RBCG (Bacillus Calmette-Guerin) expressing Brucella melitensis P39 gene as well as construction method and application of rBCG

Abstract

The invention provides rBCG (Bacillus Calmette-Guerin) expressing Brucella melitensis P39 gene. The rBCG is constructed by transferring an expression vector carrying a codon-optimized Brucella melitensis P39 gene into BCG. The cytoplasmic binding protein PBP39 (P39 as a coding gene) produced by Brucella is a T-cell antigen. BCG is the only commercial vaccine for preventing tuberculosis so far. Research proves that BCG not only has a remarkable immunoadjuvant effect, but also is an exogenous gene expression host with good performance and high safety. BCG can increase the expression quantity of the P39 gene when used for expressing the codon-optimized Brucella melitensis P39 gene, further, the constructed rBCG vaccine can simulate intracellular infection and parasitic features of Brucella and induce the organism to produce immune response more effectively, the advantages including high safety, simple preparation, low cost and the like of BCG as an expression host as well as the immunoadjuvant effect of BCG can be further realized, and rBCG is expected to be a novel vaccine for Brucella.

Description

technical field

[0001] The invention relates to the technical field of genetic engineering and vaccine preparation, in particular to an rBCG expressing the P39 gene of Brucella melis and its construction method and application. Background technique

[0002] Brucellosis, referred to as brucellosis, is a zoonotic infectious disease caused by Brucella infection. Brucella is a facultative intracellular parasite that is highly infectious and pathogenic to humans and mammals. The genus mainly includes 6 species of sheep, cattle, pig and dog. The first 3 species are mainly prevalent in China, among which Brucella melis infection is the most common. The modes of transmission of brucellosis are animal-animal and animal-human. Livestock such as sheep, cattle, and pigs are most susceptible to infection. Infection of animals can lead to inflammation of reproductive organs and fetal membranes, miscarriage, infertility, and various tissue lesions, which greatly increases the chance of t...

Examples

Embodiment 1

[0030] Embodiment 1 carries the construction of the recombinant expression vector of Brucella melii P39 gene

[0031] Include the following steps:

[0032] 1. According to the published sequence of the P39 gene of Brucella melis M5 strain (GenBank: EF189139.1), after codon optimization using Jcat software, the P39 gene after the full sequence optimization was artificially synthesized as the target gene (nucleotide The sequence is shown in SEQ ID NO: 1).

[0033] 2. Construction of the recombinant expression vector carrying the optimized M5 strain P39 gene:

[0034] The above target gene was inserted into the shuttle expression vector pMV361 through two restriction sites of PvuⅡ and EcoRI.

[0035] 3. Verification of inserting the correct recombinant expression vector:

[0036] Through nucleic acid sequence determination, it was confirmed that the recombinant expression vector carrying the optimized Brucella melii P39 gene was constructed successfully.

Embodiment 2

[0037] The construction of embodiment 2 expressing the rBCG of Brucella melis P39 gene

[0038] 1. Using BCG as the host bacterium, transform the recombinant expression vector constructed in Example 1 (full sequence shown in SEQ ID NO: 2) into BCG.

[0039] The electroporation method was used for transformation, and the experimental conditions were: 2500V, 25μF, 1000Ω, electroporation time 5ms, 0.1cm electric shock cup. The electroporation reaction system is: 3 μl of plasmid (concentration is 0.65 μg / μl), 100 μl of competent BCG bacterial solution (concentration is about 1×10 10 CFU / ml).

[0040] 2. Screening of positive clones

[0041] After electroporation, the bacterial solution was drawn and inoculated on the medium (slant) containing 50 μg / ml kanamycin for positive clone selection.

[0042] 3. Detection of target gene expression

[0043] The screened positive clones (ie, recombinant BCG) were inoculated into liquid medium for expansion culture, the culture supernatant...

Embodiment 3

[0044] Effect experiment of embodiment 3 brucellosis vaccines

[0045] Immunize female Balb / c mice aged 6-8 weeks with recombinant BCG, inject subcutaneously, and the injection dose is 4×10 8 CFU / mouse, 4 weeks after immunization, the expression of Th1 / Th2 cytokines in the serum of mice in each group was detected. The experimental results showed that, compared with the recombinant BCG (rBCG-P39(wild)) carrying the unoptimized P39 gene and untransformed BCG, the recombinant BCG carrying the codon-optimized P39 gene (rBCG-P39) could effectively induce Production of Th1 cytokines such as IL-2, IL-12 and IFN-γ. ( figure 2 )

technical field

[0001] The invention relates to the technical field of genetic engineering and vaccine preparation, in particular to an rBCG expressing the P39 gene of Brucella melis and its construction method and application. Background technique

[0002] Brucellosis, referred to as brucellosis, is a zoonotic infectious disease caused by Brucella infection. Brucella is a facultative intracellular parasite that is highly infectious and pathogenic to humans and mammals. The genus mainly includes 6 species of sheep, cattle, pig and dog. The first 3 species are mainly prevalent in China, among which Brucella melis infection is the most common. The modes of transmission of brucellosis are animal-animal and animal-human. Livestock such as sheep, cattle, and pigs are most susceptible to infection. Infection of animals can lead to inflammation of reproductive organs and fetal membranes, miscarriage, infertility, and various tissue lesions, which greatly increases the chance of t...

Examples

Embodiment 1

[0030] Embodiment 1 carries the construction of the recombinant expression vector of Brucella melii P39 gene

[0031] Include the following steps:

[0032] 1. According to the published sequence of the P39 gene of Brucella melis M5 strain (GenBank: EF189139.1), after codon optimization using Jcat software, the P39 gene after the full sequence optimization was artificially synthesized as the target gene (nucleotide The sequence is shown in SEQ ID NO: 1).

[0033] 2. Construction of the recombinant expression vector carrying the optimized M5 strain P39 gene:

[0034] The above target gene was inserted into the shuttle expression vector pMV361 through two restriction sites of PvuⅡ and EcoRI.

[0035] 3. Verification of inserting the correct recombinant expression vector:

[0036] Through nucleic acid sequence determination, it was confirmed that the recombinant expression vector carrying the optimized Brucella melii P39 gene was constructed successfully.

Embodiment 2

[0037] The construction of embodiment 2 expressing the rBCG of Brucella melis P39 gene

[0038] 1. Using BCG as the host bacterium, transform the recombinant expression vector constructed in Example 1 (full sequence shown in SEQ ID NO: 2) into BCG.

[0039] The electroporation method was used for transformation, and the experimental conditions were: 2500V, 25μF, 1000Ω, electroporation time 5ms, 0.1cm electric shock cup. The electroporation reaction system is: 3 μl of plasmid (concentration is 0.65 μg / μl), 100 μl of competent BCG bacterial solution (concentration is about 1×10 10 CFU / ml).

[0040] 2. Screening of positive clones

[0041] After electroporation, the bacterial solution was drawn and inoculated on the medium (slant) containing 50 μg / ml kanamycin for positive clone selection.

[0042] 3. Detection of target gene expression

[0043] The screened positive clones (ie, recombinant BCG) were inoculated into liquid medium for expansion culture, the culture supernatant...

Embodiment 3

[0044] Effect experiment of embodiment 3 brucellosis vaccines

[0045] Immunize female Balb / c mice aged 6-8 weeks with recombinant BCG, inject subcutaneously, and the injection dose is 4×10 8 CFU / mouse, 4 weeks after immunization, the expression of Th1 / Th2 cytokines in the serum of mice in each group was detected. The experimental results showed that, compared with the recombinant BCG (rBCG-P39(wild)) carrying the unoptimized P39 gene and untransformed BCG, the recombinant BCG carrying the codon-optimized P39 gene (rBCG-P39) could effectively induce Production of Th1 cytokines such as IL-2, IL-12 and IFN-γ. ( figure 2 )

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