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Fusion gene of Hepatitis B virus L gene and tubercle Esat6 and construction and application of expression vector thereof

An expression vector, fusion gene technology, applied in the field of biomedical engineering, can solve problems such as disseminated infection

Inactive Publication Date: 2012-11-21
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Bacillus Calmette-Guerin (BCG), as a vaccine to prevent tuberculosis, has been widely used in the vaccination of newborns in the past 100 years, but the protection rate is only about 50%, while the protection rate for adults fluctuates between 0-80% in different regions : In addition, inoculation of BCG can also cause serious complications in a small number of people, disseminated infection and death

Method used

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  • Fusion gene of Hepatitis B virus L gene and tubercle Esat6 and construction and application of expression vector thereof
  • Fusion gene of Hepatitis B virus L gene and tubercle Esat6 and construction and application of expression vector thereof
  • Fusion gene of Hepatitis B virus L gene and tubercle Esat6 and construction and application of expression vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1: The schematic diagram of the construction of the maize expression vector pCAMG-L-Esat6 comprising the fusion gene of the hepatitis B virus L gene and the tuberculosis Esat6 gene is as follows Figure 4 shown

[0091] (1) Primer design:

[0092] According to the coding sequence of the hepatitis B virus L gene, design a pair of primers for amplifying the hepatitis B virus L gene:

[0093] The upstream primer sequence is: P1 5'-ATTATT GGATCC ATGGGAGGTTGGTCTTCC-3', containing Kpn I restriction site and initiation codon ATG;

[0094] The downstream primer sequence is: P2 5'-CACTGCTGCTCTGTCATAATGTATACCCAAAAGAC-3', the sequence is reverse complementary to P3;

[0095] According to the coding sequence of the Esat6 gene of the human Mycobacterium tuberculosis H37Rv strain, design a pair of primers for amplifying the Esat6 gene of Mycobacterium tuberculosis:

[0096] The upstream primer sequence is: P3 5'-GTCTTTGGGTATACATTATGACAGAGCAGCAGTG-3', the sequence is revers...

Embodiment 2

[0161] The recombinant vector pCAMG-L-Esat6 obtained in Example 1 was extracted and screened, and transformed into Agrobacterium LBA4404. Competent Agrobacterium LBA4404 was prepared in advance and stored in a -80°C refrigerator for later use. Take 200 μL of competent cells and place them on ice for 30 minutes; add 1 μg of DNA to the competent cells, and freeze them in liquid nitrogen for more than 5 minutes; quickly thaw them in a water bath at 37°C for less than 5 minutes; add 800 μL of LB medium; 200 μL was spread on a plate containing 50 μg / mL kanamycin, and cultured on a shaker at 28°C for 2d to 3d. After the colony PCR verification, the positive clones were selected and shaken, and the plasmid was extracted and identified by double enzyme digestion. It was finally confirmed that the recombinant plasmid was transferred into LBA4404, and the strain was stored at -80°C.

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Abstract

The invention discloses fusion gene of Hepatitis B virus L gene and tubercle Esat6, a corn expression vector thereof and application thereof. The construction method comprises the following steps of: designing primers; amplifying the Hepatitis B virus L gene; amplifying the tubercle bacillus Esat6 gene; amplifying the L-Esat6 fusion gene; constructing a pEG-G vector; constructing an intermediate vector pEGG-LE; and constructing a recombinant vector pCAMG-L-Esat6, and converting agribacterium tumefaciens. In the method, a specific promoter Globulin-1 of the corn seeds, which has double advantages of driving an exogenous gene to express specificity in tissues or organs and realizing high-level expression, is selected, and the advantageous fusion gene of the Hepatitis B virus L gene and tubercle Esat6, which is controlled by the specific promoter Globulin-1 of the corn seeds, is constructed, so that the fusion gene is specifically expressed in the corn seeds efficiently. The corn expression vector of the fusion gene can be applied in the preparation of vaccines.

Description

technical field [0001] The invention belongs to the field of biomedical engineering, and particularly relates to a fusion gene of hepatitis B virus L gene and tuberculosis Esat6, a maize expression vector and application thereof. Background technique [0002] Hepatitis B virus ((Hepatitis B virus HBV) infection is a very serious global health problem. At present, 2 billion people in the world's 6 billion people have been infected with hepatitis B virus, accounting for one-third of the global population. 25% of the people will eventually transform into chronic liver disease, including liver cirrhosis and liver cancer. The main means of controlling hepatitis B transmission is to inoculate effective vaccines to prevent and block the transmission route. The current hepatitis B vaccine is mainly composed of Chinese hamster ovary cells (CHO Cells) and Hepatitis B virus surface antigen HBsAg (S protein) particles expressed by brewer's yeast. In the process of vaccination, it was fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/82A61K39/295A61P31/06A61P1/16A61P31/20A61K39/29A61K39/04
Inventor 李君武
Owner JINAN UNIVERSITY