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Genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof

A technology for genetically engineering bacteria and proteins, applied in the field of genetic engineering

Inactive Publication Date: 2012-07-18
HUAIHUA UNIV +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no relevant report on the function research of recombinant porcine bactericidal protein at home and abroad

Method used

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  • Genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof
  • Genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof
  • Genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof

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Embodiment 1

[0032] Example 1 construction

[0033] RNA was extracted from the liver tissue of Qianshao Hua pig, and then amplified by RT-PCR. Primers:

[0034] Forward Primer: 5′-GCAA GGATCC ATGGCCAGGGGCGCTGAC-3′

[0035] Reverse Primer: 5′-CCAT CTCGAG TTATTTTTTTCACTTTGGCTGTCACTGGCAG-3

[0036] Reverse transcription: Add 4 μl of Total RNA (≤5 μg), 1 μl of random hexamer primer, and DEPC-H to a 500 μl centrifuge tube 2O 7μl, mixed gently, centrifuged, total volume 12μl, incubated at 65°C for 5min, centrifuged gently, cooled on ice; then added 5×reaction buffer 4μl, Ribonuclease inhibitor (20u / μl) 1μl, 10mM dNTP mix 2μl, Rever Transcriptase (200u / μl) 1μl, total volume 20μl, mixed gently, centrifuged to precipitate, incubated at 25°C for 5min, then incubated at 42°C for 60min, then incubated at 70°C for 5min, and the synthesized cDNA was stored at -20°C for later use.

[0037] Add 7.2 μl of dd-water, 10 μl of MasterMix, 0.4 μl of Forward Primer (10 μm), 0.4 μl of Reverse Primer (10 μm...

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Abstract

The invention discloses a genetic engineering bacterium for producing recombined pig sterilization protein and construction and application thereof, which belong to the technical field of genetic engineering. An open reading frame 5' end 678bp deoxyribonucleic acid (DNA) segment of a Qianshao spotted pig sterilization protein gene is amplified by aid of the reverse transcription-polymerase chain reaction (RT-PCR) technology, inserted into an escherichia coli expression vector pET32a(+) and transformed into Esherichia coli BL21(DE3), a recombined sub-library is construed, and finally an escherichia coli BL21(DE3)-pET32a(+)-pBPIN(Esherichia coli BL21(DE3)-pET32a(+)-pBPIN)CCTCC M20111252 capable of producing recombined pig sterilization protein is obtained through screening and evaluation. External activity detection on expression products shows that the recombined pig sterilization protein has obvious sterilization effect on Gram negative bacteria.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and mainly relates to a genetically engineered bacterium producing recombinant porcine bactericidal protein, its construction method and application. Background technique [0002] my country is the world's largest pork producer, and the number of pigs on hand, the amount of slaughter, the amount of slaughter, and the total output of pork all rank first in the world. However, infectious diseases caused by pathogenic bacteria cause huge losses to pig production every year, seriously affecting my country's animal husbandry. Economic growth and export earnings of the pig and livestock industries. At present, the control of infectious diseases mainly adopts vaccination and antibiotic treatment. The use of vaccines is not always effective, and antibiotics do play an important role in the prevention and treatment of bacterial diseases in pigs, but long-term large-scale use has brought many ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P21/02C12R1/19
Inventor 魏麟刘胜贵向孙军
Owner HUAIHUA UNIV
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