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Corn expression vector of tubercle Ag85B gene and application thereof

An expression vector, pcrg-ag85b technology, applied in the field of biomedical engineering, can solve problems such as single immune response

Inactive Publication Date: 2010-09-29
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immune response stimulated by the vaccine is relatively simple, and an adjuvant must be added and co-vaccinated with BCG to induce a stronger immune response to protect the body

Method used

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  • Corn expression vector of tubercle Ag85B gene and application thereof
  • Corn expression vector of tubercle Ag85B gene and application thereof
  • Corn expression vector of tubercle Ag85B gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: The schematic diagram of the construction of the maize expression vector pCAMG-Ag85B comprising the tuberculosis Ag85B gene is as follows figure 1 shown

[0053] (1) Design of cloning gene primers:

[0054] Design a pair of primers based on the sequence of MTB-Ag85B:

[0055] The upstream primer sequence is: P1 5'-TTATCG GGATCC ATGGGAGGTTGGTCTTC-3', containing BamH I restriction site and initiation codon;

[0056] The downstream primer sequence is: P2 5'-CGCGCT GGATCCTCAAATGTATACCCAAAG-3', containing XbaI restriction site and stop codon.

[0057] (2) PCR reaction and cloning:

[0058] Extraction and purification of human Mycobacterium tuberculosis H37Rv genomic DNA: Inoculate MTB H37Rv in modified Roche medium, culture at 37°C with a rubber stopper on the mouth of the vessel, and observe once a week; Mycobacterium tuberculosis grows slowly, generally it takes 2-4 The perimeter grows into colonies visible to the naked eye; after 2-4 weeks of culture, scrap...

Embodiment 2

[0076] Extraction and screening of the recombinant expression vector pCAMG-Ag85B obtained in Example 1, transforming Agrobacterium LBA4404: Prepare Agrobacterium LBA4404 competent in advance, store in -80°C refrigerator for later use; take 200 μL of competent cells, place on ice for 30 minutes; add 1 μg of DNA to the competent In the cells, quick-freeze in liquid nitrogen for more than 5 minutes; quickly thaw in a water bath at 37°C for less than 5 minutes; add 800 μL LB medium; culture at a low speed at 28°C for 3 hours; take 200 μL and spread it on a plate containing 50 μg / mL kanamycin, at 28°C Cultivate on a shaking table for 2 days to 3 days; after colony PCR verification, select positive clones and shake the bacteria, extract the plasmid and use HindIII and Xba I double enzyme digestion for double enzyme digestion identification, and finally confirm that the recombinant plasmid has been transferred into LBA4404, and store at -80 °C strain.

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Abstract

The invention discloses a corn expression vector of tubercle Ag85B gene and application thereof. A construction method for the vector comprises the following steps of: (1) designing a pair of primers P1 and P2 according to a coding sequence of the tubercle Ag85B gene; (2) performing conventional chain polymerase reaction to amplify the tubercle Ag85B gene by using the primers P1 and P2; (3) constructing a pCRG-Ag85B vector; and (4) constructing a recombinant pCRG-Ag85B vector, and converting agrobacterium rhizogenes. The method selects a corn seed specificity promoter Globulin-1 with driving exogenous gene and with double advantages on tissue or organ expression specificity and high level expression to construct the tubercle Ag85B gene which is controlled by the corn seed specificity promoter Globulin-1 and has tubercle advantage so that the tubercle Ag85B gene is specifically and efficiently expressed in corn seeds. The corn expression vector of the tubercle Ag85B gene can be used for preparing vaccines.

Description

technical field [0001] The invention belongs to the field of biomedical engineering, in particular to a maize expression vector containing tuberculosis Ag85B gene and application thereof. Background technique [0002] Tuberculosis (TB) is one of the chronic infectious diseases with the highest morbidity and mortality among adults, caused by Mycobacteria tuberculosis (MTB). Mycobacterium tuberculosis mainly causes various organ diseases in the human body through respiratory tract infection, such as pulmonary tuberculosis and intestinal tuberculosis. Since 2006, there have been approximately 9 million new cases worldwide each year, of which an estimated 3 million have died from TB. WHO estimates that between 2010 and 2020, about 1 billion people will be infected and 35 million people will die from tuberculosis. The United Nations has listed TB, malaria, and AIDS as the three major diseases to be controlled in the 21st century. [0003] my country is one of the countries wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/31A61K39/04A61P31/06
Inventor 李君武
Owner JINAN UNIVERSITY