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Brucella shell as well as preparation method and application thereof

A Brucella and fungus shell technology, applied in the field of microorganisms, can solve the problems of human and animal body injury, serious atavism, high toxicity, etc., achieve good protection effect, broad application prospects, and small side effects

Inactive Publication Date: 2012-02-15
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the currently used Brucella vaccines are attenuated vaccines. Although these vaccines have a certain immune protection effect, they also have very obvious shortcomings, namely high toxicity, serious atavism, and will cause harm to human and animal bodies. even get sick

Method used

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  • Brucella shell as well as preparation method and application thereof
  • Brucella shell as well as preparation method and application thereof
  • Brucella shell as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: prepare the brucella shell of the present invention

[0040] With the forward primer LE-F of the nucleotide sequence shown in SEQ ID NO: 4 and the reverse primer LE-R of the nucleotide sequence shown in SEQ ID NO: 5, from phage PhiX174 (purchased from MBI company) Amplified to obtain the cleavage gene E of the nucleotide sequence shown in SEQ ID NO: 1;

[0041] The cleavage gene E with the nucleotide sequence shown in SEQ ID NO: 1 was cloned into the plasmid pBV220 (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to obtain the recombinant plasmid pBV220::E;

[0042] With the forward primer BRU1 of the nucleotide sequence shown in SEQ ID NO: 2 and the reverse primer BRU2 of the nucleotide sequence shown in SEQ ID NO: 3, amplify the recombinant plasmid pBV220::E, the amplified The product was connected between the SpeI and SalI restriction sites in the plasmid pBBR1MCS-2 to obtain the recombinant plasmid pBBR1MCS-2-E;

[0043] The recombinant plas...

Embodiment 2

[0044] Example 2: Verification of recombinant plasmid pBBR1MCS-2-E

[0045] The recombinant plasmid pBBR1MCS-2-E obtained in Example 1 was verified by SpeI / SalI double enzyme digestion, and the results are shown in figure 2 . Depend on figure 2 It can be seen that the enzyme digestion results were consistent with expectations, confirming that the recombinant plasmid pBBR1MCS-2-E was constructed correctly.

Embodiment 3

[0046] Embodiment 3: the verification of recombinant bacterial strain

[0047] The recombinant bacterial strain obtained in embodiment 1 is carried out PCR verification, see image 3 . image 3 It was confirmed that the fusion fragment of the cloned thermoregulatory sequence λpL / pR-cI857 and the cleavage gene E was amplified from the recombinant strain, indicating that the recombinant strain was constructed successfully.

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Abstract

The invention relates to the field of microorganism, and in particular discloses a Brucella shell as well as a preparation method and application thereof. The preparation method comprises the following steps: cloning a lysis gene E having a nucleotide sequence shown in SEQ ID NO:1 to a plasmid pBV220 so as to obtain a recombinant plasmid pBV220::E; subsequently, amplifying the recombinant plasmid pBV220::E, and connecting the amplifying product to a plasmid pBBR1MCS-2; then transforming to Brucella; carrying out propagation culture on the recombinant strain at the temperature of 25-30 DEG C until the OD600 value of the strain is 0.8-1.2; raising the temperature to 37-45 DEG C until the OD600 value is stable; then centrifuging and collecting strain; and washing and then carrying out freeze thawing with a hypertonic solution so as to obtain the Brucella shell. According to the invention, the Brucella shell with high safety and good protection effect is prepared by transforming a fusion sequence of a temperature-controlling regulation sequence and the lysis gene E to the Brucella through adopting biotechnology, and the obtained Brucella shell has an important significance in control of epidemic and propagation of Brucella disease.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a Brucella shell and its preparation method and application. Background technique [0002] Brucella (Brucella spp.) is an intracellular parasitic, zoonotic pathogenic bacteria, and Brucellosis (brucellosis) refers to a widespread, highly contagious, harmful disease caused by Brucella It is a zoonotic infectious disease that is extremely large and difficult to cure after infection. The main symptoms of animal brucellosis are fever, miscarriage, infertility, chronic arthritis and nerve damage, etc., which can cause miscarriage in pregnant female animals in the late pregnancy, as well as orchitis and epididymitis in male animals; human brucellosis mainly passes through the skin and mucous membranes. , digestive tract and respiratory tract infection, its clinical manifestations are long-term fever, accompanied by hyperhidrosis, arthralgia, hepatosplenomegaly, and reproductive system da...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/33C12N15/63A61K39/10A61P31/04
Inventor 刘军刘爽冯书章
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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