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

A gene and encoding technology, applied to the core domain of VirB protein and its encoding gene and application field, can solve the problem of bacterial metabolic process burden and so on

Inactive Publication Date: 2014-01-01
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] Example 1. Preparation of VirB core fragments

[0039] 1. Construction of recombinant plasmid

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

[0041] 2. Preparation of VirB core fragments

[0042] 1. The recombinant plasmid pET28a-VirB core obtained in step 1 is introduced into competent cells of Escherichia coli Rosseta to obtain recombinant bacteria.

[0043] 2. In...

Embodiment 2

[0053] Example 2. Preparation of VirB core fragments

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

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

[0056] 3. Take the bacterial solution obtained in step 2 and 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 pellet by centrifugation.

[0057] 4. Preparation of selenomethionine medium: (1) Weigh 86.4 grams of medium base from the reagent kit (Molecular Dimensions Company, Catalog No. MD12-501B), dissolve it in 4L ultrapure water, and autoclave at 121°C for 15 minutes , Keep it at 4℃ after cooling; (2) Weigh 20.4g of nutrient mix (Molecular Dimensions Company, Product No. MD12-502) f...

Embodiment 3

[0062] Example 3. Crystal analysis of the complex formed by VirB core fragments and DNA molecules

[0063] 1. The assembly of the complex

[0064] 1. Synthesize single-stranded DNA molecule A and single-stranded molecule B separately. Dissolve each single-stranded DNA molecule in a pH 8.5, 100 mM Tris buffer and make the concentration 100 μM, and then combine the two single-stranded DNA molecules The solutions were mixed in equal volumes, 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 an equimolar ratio of protein and DNA, and incubate on ice for 3 hours.

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

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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 structure domain of VirB protein and its coding gene and application. Background technique [0002] Shigella, also known as Shigella, is a kind of gram-negative facultative anaerobic bacilli that is highly infectious and seriously harmful. According to WHO's statistics in 2009, there are about 100 million people in the world who are sick, and the death toll has reached 1 million. Approximately 99% of the cases occurred in developing countries. In some places with poor sanitation and water sources, large-scale intestinal diseases are more likely to occur. [0003] Shigella can cause extremely infectious bacillary dysentery. Shigella flexneri can cause disease at 10-100 bacterial inoculums. Bacterial dysentery is widespread in my country and other developing countries, and remains one of the primary threats to public health worldwide. Bacterial dysentery causes hundreds of thousands of deaths every year, and it is ...

Claims

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Application Information

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Patent Type & Authority Applications(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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