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Recombinant bacillus calmette guerin vaccine, and applications thereof

A recombinant BCG vaccine and BCG vaccine technology, applied in the application, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problems of uneven results, the safety of BCG immunity, and the limited effect of adult pulmonary tuberculosis protection, etc. Strong protective effect, improve anti-infection protection effect, improve vaccine safety effect

Active Publication Date: 2018-12-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been used for nearly 100 years, BCG has two main defects: First, although BCG has a certain protective effect on miliary tuberculosis and tuberculous meningitis in children, its protective effect on adult tuberculosis is very limited. The results are mixed (0-80%); the second is the safety of BCG in immunocompromised populations
[0008] After searching, there is no report or patent application for recombining the RD4-related region of Mycobacterium tuberculosis into BCG to construct a new recombinant BCG that expresses or overexpresses related genes

Method used

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  • Recombinant bacillus calmette guerin vaccine, and applications thereof
  • Recombinant bacillus calmette guerin vaccine, and applications thereof
  • Recombinant bacillus calmette guerin vaccine, and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Construction and identification of recombinant plasmids pMV306-Rv1501-Rv1508c and pMV306-Rv1501-Rv1516c

[0078] The integrative vector pMV306 was used for molecular cloning. Molecular biology techniques are routinely performed:

[0079] (1) First construct pMV306-Rv1501-Rv1502

[0080] The Rv1501-Rv1502 gene was amplified from the Mycobacterium tuberculosis H37Rv genome by PCR technology, (upstream primer 5'-CACTGGTCGACAATGTCACTTCATTTAGCAAC-3'(SEQ ID NO.3); downstream primer 5'-CATGAAAGCTTCGAATCATTGGAACAGCGG-3'(SEQ ID NO. 4)), the amplification conditions are: 98°C for 5min, [98°C for 10s, (Tm-5)°C for 10s, 72°C for 1min / kbp] 30 cycles, and 72°C for 10min. PCR products were recovered using the AxyPrep PCR Product Recovery Kit (Axygen). The gene fragment and the pMV306 plasmid were digested with SalI and HindIII, and the digested fragment was recovered with the AxyPrep DNA Gel Extraction Kit and ligated to form the recombinant plasmid pMV306-Rv1501-Rv1502. ...

Embodiment 2

[0086] Example 2: Establishment and verification of recombinant BCG rBCG::RD4

[0087] (1) Preparation of BCG Competent Cells:

[0088] Get 1ml of BCG-China and BCG-Japan strains in logarithmic growth phase, aseptically inoculate in 50ml Middlebrook 7H9 liquid medium (Difco™) that has added 10% ADC (DIFCO, Bection-Dickinson), 0.2% glycerol and 0.05% Tween80 In 37 ° C static culture to OD600 = 0.8-1.0. 4 ° C centrifugation to collect bacteria. After resuspending with 10% glycerol, wash 3 times with 1 / 2, 1 / 10 and 1 / 50 of the original culture volume of glycerol, and finally resuspend with 1ml pre-cooled glycerol, aliquot into 100μL tubes, and store at -80°C spare.

[0089] (2) Electrotransformation of BCG:

[0090] Add 5 μL of high-concentration plasmids (such as pMV306, pMV306-Rv1501-Rv1508c, pMV306-Rv1501-Rv1516c) and 200 μL of BCG competent cells to the labeled 0.2 cm Bio-Rad electroporation cuvette, gently blow and mix, and use Bio -rad GenePulser electroporator electropo...

Embodiment 3

[0112] Example 3: Safety Evaluation of Recombinant BCG rBCG::RD4

[0113] Six-week-old female SCID mice were randomly divided into 20 groups. Each mouse was injected with 100 μL (1×107 CFU) of recombinant BCG rBCG::RD4 and rBCG::306 through the tail vein, and PBS was used as a negative control. On the second day after infection, 2 mice in each group were sacrificed, the lungs and spleens were aseptically isolated and the bacterial load was counted to determine the infection dose. The remaining mice were observed for a long time, their body weight changes and death conditions were recorded, and the survival curve of the mice was drawn.

[0114] Statistics found that the half-life time of wild-type BCG (i.e. BCG::pMV306) group, BCG::Rv1501-1508c group and BCG::Rv1501-1516c group were 63, 77 and 67.5 days respectively (attached image 3). Log-rank statistical analysis showed that the survival period of the mice in the BCG::Rv1501-1508c group was significantly longer than that ...

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Abstract

The invention belongs to the technical field of gene engineering and tuberculosis vaccine, and provides a recombinant bacillus calmette guerin (BCG) vaccine containing mycobacterium tuberculosis genome RD4 zone related coding genes. According a preparation method, the recombinant bacillus calmette guerin vaccine is produced through transformation of recombinant Escherichia coli-mycobacterium shuttle plasmids containing genes used for coding of mycobacterium tuberculosis genome RD4 zone proteins into bacillus calmette guerin vaccine. The recombinant bacillus calmette guerin vaccine is capable of realizing RD4 zone gene and protein expression. After immunization of animals with the recombinant bacillus calmette guerin vaccine, the safety of recombinant BCG bacterial strain containing complete RD4 zone is not reduced, the safety of recombinant BCG bacterial strain containing a part of RD4 zone (Rv1501-Rv1508c) is increased obviously. The recombinant BCG bacteria strain containing the complete or a part of RD4 zone genes possess better anti-infection protection effects. The recombinant bacillus calmette guerin vaccine can be used in prevention or treatment of tuberculosis.

Description

technical field [0001] The invention belongs to the fields of genetic engineering vaccines and novel tuberculosis vaccines. Specifically, the present invention provides a novel recombinant BCG against pathogenic mycobacteria and its application in preventing and / or treating infections caused by pathogenic mycobacteria. Background technique [0002] Tuberculosis is a respiratory disease caused by Mycobacterium tuberculosis (Mtb), and it is the infectious disease with the highest mortality rate caused by a single cause worldwide. According to WHO statistics, one-third of the world (about 2 billion people) is infected with Mycobacterium tuberculosis, which represents a huge hidden danger of active tuberculosis. In 2015, 1.4 million people died of tuberculosis worldwide, and 10.4 million people were newly infected with tuberculosis (nearly 63% of whom had symptoms of tuberculosis). China is one of the 22 high-risk countries and regions for tuberculosis in the world. The number...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/74C12N1/21A61K39/04A61P31/06A61P31/04C07K14/35C12R1/32
CPCA61K39/04A61K2039/523C07K14/35C12N15/74
Inventor 刘军张鹭
Owner FUDAN UNIV
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