Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Antituberculous recombinant bacillus calmette-guerin with mazG gene deleted

A technology for recombinant BCG and gene deletion, which is applied in the field of microorganisms and bioengineering, and can solve difficult problems

Active Publication Date: 2016-11-23
SHANGHAI PUBLIC HEALTH CLINICAL CENT
View PDF2 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

BCG vaccine is the most widely used vaccine in the world. Although it has defects, it is cheap and safe. It is estimated that it will be difficult to completely replace it in the future

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Antituberculous recombinant bacillus calmette-guerin with mazG gene deleted
  • Antituberculous recombinant bacillus calmette-guerin with mazG gene deleted
  • Antituberculous recombinant bacillus calmette-guerin with mazG gene deleted

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Construction and identification of mazG gene-deleted recombinant BCG (BCGΔmazG)

[0044] According to the mazG gene and its flanking sequences in Sequence 2, primer pairs for amplifying the upstream and downstream homology arms were respectively designed. The sequences are as follows, where the boldface shows the paired sequence with the genome, the underlined line is the added restriction site, and others For protected bases:

[0045] Upstream homology arm F1: CTCTC CTTAA

[0046] Upstream homology arm R1:CTCTC TCTAGA

[0047] Downstream homology arm F2: CTCTC AAGCTT

[0048] Downstream homology arm R2: CTCTC CTCGAG

[0049] The 5' ends of the upstream and downstream primers of the upstream homology arm were respectively introduced with Afl II / Xba I restriction sites, and the 5' ends of the upstream and downstream primers of the downstream homology arms were respectively introduced with Hind III / Xho I restriction sites. Then, using BCG Da...

Embodiment 2

[0050] Example 2. Preparation of mazG gene-deleted recombinant BCG (BCGΔmazG) and mouse immunization and infection

[0051] MazG gene-deleted recombinant Bacillus Calmette-Guerin (BCGΔmazG) and its wild-type BCG Danish strain were grown to log phase in Middlebrook 7H9+10% OADC medium, collected by centrifugation at 4,000×g, and resuspended in PBS containing 15% glycerol- T80 buffer solution (1×PBS, pH7.4, 0.05% Tween80), stored in -80°C refrigerator for later use.

[0052] SPF grade female BALB / c mice aged 6-8 weeks were purchased from Shanghai Slack Experimental Animal Co., Ltd., 10 mice / group, a total of 3 groups, subcutaneously inoculated (s.c.) 10 6 CFU / 0.1mL BCG or BCGΔmazG, the blank control group was inoculated with 0.1mL PBS-T80. Four mice were killed after 8 weeks of immunization, the spleen was removed under aseptic conditions, and lymphocytes were isolated for T cell immunoassay; the remaining mice were aerosol challenged with 100-200 CFU / lung of Mycobacterium tube...

Embodiment 3

[0053] Example 3. Preparation of mouse spleen lymphocytes and IFN-γ ELISPOT assay for T cell responses in immunized mice

[0054] After 8 weeks of immunization, the mice were sacrificed by cutting off the carotid artery, and the spleen was isolated aseptically, and the spleen of each group of mice was collected in a 200-mesh nylon mesh bag and ground together to prepare the spleen lymphocytes of each group, and the mouse ELISPOT kit was used to (eBioscience Inc.) Quantification of the number of antigen-specific IFN-γ-secreting T lymphocytes. First, the capture antibody (clone AN-18, eBioscience Inc.) with a final concentration of 4 μg / mL was used for antibody coating overnight, and the next day, 1640 complete culture medium containing 10% fetal bovine serum was added to block the non-specific binding antigen site . Then add isolated lymphocytes 2.5×10 5 / well, and added antigen stimulators to the final concentrations of rAg85A 2 μg / mL and PPD 10 μg / mL to continue culturing t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides antituberculous recombinant bacillus calmette-guerin (BCG delta mazG) with a mazG gene deleted. The mazG gene in a genome of the antituberculous recombinant bacillus calmette-guerin with the mazG gene deleted is replaced with a hygromycin resistant gene. The invention further provides a preparation method of the BCG delta mazG and application thereof in preparing a recombinant vaccine for preventing tuberculosis. After the BCG delta mazG is inoculated subcutaneously into a mouse, a T cell immune response is obviously improved, after the immunized mouse is infected with a mycobacterium tuberculosis standard strain H37Rv, the bacterial load of the infected animal lung and spleen is significantly decreased, the pathological degree is relieved, and after the immunized mouse is infected for five weeks, a T cell secondary immune response is significantly improved. It is found through bacterial in-vivo survival tests that after high-dose intravenous inoculation is conducted, the BCG delta mazG can survive on viscera of the spleen, the lung, the liver and the kidney of the mouse continuously for 20 w or above, and the amount of bacteria of a BCG delta mazG recombinant strain is obviously increased compared with a wild strain. It is proved through experiments that the BCG delta mazG recombinant strain has antituberculous immunogenicity, protective efficacy and in-vivo persistence activity which are significantly improved, and the antituberculous recombinant bacillus calmette-guerin (BCG delta mazG) with the mazG gene deleted is expected to become one of the novel candidate vaccines for preventing tuberculosis infection.

Description

technical field [0001] The invention belongs to the field of microorganisms and bioengineering. It specifically relates to a gene knockout recombinant vaccine, and more specifically relates to a mazG gene deletion recombinant BCG and its preparation method, as well as its use in a preventive vaccine against Mycobacterium tuberculosis infection. Background technique [0002] Tuberculosis is a major public infectious disease of worldwide concern. As early as 1993, the World Health Organization (WHO) declared a state of emergency for the tuberculosis epidemic because of the increasing number of tuberculosis cases worldwide. At present, about 1 / 3 of the world's population (1.86 billion) carries Mycobacterium tuberculosis, with about 8 million new cases and 2 million deaths from tuberculosis every year; there are about 6 million active tuberculosis patients in my country, and there are about 6 million active tuberculosis patients in China. 250,000 people died from tuberculosis. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/56A61K39/04A61P31/06C12R1/32
Inventor 范小勇吕亮东赵国屏马辉吴康
Owner SHANGHAI PUBLIC HEALTH CLINICAL CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com