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Construction method of yeast two-hybird bait vectors for screening ASICs interaction proteins

A yeast two-hybrid, protein-acting technology, applied in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve problems such as lack of understanding of protein interactions and regulatory mechanisms

Inactive Publication Date: 2017-11-24
湖南甲骨文生物医药有限公司
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Problems solved by technology

Studies have shown that ASICs play an important role in pain, nociception, sour taste and other physiological functions caused by inflammation. The research on its molecular structure and distribution characteristics has been relatively sufficient, but there is still a lack of understanding of the interaction and regulation mechanism between its proteins

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  • Construction method of yeast two-hybird bait vectors for screening ASICs interaction proteins
  • Construction method of yeast two-hybird bait vectors for screening ASICs interaction proteins
  • Construction method of yeast two-hybird bait vectors for screening ASICs interaction proteins

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Embodiment

[0039] Step 1: PCR reaction amplifies target gene fragments and product identification and recovery

[0040] 1. PCR reaction to amplify the target gene fragment

[0041]According to the gene sequences of ASIC1a and ASIC2a subunits queried in GenBank (ASIC1a extracellular loop sequence is 1086bp, and ASIC2a extracellular loop sequence is 1086bp), use Primer Premier5.0 software to analyze and design PCR primers. The primer sequences are shown in Table 1. Primer synthesis Completed by Nanjing Jinsite Biotechnology Co., Ltd. The PCR reaction system is shown in Table 2. The ASIC1a and ASIC2a subunit DNA templates were provided by the National Key Laboratory of Proteomics and Developmental Biology, Hunan Normal University, and the amplification conditions were: initial denaturation at 98°C for 5 minutes, Then the following reaction conditions were implemented: 98°C for 10s, 56°C for 15s, 72°C for 1.5min, 35 cycles, and finally 72°C for 5min.

[0042] Table 1 Primer sequence list ...

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Abstract

The invention discloses a construction method of yeast two-hybird bait vectors for screening ASICs interaction proteins, belonging to the technical field of biology. According to the construction method, subunit extracellular loop genes ASIC1a and ASIC2a in acid-sensitive ion channels (ASICs) are respectively cloned to a yeast two-hybird bait vector pGBKT7 by virtue of a gene engineering technique so as to successfully construct two yeast two-hybird bait vectors pGBKT7-ASIC1a and pGBKT7-ASIC2a, the expression and identification of the constructed vectors are realized, and the recombinant bait vectors are proved to be applicable to a yeast two-hybird system, so that an important experimental foundation is laid for screening the ASICs interaction proteins by virtue of the yeast two-hybird system and further researching and establishing an ASICs regulation and control machine, and a new direction is opened up for the treatment of inflammatory pain.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the construction and expression of yeast two-hybrid bait vectors pGBKT7-ASIC1a and pGBKT7-ASIC2a of ASIC1a and ASIC2a subunit extracellular ring genes in acid-sensitive ion channels (ASICs). Background technique [0002] Acid-sensing ion channels (ASICs) are a class of cation channels activated by extracellular acidification, and belong to the ENaC (Na+Channel) / DEG (degener-in) channel family. The common feature of ENaC / DEG members is that they are non-voltage-gated ion channels with high permeability to Na+. These channels are usually composed of several subunits, and different subunits have similar topological structures. Seven subunits of ASICs encoded by four genes have been cloned so far, namely ASIC1a, 1b, 1b2, 2a, 2b, 3 and 4. The subunit of ASICs is composed of more than 500 amino acids, and its structure includes two hydrophobic transmembrane regions, namely transmembrane 1 r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/66
CPCC12N15/66C12N15/81
Inventor 霍林巨罗玉娇张常昕夏艳东司品法张允雷刘一佑金伟雷兰婷
Owner 湖南甲骨文生物医药有限公司
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