MYCOBACTERIUM TUBERCULOSIS (Mtb) ADENOSINE TRIPHOSPHATE (ATP) SYNTHASE-EXPRESSING RECOMBINANT BACTERIUM, AND CONSTRUCTION METHOD AND EXPRESSION METHOD THEREOF

Patent metadata

Field	Value
Publication number	US-2024360459-A1
Application number	US-202418437991-A
Country	US
Kind code	A1
Filing date	Feb 9, 2024
Priority date	Apr 27, 2023
Publication date	Oct 31, 2024
Grant date	—

Abstract

The present disclosure provides a Mycobacterium tuberculosis (Mtb) adenosine triphosphate (ATP) synthase-expressing recombinant bacterium, and a construction method and an expression method thereof, and belongs to the technical field of exogenous expression. The present disclosure provides a construction method of an Mtb ATP synthase-expressing recombinant bacterium, including the following steps: using a Mycobacterium smegmatis (Msm) competent cell containing an auxiliary gene knockout plasmid as a basal cell; transferring an Mtb ATP synthase gene cluster with an affinity purification tag into the basal cell, knocking out a Msm ATP synthase genome, and collecting a strain without the auxiliary gene knockout plasmid after conducting repeated subculture; and transferring the Mtb ATP synthase gene cluster into the strain by prokaryotic expression to obtain the Mtb ATP synthase-expressing recombinant bacterium.

First claim

What is claimed is:

1 . A construction method of a Mycobacterium tuberculosis (Mtb) adenosine triphosphate (ATP) synthase-expressing recombinant bacterium, comprising the following steps:
(1) transferring an Mtb ATP synthase gene cluster with an affinity purification tag into a Mycobacterium smegmatis (Msm) competent cell to obtain a strain a; wherein the Msm competent cell carries an auxiliary gene knockout plasmid; (2) knocking out a Msm ATP synthase genome in the strain a to obtain a strain b; (3) subjecting the strain b to repeated subculture on a kanamycin resistance-free plate medium to obtain a strain c without the auxiliary gene knockout plasmid; and (4) transferring a prokaryotic expression vector with the Mtb ATP synthase gene cluster into the strain c to obtain the Mtb ATP synthase-expressing recombinant bacterium.

2 . The construction method according to claim 1 , wherein the Mtb ATP synthase gene cluster in step (1) is inserted into a sodC gene locus of the Msm competent cell using streptomycin as a selection marker.

3 . The construction method according to claim 1 , wherein the Mtb ATP synthase gene cluster in step (1) has a nucleotide sequence shown in SEQ ID NO: 1.

4 . The construction method according to claim 2 , wherein the Mtb ATP synthase gene cluster in step (1) has a nucleotide sequence shown in SEQ ID NO: 1.

5 . The construction method according to claim 1 , wherein the knocking out in step (2) is conducted using hygromycin as a selection marker.

6 . The construction method according to claim 1 , wherein the Msm ATP synthase genome in step (2) has a nucleotide sequence shown in SEQ ID NO: 2.

7 . The construction method according to claim 5 , wherein the Msm ATP synthase genome in step (2) has a nucleotide sequence shown in SEQ ID NO: 2.

8 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 1 .

9 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 2 .

10 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 3 .

11 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 4 .

12 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 5 .

13 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 6 .

14 . An Mtb ATP synthase-expressing recombinant bacterium constructed by the construction method according to claim 7 .

15 . A method for expressing an Mtb ATP synthase using the Mtb ATP synthase-expressing recombinant bacterium according to claim 8 , comprising: culturing the Mtb ATP synthase-expressing recombinant bacterium to allow induced expression of the Mtb ATP synthase with acetamide.

16 . A method for expressing an Mtb ATP synthase using the Mtb ATP synthase-expressing recombinant bacterium according to claim 9 , comprising: culturing the Mtb ATP synthase-expressing recombinant bacterium to allow induced expression of the Mtb ATP synthase with acetamide.

17 . A method for expressing an Mtb ATP synthase using the Mtb ATP synthase-expressing recombinant bacterium according to claim 10 , comprising: culturing the Mtb ATP synthase-expressing recombinant bacterium to allow induced expression of the Mtb ATP synthase with acetamide.

18 . The method according to claim 15 , wherein the culturing is conducted at 37° C. with shaking.

19 . The method according to claim 15 , wherein an obtained cultured bacterial solution is cooled to 16° C. to allow the induced expression with the acetamide.

20 . The method according to claim 15 , further comprising the following steps after the induced expression is conducted with the acetamide: collecting a bacterial cell to allow cell disruption, and purifying an obtained Msm cell membrane total protein solution.

Assignees

• Univ Nankai

Inventors

• Gong hongri
• Zhang Yuying
• Rao Zihe

Classifications

• C12N9/14

  Hydrolases (3) · CPC title

• C12R2001/34

  Mycobacterium smegmatis · CPC title

• C12R2001/32

  Mycobacterium · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C12N15/74 Primary

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title