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Yeast two-hybrid method

A yeast two-hybrid and yeast technology, applied in the field of genetic engineering, can solve the problems of false negative screening results and wrong conformation of fusion proteins, and achieve the effects of increasing the positive clone rate, improving the interaction, and increasing the detection rate.

Active Publication Date: 2019-07-12
FOSHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, for special proteins such as extracellular proteins, membrane protein receptors, etc., they cannot enter the nucleus because they must undergo post-translational modification and folding assembly processes in organelles such as the endoplasmic reticulum, or the addition of fusion proteins will produce wrong conformations. False Negative Screening Results

Method used

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Embodiment 1

[0024] A yeast two-hybrid method, the specific steps comprising:

[0025] 1) The library containing three reading frames is preserved in the form of yeast Y187. Activate before use. Dilute 2ml of the AD-cDNA library from tissues or cells 10 times with SD / -LEU liquid medium, spread evenly to SD / -LEU solid medium (100 plates) and culture for 5 days, use a glass rod Scrape and mix all the yeasts and divide into centrifuge tubes to obtain activated yeast library, add glycerol and store at -80°C. The titer of the yeast library was measured by 10-fold serial dilution, and the volume of the original library bacterial solution required to amplify the library was calculated; two proteins interacted as: ZFP36 and OsLEA5, and the AD-ZFP36 fusion vector was transformed by electroporation into yeast Y187 and preserved in SD / -LEU;

[0026] 2) Transform the bait plasmid BD-OsLEA5 into yeast cell Y2H Gold by electroporation to obtain recombinant yeast expressing the bait protein BD-OsLEA5,...

Embodiment 2

[0029] The comparison group is a yeast two-hybrid method, the steps of which are basically the same as in Example 1, and the medium used in step (3) is the same as that of Example 1 except that it does not contain zinc ions.

[0030] The blue single bacterium colony obtained in embodiment 1 is compared with the blue single bacterium colony obtained by the contrast group and finds, as figure 1 , a is a comparison group, and b is Example 1. It can be seen that the colony spots on the b diagram are obviously more than that of the a diagram, which shows that the positive clone rate obtained by a yeast two-hybrid method described in the present disclosure is obviously higher Compared with the comparison group, it shows that the method of the present disclosure can improve the positive cloning rate, and further confirm the positive results at the end, such as figure 2 , a is the comparison group, and b is Example 1. It can be seen that the method of the present disclosure can reduc...

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Abstract

The invention provides a yeast two-hybrid method. The yeast two-hybrid method is characterized by comprising the steps that 1, library yeast is activated, and an AD-Y fusion vector is transformed intothe library yeast; 2, bait plasmids BD-X are transformed into yeast cells Y2H Gold; 3, recombined yeast of BD-X and recombined yeast containing the AD-Y are fused into a diploid, the diploid yeast iscultured on an amplification culture medium containing zinc ions to obtain a bacterial colony, and then positive clone screening is conducted. GAL4 protein in the yeast cells Y2H Gold used in the method is a dependent transcription factor of the zinc ions, therefore, the zinc ions are added into the amplification culture medium used in the method, interactions between low-abundance proteins are improved, the detection rate of low-interaction proteins is improved, and the positive clone rate is improved.

Description

technical field [0001] The disclosure relates to the field of genetic engineering, in particular to a yeast two-hybrid method. Background technique [0002] Yeast two-hybrid is a common method for protein function research. This method can quickly verify the interaction between known proteins and find new interacting proteins, especially when looking for protein interaction regions. Using yeast two-hybrid to obtain the pseudo-interacting protein of the target protein is the preferred method to find the target protein of the target protein and study its function, and is widely used in scientific research. There are many kinds of yeast two-hybrid methods currently used, such as the Y2H Gold Matchmaker GAL4 Two-Hybrid System series from Clontech (Takara), and the ProQuestTwo-Hybrid System from Invitrogen. The yeast two-hybrid system was proposed by Fields and Song as early as 1989 and 1994 respectively. According to the structural characteristics of eukaryotic cell transcripti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/04
CPCC12N15/81C12N15/04
Inventor 黄丽萍王华阳任银彩喻敏
Owner FOSHAN UNIVERSITY
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