Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers

A technology without resistance marker and expression cassette

Inactive Publication Date: 2013-12-18
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The total length of the three fragments has a certain limit. If it is too long, it will not be packaged into phage, and the phagemid for gene knockout will not be obtained.
However, using dif In the study of sequence system, the used Hyg The resistance gene has a long fragment (1.7kb), with a complex secondary structure at the 3' end, and there are common enzyme cutting sites inside, and there are few available enzyme cutting sites at both ends, and the resistance expression cassette constructed cannot " Universal"

Method used

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  • Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers
  • Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers
  • Resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of plasmid pUCDHmKE

[0036] The schematic diagram of the structure of plasmid pUCDHmKE is shown in figure 1 , see restriction site map figure 2 , which contains the above dif- ΩHYG -dif Resistance expression cassette. Depend on figure 1 It can be seen that the plasmid pUCDHmKE contains in a clockwise direction: Escherichia coli origin of replication (pMB1 ori), dif- ΩHYG -dif Resistance expression cassette, promoter P (BLA) (for promoting the ampicillin resistance gene Amp ( which is bla) , ampicillin resistance gene Amps Can be used for plasmid screening.

[0037] said dif- ΩHYG -dif The resistance expression cassette contains a simplified modified hygromycin resistance gene ( Hyg ), as described in SEQ ID NO:1, the 24th to 73bp on the gene is the promoter sequence (Pr), which is used to start Hyg gene expression. Hyg DNA sequences joined at both ends dif The sequences are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0...

Embodiment 2

[0049] Example 2 Construction and Application of Integrated Plasmid pMH94DHmKE

[0050] The schematic diagram of the structure of the integrated plasmid pMH94DHmKE is shown in Figure 4 , the plasmid pMH94DHmKE contains in a clockwise direction: LacZ promoter (can be used for blue and white screening, and to promote the expression of the entire plasmid backbone behind), phage integration site attP , integrase gene Int (can express integrase, allowing plasmids to pass through attP sites integrated into the mycobacterial genome), dif- ΩHYG -dif Resistance expression cassette, E. coli origin of replication ( oriE ), the ampicillin resistance gene Amps (for plasmid screening). dif- ΩHYG -dif The two ends of the resistance expression cassette were connected sequentially Hin dIII restriction site, xho I restriction site, can be used to cut out the dif- ΩHYG -dif Resistance expression cassette.

[0051] For the construction procedure of the integrated plasm...

Embodiment 3

[0058] Example 3 Construction and application of integrated plasmid pblDHCiGn

[0059] In the integrated plasmid pblDHCiGn, the resistance expression cassette exists in the form of double restriction sites on both sides. The construction of this plasmid is to prove that the resistance expression cassette is still effective in this form. Plasmid structure see Image 6 , in a clockwise direction, which in turn contains: promoter P(BLA) (used to start the following Amps expression), ampicillin resistance gene Amps (can be used for plasmid screening), E. coli origin of replication ( OriE ), phage integration site ( attP ), integrase gene Int (can express integrase, allowing plasmids to pass through attP integrated into the mycobacterial genome), dif- ΩHYG -dif Resistance expression cassette, enhanced green fluorescent protein gene eGFP . dif- ΩHYG -dif The two ends of the resistance expression cassette are connected with different enzyme cutting sites.

[0...

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Abstract

The invention discloses a resistance expression cassette for efficiently constructing recombinant mycobacterium without resistance markers. The resistance expression cassette contains a simplified hygromycin resistance gene (Hyg) (as shown in SEQ ID NO: 1) and dif1 and dif2 sequences located at two ends of the Hyg. The sequence of the simplified Hyg is shortened from the original 1.7kb to about 1kb, and the complex structures such as a transcription terminator, a longer natural promoter and the like are removed, so that the PCR (Polymerase Chain Reaction) amplification is convenient, and the internal restriction sites are fewer (the common restriction sites are mutated by fixed sites); and meanwhile, the Hyg has short artificial promoters, so that expression in both Escherichia coli and mycobacterium can be realized, and the transcription and the translation in a downstream gene are not affected during oriented insertion. According to the resistance expression cassette disclosed by the invention, the dif sequences are added at two ends of the simplified Hyg and can automatically dissociate in the mycobacterium. After the Hyg is lost, the damaged gene can still continue to express shortened small peptides, so that the translation of the downstream genes is not affected, and furthermore, the polar effect can be effectively avoided.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a resistance expression cassette for efficiently constructing recombinant mycobacteria without resistance markers and an application thereof. Background technique [0002] Mtb is the pathogenic bacterium that causes tuberculosis, which can invade various organs of the body, but tuberculosis is the most common. Tuberculosis is still an important infectious disease, and its diagnosis, prevention and treatment are progressing slowly. These three aspects are all related to the study of the function of Mtb gene, and the genetic modification of Mtb plays a pivotal role in the study of Mtb. Mycobacterium tuberculosis grows slowly, it is difficult to carry out genetic manipulation on it, and requires an expensive level 3 biosafety laboratory with negative pressure. Long-term cultivation not only takes up space but is easy to contaminate, etc., making the research on Mycoba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63
Inventor 张天宇杨峰邹文英
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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