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Preparation method for Brucella shell vaccine strain

A Brucella and vaccine strain technology, applied in the fields of molecular biology and microbiology, can solve the problems of unsuitability for large-scale production and high cost, achieve good safety and immunogenicity, good genetic stability, and avoid biological Effects of security risks

Inactive Publication Date: 2014-07-30
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

[0005] In order to solve the problem that the cost of the existing fungus shell preparation method is too high and not suitable for large-scale production, the invention provides a preparation method of Brucella fungus shell vaccine strain

Method used

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  • Preparation method for Brucella shell vaccine strain
  • Preparation method for Brucella shell vaccine strain
  • Preparation method for Brucella shell vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Such as figure 1 Shown, the method for the preparation Brucella shell vaccine strain of taking Brucella canis RM6 / 66 bacterial strain as example comprises three steps altogether:

[0044] 1. Construction of temperature-controlled cleavage suicide plasmid pBK-CMV-SacB-ΔB0419-TLC

[0045] (1) PCR amplification of upper and lower homology arm sequences of specific genes in the genome of Brucella canis RM6 / 66 strain

[0046] 1) Primer design

[0047] Upstream homology arm primer B0419 upstream primer:

[0048] 5′- GGATCC GTGATTATCTCCGACGTTTCATC-3′ (with BamHI site),

[0049] Upstream homology arm primer B0419 downstream primer:

[0050] 5′- CTCGAG GGTTTGCAGCCTTGCCTGCGCGGT-3′ (with XhoI site);

[0051] Downstream homology arm primer B0419 upstream primer:

[0052] 5′- CTCGAG AGGCCAGCCCAGACGACCGCGCT-3′ (with XhoI site),

[0053] Downstream homology arm primer B0419 downstream primer:

[0054] 5′- TCTAGA CTATGCCTGTTCTTCCATCGG-3' (with XbaI site).

[0055] 2) G...

Embodiment 2

[0087] The resistance detection of embodiment 2 canine brucella capsid vaccine strains

[0088] The brucella shell vaccine strain that the present invention constructs is inoculated in containing kanamycin resistance Kan R (100μg / mL) TSB liquid medium, cultured at 28°C for 3 days, or coated with kanamycin-resistant Kan R (100μg / mL) TSA plate, cultured at 28°C for 7 days; observe whether the liquid is turbid or a single colony grows on the plate.

[0089] The result shows: the TSB liquid culture medium is clear and there is no single bacterium colony growth on the TSA flat plate, illustrates that the Brucella bacterial shell vaccine strain that the present invention builds can't contain kanamycin resistance Kan R Growth in the culture medium further indicates that the Brucella capsid vaccine strain is a non-resistance strain, which avoids the existence of resistance genes and the biosafety risk of horizontal transfer of resistance genes in the host.

Embodiment 3

[0091] Genetic Stability Detection of Capsid Vaccine Strains of Brucella canis

[0092] The brucella shell vaccine strain that the present invention constructs is inoculated on TSB liquid culture medium and is passed down continuously, records and saves each generation bacterium liquid, confirms the stability of its passage, with specific primer (upstream homologous arm primer B0419 upstream Primers and downstream homology arm primer B0419 downstream primer) for PCR verification.

[0093] The result is as Figure 9 As shown, the 1st to 30th generation BCG-ΔB0419 strains were amplified by B0419 gene primers to obtain a 3647bp band, while the genomic DNA of the control group Brucella canis only amplified a 2190bp band. It shows that after the temperature-controlled lysis component is inserted into the Brucella canis B0419 gene sequence, there is no loss of specific fragments or reverse mutation of the original B0419 gene, which further indicates that the Brucella capsid vaccine...

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Abstract

A preparation method for Brucella shell vaccine strain is disclosed, relates to the field of molecular biology and microbiology, and helps to solve the problem that conventional bacteria shell preparation method is high in cost and not suitable for large-scale production. The method comprises: respectively cloning PCR products of homogenous arms at the upstream and at the downstream of a specific gene of Brucella strain genome to a pMD18-T Simple vector to construct recombinant plasmids, performing double digestion on the recombinant plasmids, successively connecting with plasmid pBK-CMV-SacB subjected to digestion processing for constructing a suicide plasmid, performing PCR amplification on a temperature-control lysis part of a temperature-control expression plasmid pBV220-E, performing digestion processing on the amplification product, inserting into the suicide plasmid; and transforming the temperature-control lysis type suicide plasmid into competent Brucella, and performing positive and negative screening by utilizing kanamycins resistance and fructose sucrase gene, so as to obtain the Brucella shell vaccine strain. The Brucella shell vaccine strain has good heredity stability, security, effectiveness and immunogenicity.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and microbiology, in particular to a preparation method of a Brucella capsid vaccine strain. Background technique [0002] Brucella is a facultative anaerobic intracellular parasite. Brucellosis (hereinafter referred to as brucellosis) caused by this bacterium is currently the most prevalent and most harmful zoonosis in the world. one of the infectious diseases. After the 1990s, the human and animal brucellosis epidemics in my country continued to rise rapidly, and some areas showed an outbreak and epidemic trend, which brought a huge threat to my country's public health security. In my country, mainly cattle, sheep and suis Brucella are widely prevalent, and now there are also reports of canine Brucella infection. The current main method to control and eradicate brucellosis is the vaccine, and there are many problems in the safety and effectiveness of the commonly used vaccines. The...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12R1/01
Inventor 王兴龙钱晶郎需龙卜昭阳王秀然陈思
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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