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Recombinant expression vector for improving soluble expression of pathogenesis-related proteins in Mongolia astragaloside

An expression vector, the technology of Astragalus mongolica, applied in the field of genetic engineering, can solve the problems of high cost, low protein yield, inconvenient research, etc., and achieve the effect of saving cost and increasing production

Pending Publication Date: 2020-10-02
SHANXI UNIV OF CHINESE MEDICINE
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Problems solved by technology

Therefore, it is of great significance to study the performance of AmPR-10 in vitro to further explore the growth and development of Astragalus membranaceus and the mechanism of disease resistance. However, the traditional method of extracting protein from natural Astragalus membranaceus is costly and the protein yield is low, which makes subsequent research inconvenient.

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  • Recombinant expression vector for improving soluble expression of pathogenesis-related proteins in Mongolia astragaloside
  • Recombinant expression vector for improving soluble expression of pathogenesis-related proteins in Mongolia astragaloside
  • Recombinant expression vector for improving soluble expression of pathogenesis-related proteins in Mongolia astragaloside

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Embodiment 1

[0029] Example 1 Recombinant construction.

[0030] (1) Total RNA extraction of Astragalus mongolica: Weigh 0.2 g leaves of Astragalus mongolica, grind on ice until homogenized, and transfer to a centrifuge tube. Add 1ml Trizol reagent and place at room temperature for 5min. Add chloroform and isopropanol to extract by centrifugation, dry at room temperature and add buffer to dissolve RNA.

[0031] (2) Target gene cloning: using the total RNA of Astragalus mongolica as a template, the PCR product of AmPR-10 was obtained by RT-PCR kit, which was composed of 158 amino acids, and the protein size was 16.8kD; the genome of Escherichia coli K12 was extracted, and its As a template, the PCR product of Escherichia coli molecular chaperone skp was obtained, which consisted of 161 amino acids and had a protein size of 17 kD. The target gene was cloned using Escherichia coli expression vectors pET-28a and pET-30a, and three recombinants were constructed. The corresponding primers wer...

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Abstract

The invention discloses a recombinant expression vector for improving soluble expression of pathogenesis-related proteins in Mongolia astragaloside. The recombinant expression vector is a recombinantpET30a-skp-AmPR-10 constructed by using pET30a as a vector and modifying AmPR-10 by utilizing an escherichia coli molecular chaperone skp. According to the invention, by a method of replacing the vector and carrying out molecular chaperone modification, the soluble expression of target proteins is improved so as to fulfill the aims of saving cost and improving yield; the exogenously expressed AmPR-10 is further subjected to activity measurement so as to show that the exogenously expressed proteins also have the nuclease activity and provide reference strategies for in-vitro expression of otherhomologous related proteins; and the activity may have the important significance for researching the RNA virus resistance ability of radix astragali.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant expression vector for increasing the soluble expression level of a disease course-related protein of Astragalus mongolica. Background technique [0002] Plant pathogenesis-related proteins (PR) are produced when plants are attacked by pathogens and subjected to external pressure, and they are of great significance to the plant's own disease resistance and immunity. It is mainly divided into 14 families, including PR-1 to PR-14. Studies have shown that the disease process-related protein PR-10 is not only involved in the plant's disease resistance mechanism, but also plays a role in the plant's own development (Walter M H, Liu J W, Wunn J, et al.Beanribonuclease-like th nes related protein genes (YPR-10) display complex patterns of developmental, dark-induced and exogenous stimulus-dependent expion [J]. Eur J Biochem, 1996, 239: 281-293). In recent ye...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/29C12N15/67C07K14/415C07K1/22
CPCC12N15/70C12N15/67C07K14/415
Inventor 王珏
Owner SHANXI UNIV OF CHINESE MEDICINE
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