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Building method of lactococcus lactis genetic engineering bacterial strain loaded with clfA gene

A technology of genetically engineered strains, Lactococcus lactis, applied in the field of construction of Lactococcus lactis genetically engineered strains, can solve the problems of restriction, vaccine invasiveness, toxicity, etc.

Inactive Publication Date: 2012-07-11
INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the vaccine developed using it as a carrier has the possibility of maintaining invasiveness and toxicity, which is limited when it is applied to children, the elderly and immunocompromised persons

Method used

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  • Building method of lactococcus lactis genetic engineering bacterial strain loaded with clfA gene
  • Building method of lactococcus lactis genetic engineering bacterial strain loaded with clfA gene
  • Building method of lactococcus lactis genetic engineering bacterial strain loaded with clfA gene

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specific Embodiment approach 1

[0011] Specific embodiment one: the construction method of the Lactococcus lactis genetically engineered bacterial strain that introduces clfA gene in this embodiment, carry out according to the following steps: 1, extract the genomic DNA of Staphylococcus aureus; 2, take Staphylococcus aureus genomic DNA as template, MG-CLaF and MG-CLaR2 were used as primers to perform PCR amplification; 3. The PCR amplification product was detected by 1% agarose gel electrophoresis, and then purified using a gel recovery kit to obtain the clfA gene; 4. The clfA gene was obtained by using a plasmid The extraction kit extracts the plasmid pMG36c stored in Escherichia coli JM109; 5. Carry out double digestion of the clfA gene and the plasmid pMG36c respectively, and connect the digested clfA gene and the plasmid pMG36c with a ligation kit to obtain a ligation product; 6. 1. Transform the ligation product into Escherichia coli JM109 competent cells, use chloramphenicol to screen the transformants...

specific Embodiment approach 2

[0045] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the reaction system of PCR amplification in step 2 is a 20 μL reaction system, which consists of the following components:

[0046]

[0047] PCR amplification conditions were: denaturation at 94°C for 2 min, denaturation at 94°C for 15 s, annealing at 58°C for 30 s, extension at 68°C for 90 s, a total of 35 cycles, extension at 68°C for 5 min, and incubation at 4°C. Others are the same as in the first embodiment.

specific Embodiment approach 3

[0048] Specific embodiment three: the difference between this embodiment and specific embodiment one is: the system of clfA gene double-enzyme digestion in step five is as follows:

[0049]

[0050] Enzyme digestion reaction conditions: 37°C water bath, 1h. Others are the same as in the first embodiment.

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Abstract

The invention provides a building method of a lactococcus lactis genetic engineering bacterial strain loaded with a clfA gene, and relates to a building method of a lactococcus lactis genetic engineering bacterial strain loaded with a clfA gene. The method provided by the invention can be used for solving the problem that the research of a genetic engineering vaccine has invasion and toxicity since the existing escherichia coli is taken as a host expression clFA gene. The method comprises the following steps of: extracting genome DNA (deoxyribonucleic acid) of staphylococcus aureus; carrying out PCR (polymerase chain reaction) amplification by taking the genome DNA of the staphylococcus aureus as a template, so as to obtain the clFA gene; extracting plasmid pMG36c stored in escherichia coli JM09; carrying out double digestion on the clFA gene and the plasmid pMG36c respectively, and connecting a product obtained by means of enzyme digestion; transforming a connected product into an escherichia coli JM09 competent cell, and screening a transformant by use of chloramphenicol, so as to build a recombinant plasmid; and electrically transforming the recombinant plasmid into lactococcus lactis. The building method can be used for researching the genetic engineering bacterial, and is safe and free of pathogenicity.

Description

technical field [0001] The invention relates to a method for constructing a genetically engineered strain of Lactococcus lactis into which a clfA gene is introduced. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is an important pathogenic bacterium of zoonosis, and it is the most virulent pathogenic bacterium. It can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and even systemic infection such as sepsis and sepsis. At the same time, Staphylococcus aureus is also one of the most common bacterial food poisoning pathogens at home and abroad. According to the U.S. Centers for Disease Control and Prevention report, food poisoning caused by Staphylococcus aureus is second only to Escherichia coli, accounting for 33% of bacterial food poisoning. In my country, food poisoning caused by Staphylococcus aureus accounts for about 25% of bacterial food poisoning incidents, and in developed countries such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/01
Inventor 曲晓军崔艳华张超孙建华夏海华王金英于冲
Owner INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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