Method for producing blattella germanica allergen Bla g 7 protein in bacilliform virus-insect expression system

Abstract

The invention discloses a method for producing German cockroach allergen Bla g 7 protein in a rhabdovirus-insect expression system, comprising the following steps: total RNA is extracted from the tissue of the German cockroach, the total RNA is reversely transcripted into cDNA, a coding gene of German cockroach allergen Bla g 7 is obtained by the method of PCR; the coding gene is cloned to a rhabdovirus carrier, insect cells are transformed for expression; the method of affinity chromatography is adopted for purification, thus obtaining the German cockroach allergen Bla g 7 protein. The protein is different from Bla g 7 protein of foreign sources in terms of 2 amino acid locuses. In the invention, the rhabdovirus-insect expression system is adopted to produce German cockroach allergen Bla g 7 protein, thus avoiding influence on Bla g 7 allergen structures in different expression systems (such as bacterial and microzyme); in addition, the invention provides the method of affinity chromatography, thus solving the problem that the Bla g 7 allergen has complex purification process.

Description

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for producing Bla g 7 protein of the German cockroach allergen in a baculovirus-insect 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. Taking allergic asthma (asthma) as an example, there are currently 5.2 million asthmatic patients in the UK: 1.1 million children (1 / 10 of the total population of children) and 4.1 million adults (1 / 12 of the total population of adults), with an average of 1,...

Examples

Embodiment 1

[0020] Example 1: Extraction of total RNA of Blattella germanica.

[0021] 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

[0022] Example 2: The gene of the allergen Bla g 7 of the German cockroach was amplified by PCR and cloned.

[0023]The extracted total RNA was reverse transcribed into cDNA. Primers were designed according to the published Bla g 7 gene sequence of the German cockroach allergen: 5'-ATGGATGCCATCAAGAAGATG-3' and 5'-TTAGTTGCCAATAAGTTCGGT-3', using the PCR method (after 10 minutes of pre-denaturation at 94°C, 94°C for 30 seconds, 55°C for 45 seconds, 72°C for 1 minute, a total of 35 cycles), the Bla g 7 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 German cockroach allergen Bla g 7 gene was obtained, the nucleotide sequence of which is shown in SEQ ID No:1.

Embodiment 3

[0024] Example 3: Construction of the allergen Bla g 7 baculovirus vector pFastBacHTA of Blattella germanica.

[0025] The obtained Bla g 7 gene was cloned into the plasmid pFastBacHTA through EcoR I and Sal I restriction sites, and transformed into competent JM109. Positive clones were screened out.

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for producing Bla g 7 protein of the German cockroach allergen in a baculovirus-insect 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. Taking allergic asthma (asthma) as an example, there are currently 5.2 million asthmatic patients in the UK: 1.1 million children (1 / 10 of the total population of children) and 4.1 million adults (1 / 12 of the total population of adults), with an average of 1,...

Examples

Embodiment 1

[0020] Example 1: Extraction of total RNA of Blattella germanica.

[0021] 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

[0022] Example 2: The gene of the allergen Bla g 7 of the German cockroach was amplified by PCR and cloned.

[0023]The extracted total RNA was reverse transcribed into cDNA. Primers were designed according to the published Bla g 7 gene sequence of the German cockroach allergen: 5'-ATGGATGCCATCAAGAAGATG-3' and 5'-TTAGTTGCCAATAAGTTCGGT-3', using the PCR method (after 10 minutes of pre-denaturation at 94°C, 94°C for 30 seconds, 55°C for 45 seconds, 72°C for 1 minute, a total of 35 cycles), the Bla g 7 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 German cockroach allergen Bla g 7 gene was obtained, the nucleotide sequence of which is shown in SEQ ID No:1.

Embodiment 3

[0024] Example 3: Construction of the allergen Bla g 7 baculovirus vector pFastBacHTA of Blattella germanica.

[0025] The obtained Bla g 7 gene was cloned into the plasmid pFastBacHTA through EcoR I and Sal I restriction sites, and transformed into competent JM109. Positive clones were screened out.

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