rbcg expressing brucella melis p39 and l7/l12 fusion gene and its construction method

Abstract

The invention provides rBCG for expression of Br. Melitensis P39 and L7 / L12 fusion gene. The rBCG is constructed by transferring an expression vector carrying codon-optimized Br. Melitensis P39 and L7 / L12 fusion gene into BCG. Brucellosis-generated cytoplasm binding protein PBP39 (coding gene is P39) and Brucellosis ribosomal protein L7 / L12 are both T-cell antigen. Bacillus Calmette-Guerin (BCG) vaccine is the only one commercial vaccine for preventing tuberculosis so far. The BCG vaccine has a remarkable immunologic adjuvant effect and is an exogenous gene expression host with good performance and high safety. By BCG expression of the codon-optimized Brucellosis P39 and L7 / L12 fusion gene, expression quantity of the target gene can be increased. The rBCG vaccine can simulate intracellur infection and parasitic characteristics of Brucellosis to more effectively induce body to generate immune response, can perform advantages of high safety, simple preparation, low cost, etc. of BCG as the expression host as well as the immunologic adjuvant effect of the BCG itself, and is expected to become a novel Brucellosis vaccine.

Description

technical field

[0001] The invention relates to the technical field of genetic engineering and vaccine preparation, in particular to an rBCG expressing P39 and L7 / L12 fusion genes of Brucella melis and a construction method thereof. 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...

Examples

Embodiment 1

[0032] Embodiment 1 carries the construction of the recombinant expression vector of Brucella melii P39 and L7 / L12 fusion gene

[0033] Include the following steps:

[0034] 1. According to the published sequences of genes P39 and L7 / L12 of Brucella melis M5 strain (GenBank: EF189139.1; GenBank: EF173477.1), after codon optimization using Jcat software, artificially synthesized full sequence optimization The latter P39-L7 / L12 fusion gene was used as the target gene. (The nucleotide sequence of the fusion gene is shown in SEQ ID NO:1).

[0035] 2. Construction of a recombinant expression vector carrying the optimized M5 strain P39-L7 / L12 fusion gene: the above target gene was inserted into the shuttle expression vector pMV361 through two restriction sites of PvuII and BstBI.

[0036] 3. Verification of inserting the correct recombinant expression vector.

[0037] Through nucleic acid sequence determination, it was confirmed that the recombinant expression vector carrying the...

Embodiment 2

[0038] Example 2 Construction of rBCG expressing Brucella melis P39 and L7 / L12 fusion gene

[0039] 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.

[0040] 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 0.68 μg / μl), 100 μl of competent BCG bacterial solution (concentration is about 1×10 10 CFU / ml).

[0041] 2. Screening of positive clones

[0042] After electroporation, they were inoculated on a medium (slant) containing 50 μg / ml kanamycin for positive clone selection.

[0043] 3. Detection of target gene expression

[0044]The screened positive clones (ie, recombinant BCG) were inoculated into liquid medium for expansion culture, the culture supernatant was collected, and the ex...

Embodiment 3

[0045] Effect experiment of embodiment 3 brucellosis vaccines

[0046] Immunize female Balb / c mice aged 6-8 weeks with recombinant BCG and inject subcutaneously at a dose of 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 recombinant BCG carrying unoptimized P39-L7 / L12 fusion gene (rBCG-P39-L7 / L12(wild)) and untransformed BCG, carrying codon-optimized P39-L7 / L12 The recombinant BCG with fusion gene (rBCG-P39-L7 / L12) can effectively induce the 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 P39 and L7 / L12 fusion genes of Brucella melis and a construction method thereof. 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...

Examples

Embodiment 1

[0032] Embodiment 1 carries the construction of the recombinant expression vector of Brucella melii P39 and L7 / L12 fusion gene

[0033] Include the following steps:

[0034] 1. According to the published sequences of genes P39 and L7 / L12 of Brucella melis M5 strain (GenBank: EF189139.1; GenBank: EF173477.1), after codon optimization using Jcat software, artificially synthesized full sequence optimization The latter P39-L7 / L12 fusion gene was used as the target gene. (The nucleotide sequence of the fusion gene is shown in SEQ ID NO:1).

[0035] 2. Construction of a recombinant expression vector carrying the optimized M5 strain P39-L7 / L12 fusion gene: the above target gene was inserted into the shuttle expression vector pMV361 through two restriction sites of PvuII and BstBI.

[0036] 3. Verification of inserting the correct recombinant expression vector.

[0037] Through nucleic acid sequence determination, it was confirmed that the recombinant expression vector carrying the...

Embodiment 2

[0038] Example 2 Construction of rBCG expressing Brucella melis P39 and L7 / L12 fusion gene

[0039] 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.

[0040] 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 0.68 μg / μl), 100 μl of competent BCG bacterial solution (concentration is about 1×10 10 CFU / ml).

[0041] 2. Screening of positive clones

[0042] After electroporation, they were inoculated on a medium (slant) containing 50 μg / ml kanamycin for positive clone selection.

[0043] 3. Detection of target gene expression

[0044]The screened positive clones (ie, recombinant BCG) were inoculated into liquid medium for expansion culture, the culture supernatant was collected, and the ex...

Embodiment 3

[0045] Effect experiment of embodiment 3 brucellosis vaccines

[0046] Immunize female Balb / c mice aged 6-8 weeks with recombinant BCG and inject subcutaneously at a dose of 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 recombinant BCG carrying unoptimized P39-L7 / L12 fusion gene (rBCG-P39-L7 / L12(wild)) and untransformed BCG, carrying codon-optimized P39-L7 / L12 The recombinant BCG with fusion gene (rBCG-P39-L7 / L12) can effectively induce the production of Th1 cytokines such as IL-2, IL-12 and IFN-γ. ( figure 2 )

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