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Method for producing and recombining main allergic protein Hum j 3 of Humulus scandens by induced secretion expression

A technology of secretory expression and inducible expression, applied in the fields of genetic engineering drugs and medical diagnostic preparations, can solve the problem of not obtaining full sequence identification, and achieve the effect of simple separation and purification steps

Active Publication Date: 2011-03-30
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

The sequence is very close to the 10kDa main allergenic protein discovered by Korean Park JW in 1999 in terms of molecular weight, isoelectric point, serum sIgE binding rate of allergic patients, protein N-terminal sequence, etc., but the full sequence identification has not yet been obtained

Method used

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  • Method for producing and recombining main allergic protein Hum j 3 of Humulus scandens by induced secretion expression
  • Method for producing and recombining main allergic protein Hum j 3 of Humulus scandens by induced secretion expression
  • Method for producing and recombining main allergic protein Hum j 3 of Humulus scandens by induced secretion expression

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Embodiment 1

[0035] 1. Extraction of total RNA from Humulus pollen and synthesis of cDNA

[0036] (1). Extraction of total RNA from Humulus japonicus pollen. In September 2007, fresh humulus pollen was collected in the suburbs of Beijing, dried in the sun on a sunny day, filtered through a No. 9 sampling sieve, degreased with acetone, air-dried, immediately put into bottles, and stored at -80°C. use Plant Mini kits (QIAGEN TM , Catalog No.74903) kit to extract total RNA from pollen of Humulus japonicus, and extract according to the operating procedure of the kit. When extracting, put about 75mg of pollen into a liquid nitrogen tank and grind repeatedly, and use the Buffer RLT in the kit to destroy the pollen tissue. The final amount of total RNA obtained is about 20-60 μg / 75mg pollen, A260 / A80=1.81, and formaldehyde agarose gel electrophoresis (1.2% agarose) shows that rRNA has clear bands such as 16S, 18S, 23S, 25S, etc., showing that the total The RNA quality was intact, and the fin...

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Abstract

The invention provides a method for recombining main allergic protein Hum j 3 of Humulus scandens by utilizing induced expression in a yeast system, comprising the following steps of: constructing a recombined expression vector for expressing an amino acid sequence shown in the SEQ ID NO.2; linearizing the recombined expression vector and transforming competence yeast cells; screening positive clone to obtain a recombined bacterial strain and expanding cultivation; recombining the induced expression of the bacterial strain and screening; carrying out secretion expression induced by alpha-factor signal peptide protein in yeast on Hum j 3 protein; carrying out induced expression recombination of the Hum j 3 protein in high density by using high-activity recombined bacterial strain as a seed bacterium; and collecting cultivated supernatants and separating and purifying the Hum j 3. The invention internationally confirms the full-gene sequence of the main allergic protein of the Humulus scandens for the first time, successfully carries out yeast secretion expression for the first time and affirms that the combined protein has immunological competence, thereby providing brand-new protein for molecular diagnosis and clinical immunization treatment on patients with Humulus scandens pouen allergy.

Description

technical field [0001] The invention relates to a method for inducing, secreting and expressing the main allergenic protein of Humulus pollen in a yeast system at high density, in particular to a method for inducing, secreting and expressing the main allergenic protein of Humulus pollen in a Pichia pastoris (KM71H) system with high density The method of Hum j 3 and the method of engineering bacteria. The invention belongs to the field of genetic engineering drugs and medical diagnostic preparations, and the protein may play an important role in clinical molecular diagnosis of humulus pollen allergic disease, allergen immunotherapy, industrial production and quality control. Background technique [0002] Humulus japonicus or Humulus scandens is an annual twining herb of the Rosaceae Cannabis family. It is native to my country and widely distributed in the Far East. It is distributed in all provinces and regions except Xinjiang and Qinghai in my country. East Asian countries (...

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Application Information

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IPC IPC(8): C12N15/81C12P21/02C12N1/19G01N33/68A61K39/00A61P37/08C12R1/84
Inventor 尹佳周俊雄程璇
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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