an allergen of the German cockroach, in a Pichia pastoris expression system. Background technique [0002] Allergic diseases are one of the major human diseases. Its incidence rate is currently estimated to account for 30-40% of the world's population, and is increasing at a rate of more than 1% per year, with the most obvious increase in the incidence rate of children. Over the past few decades, the incidence of allergic diseases has increased significantly and rapidly globally, more in Western than in developing countries, and more in urban than in rural areas. The current method of treating allergic diseases is mainly reflected in alleviating symptoms. Patients often take some antihistamines and steroids to relieve symptoms, but these drugs do not suppress the production of IgE antibodies and often cause side ef...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/81C12N9/16
Inventor 何韶衡魏继福
Owner 何韶衡
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