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Corn expression carrier of tuberculosis Hsp65 antigen gene and application thereof

A technology of antigen gene and expression vector, applied in the field of biomedical engineering, can solve the problems of wasting nutrients, affecting the normal development of plants, and changing the shape of plants

Inactive Publication Date: 2009-12-30
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for genes that express special nutrients, such as the expression of hepatitis B antigen genes, the continuous and efficient expression of foreign genes in plants will not only cause waste of nutrients, but also often cause changes in plant morphology, and in severe cases, it will also affect plants. normal development of

Method used

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  • Corn expression carrier of tuberculosis Hsp65 antigen gene and application thereof
  • Corn expression carrier of tuberculosis Hsp65 antigen gene and application thereof
  • Corn expression carrier of tuberculosis Hsp65 antigen gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0062] Embodiment 1: Cloning of Hsp65 gene and construction of plasmid vector

[0063] (1) Design of cloning gene primers:

[0064] A pair of primers for the antigen gene Hsp65 were designed according to the coding sequence of the Hsp65 gene of the human Mycobacterium tuberculosis H37Rv strain:

[0065] The sequence of the upstream primer is: P15'TT AGATCT GCAATGGCCAAGACAATTGCGT-3', containing the BglII restriction site and the initiation codon ATG;

[0066] The downstream primer sequence is: P25'-CC TCTAGA TCAGAAATCCATGCCACCCATG-3', containing Xba I restriction site and stop codon CTA;

[0067] (2) PCR reaction and cloning:

[0068] Using the plasmid pEGFPHsp65-Esat6 as a template, use the primers P1 and P2 designed in step (1) to perform a conventional chain polymerase reaction to amplify Hsp65; the specific steps of the conventional chain polymerase reaction are as follows: pre-denature the mixture at 95°C 5min, then enter the following cycle: 95°C, 50s, 59°C, 50s, 72°C,...

Embodiment 2

[0089] The recombinant expression vector pCAMBIA1300GHsp65 obtained in Example 1 was extracted and screened, and transformed into Agrobacterium LBA4404 by electric shock method, and the method was referred to Lu Shengdong "Modern Molecular Biology Experimental Technology". The extracted plasmid was double digested with HindIII and XbaI, identified, and finally confirmed that the recombinant plasmid pCAMBIA1300G Hsp65 was transferred into LBA4404.

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Abstract

The invention discloses a corn expression carrier containing a tuberculosis Hsp65 antigen gene and an application thereof. The construction method of the expression carrier comprises the following steps: (1) fusing a pair of primers P1 and P2 of the antigen gene Hsp65 according to a coded sequence of a human tuberculosis mycobacterium H37Rv plant Hsp65 gene; (2) using a plasmid pEGFPHsp65 as a template and using the primers P1 and P2 to increase the Hsp65 by a general chain polymerase reaction; (3) constructing a pCR-G carrier; (4) constructing a pCAMBIA1300-nos-bar carrier; (5) cloning and constructing an intermediate carrier pCRGHsp65; and (6) cloning and constructing a destination carrier pCAMBIA1300GHsp65. The expression carrier of the tuberculosis Hsp65 antigen gene can be used for preparing vaccines.

Description

technical field [0001] The invention relates to a maize expression vector containing tuberculosis Hsp65 antigen gene and application thereof, belonging to the field of biomedical engineering. Background technique [0002] Tuberculosis is an infectious disease that seriously endangers human health and is caused by Mycobacterium tuberculosis infection. About one-third of the people in the world have been infected with tuberculosis (about 2.1 billion), and 10 million people are newly diagnosed every year. As a vaccine to prevent tuberculosis, BCG (BCG) has been widely used in the vaccination of newborns in the past 100 years. The protection rate for adults is only about 50%, while the protection rate for adults fluctuates between 0 and 80% in different regions; in addition, BCG vaccination can also cause serious complications in a small number of people, disseminated infection and death. In view of the serious situation of tuberculosis, strengthening the prevention and control...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/31A61K39/04A61P31/06
Inventor 李君武胡建广
Owner JINAN UNIVERSITY