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

A German cockroach and expression system 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: 2012-06-20
何韶衡
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 from Periplaneta germanium.

[0023] Frozen German cockroaches (from Jiangsu Provincial Center for Disease Control) were taken and ground in liquid nitrogen, and Trizol reagent was added according to 100 mg / mL Trizol reagent. The total RNA of German cockroach was extracted.

Embodiment 2

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

[0025] The extracted total RNA was reverse transcribed into cDNA. The primers 5'-ATGGTGGATGCCGCAGTTCT-3' and 5'-CAGGGAGCTCTCAATCTTGAT-3' were designed according to the gene sequence of the German cockroach allergen Blagarg registered in genbank. After pre-denaturation at 94°C for 10 minutes, 94°C for 30 seconds, and 50°C for 45 seconds , 72°C for 1 minute for a total of 35 cycles) to amplify the Blagarg gene, clone it into the pMD18-T vector, and transform it into E. coli JM109 competent cells. Plates were incubated overnight. The positive bacteria were screened, plasmids were extracted and identified by PCR and sequencing. The German cockroach allergen Blagarg gene was obtained, and its nucleotide sequence is shown in SEQ ID No: 1.

Embodiment 3

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

[0027] The obtained Blagarg gene was cloned into plasmid pGAPaA through XhoI and Not I restriction sites, and transformed into competent JM109. Positive clones were screened.

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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 of the German cockroach allergen in a Pichia pastoris expression system. Background technique [0002] Allergic disease is one of the major human diseases. Its incidence is currently estimated to account for about 30 to 40% of the world's population, and is increasing at a rate of more than 1% per year, especially in children. Over the past few decades, the incidence of allergic diseases has increased significantly and rapidly globally, more in Western countries than in developing countries, and more in cities than in rural areas. The current methods of treating allergic diseases are mainly reflected in the alleviation of symptoms. Patients often take some antihistamines and steroids to relieve symptoms, but these drugs do not suppress the production of IgE antibodies and often bring side effects. In recent years, ...

Claims

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Application Information

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