Core structural domain of VirB protein, and coding genes and applications thereof

A protein and residue technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve problems such as bacterial metabolic process burden

Inactive Publication Date: 2015-07-22
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the invasion of intestinal epithelial cells by Shigella requires the synthesis of a large number of proteins, which imposes a great burden on the metabolic process of bacteria.

Method used

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  • Core structural domain of VirB protein, and coding genes and applications thereof
  • Core structural domain of VirB protein, and coding genes and applications thereof
  • Core structural domain of VirB protein, and coding genes and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, the preparation of VirB core fragment

[0039] 1. Construction of recombinant plasmids

[0040] Insert the double-stranded DNA molecule shown in the 64th to 429th nucleotides of the sequence 2 of the sequence listing from the 5' end between the Nde1 and Xhol restriction sites of the plasmid pET-28a(+), to obtain the recombinant plasmid pET28a-VirB core. In the recombinant plasmid pET28a-VirB core, the foreign gene and some nucleotides in the plasmid pET-28a (+) form the fusion gene shown in the sequence 2 of the sequence table, and express the fusion protein shown in the sequence 1 of the sequence table (sequence In 1, the 5th to 10th amino acid residues from the N-terminal form the His tag, and the 22nd to 143rd amino acid residues form the VirB core fragment)

[0041] 2. Preparation of VirB core fragments

[0042] 1. Introduce the recombinant plasmid pET28a-VirB core obtained in step 1 into competent cells of Escherichia coli Rosseta to obtain recombi...

Embodiment 2

[0053] Embodiment 2, the preparation of Selenium VirB core fragment

[0054] 1. Transform Escherichia coli B834 with the recombinant plasmid pET28a-VirB core obtained in step 1 of Example 1 to obtain recombinant bacteria.

[0055] 2. Inoculate the single clone of the recombinant bacteria obtained in step 1 into 3 ml of LB liquid medium containing 50 μg / μL kanamycin, and cultivate overnight at 37° C. and 250 rpm with shaking.

[0056] 3. Take the bacterial liquid obtained in step 2, inoculate it into 200 mL of LB liquid medium containing 50 μg / μL kanamycin, incubate at 37°C and 250 rpm for 12 hours, and collect the bacterial precipitate by centrifugation.

[0057] 4. Preparation of selenomethionine medium: (1) Weigh 86.4 grams of medium base from the reagent kit (Molecular dimensions company, product number MD12-501B), dissolve it in 4L of ultrapure water, and autoclave at 121°C for 15 minutes , stored at 4°C after cooling; (2) Weigh 20.4 grams of nutrient mix (Molecular dimen...

Embodiment 3

[0062] Example 3, Crystal Analysis of the Complex Formed by VirB core Fragment and DNA Molecule

[0063] 1. Assembly of the complex

[0064] 1. Synthesize single-stranded DNA molecule A and single-stranded molecule B respectively, dissolve each single-stranded DNA molecule in pH 8.5, 100mM Tris buffer and make the concentration 100μM, and then mix the two single-stranded DNA molecules Equal volumes of the solutions were mixed, incubated at 95°C for 10 minutes, and naturally cooled to 4°C to obtain a double-stranded DNA molecule solution (icsB solution).

[0065] Single-stranded DNA molecule A: 5'-AAA CTCGTTTCATCATGAAATCCCAC-3';

[0066] Single-stranded DNA molecule B: 3'-GAGCAAAGTAGTACTTTAGGGTG TTT-5'.

[0067] 2. Mix the VirB core fragment solution obtained in Example 1 and the double-stranded DNA molecule solution obtained in step 1 according to the equimolar ratio of protein and DNA, and incubate on ice for 3 hours.

[0068] 3. Synthesize single-stranded DNA molecule C a...

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Abstract

The invention discloses a core structural domain of VirB protein, and coding genes and applications thereof. The invention provides VirB core segments or fusion proteins containing the VirB core segments; the VirB core segments are described as (a) or (b). (a) a protein composed of amino acid residues from the 22nd site to the 143rd site in the sequence table sequence No.1; (b) proteins which are derived from the proteins of (a), wherein one or a plurality of amino acid residues are substituted and / or lost and / or added, and the proteins of (b) possess DNA binding activity. The invention also discloses the applications of the VirB core segments or the fusion proteins in preparation of products used for binding with DNA, and in preparation of products used for identifying DNA. The invention provides the core structural domain of VirB protein, and operating principles of VirB protein are revealed at the molecular level, so that accurate and all-around structural biology information is provided for the design of structure oriented drugs.

Description

technical field [0001] The present invention relates to the core domain of VirB protein and its coding gene and application. Background technique [0002] Shigella, also known as Shigella, is a highly contagious and serious Gram-negative facultative anaerobic bacilli. According to the statistics of WHO in 2009, there are about 100 million people in the world who are sick, and the death toll reaches 1 million. About 99% of these cases occur in developing countries. Large-scale intestinal diseases are more likely to occur in some places with poor sanitation and water sources. [0003] Shigella causes bacillary dysentery, which is highly contagious. Shigella flexneri can cause disease at inoculums of 10-100 bacteria. Bacillary dysentery is widely prevalent in my country and other developing countries, and is still one of the most important threats to public health worldwide. Bacillary dysentery causes hundreds of thousands of deaths every year, especially the greatest thre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/25C07K19/00C12N15/31C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21
CPCC07K14/25
Inventor 崔胜高小攀邹婷婷牟志霞秦博
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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