阅读说明：本技术 一种布鲁氏菌L7/L12和GroES真核表达载体的构建方法及其应用 (Construction method and application of Brucella L7/L12 and GroES eukaryotic expression vector ) 是由 吴同垒 肖丽荣 史秋梅 张志强 周诗淼 冀梦瑶 于 2021-08-06 设计创作，主要内容包括：本发明公开了一种布鲁氏菌L7/L12和GroES真核表达载体的构建方法及其应用,属于生物技术领域。本发明公开的一种布鲁氏菌L7/L12和GroES真核表达载体的构建方法,利用OverlapPCR方法连接基因L7/L12和GroES,利用pcDNA3.1构建真核表达载体pcDNA3.1-LG。真核表达载体pcDNA3.1-LG用于制备布鲁氏菌疫苗。本发明通过构建L7/L12和GroES的融合表达真核载体,免疫小鼠后,机体产生了良好的体液和细胞免疫力,为布鲁氏菌核酸疫苗的开发奠定了重要基础。(The invention discloses a construction method and application of Brucella L7/L12 and GroES eukaryotic expression vectors, and belongs to the technical field of biology. The invention discloses a construction method of Brucella L7/L12 and GroES eukaryotic expression vectors, which comprises the steps of connecting genes L7/L12 and GroES by using an overlapPCR method, and constructing the eukaryotic expression vectors pcDNA3.1-LG by using pcDNA3.1. The eukaryotic expression vector pcDNA3.1-LG is used for preparing brucella vaccine. According to the invention, through constructing the fusion expression eukaryotic vector of L7/L12 and GroES, after a mouse is immunized, an organism generates good humoral and cellular immunity, and an important foundation is laid for the development of a Brucella nucleic acid vaccine.)

1. A construction method of Brucella L7/L12 and GroES eukaryotic expression vectors is characterized by comprising the following specific steps:

(1) abortus 2308 genomic DNA was extracted;

(2) L7/L12 gene is amplified by PCR by using the primers LG-1 and LG-2, and GroES gene is amplified by PCR by using LG-3 and LG-4; after purification of the amplification product 1:1, uniformly mixing the mixture as a template of overlapped PCR, amplifying by using primers LG1 and LG4, and connecting two genes of L7/L12 and GroES to obtain L7/L12-GroES;

wherein the primer sequences of LG-1 and LG-2, and LG-3 and LG-4 are as follows:

LG-1：5’-GCGGATCCACCATGGTGGCTGATCTCGCAAAGATCGTT-3’；SEQ ID NO.1；

LG-2：5’-GCGCTCGGGCAGTATCATCATTCCTCCCTTGAGTTCAACCTTGGCGCCAGCA-3’；SEQ ID NO.2；

LG-3：5’-GGCGCCAAGGTTGAACTCAAGGGAGGAATGATGATACTGCCCGAGCGCCT-3’；SEQ ID NO.3；

LG-4：5’-GCCTCGAGTCAGACAAGCGCATGTAGAGAAGCA-3’；SEQ ID NO.4；

(3) carrying out double enzyme digestion, connection and transformation on the purified L7/L12-GroES and plasmid pcDNA3.1, and coating an Amp plate for overnight culture; selecting a single colony for PCR identification to obtain positive clone, extracting plasmid, and obtaining the eukaryotic expression vector pcDNA3.1-LG through sequencing verification.

2. The method for constructing eukaryotic expression vectors of Brucella L7/L12 and GroES according to claim 1, wherein the reaction system of PCR and overlap PCR in step (2) is: 1 mul of each upstream and downstream primer, 1 mul of DNA template, 25 mul of PCR mix and 50 mul of distilled water; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 60s at 94 ℃, 30s at 50 ℃, 60s at 72 ℃ for extension, 32 cycles; final extension at 72 ℃ for 8 min.

3. The use of Brucella L7/L12 and GroES eukaryotic expression vector of claim 1 in the preparation of Brucella vaccine.

Technical Field

The invention relates to the technical field of biology, in particular to a construction method and application of Brucella L7/L12 and GroES eukaryotic expression vectors.

Background

Brucellosis is a zoonosis infectious disease caused by brucella, causes huge economic loss to the cattle and sheep breeding industry, and seriously threatens human health. Vaccine immunization is a more effective method for preventing brucellosis, and commercial brucella vaccines are A19, S2, Rev1, M5, RB51 and the like, which are all low-toxicity vaccines, but have the defect of insufficient safety, for example, A19 can cause abortion of pregnant animals, has the risk of detoxification, and can mostly infect people and cause transient diseases. The DNA vaccine has high safety, and the existing research shows that certain genes can stimulate organisms to generate stronger protective force, so that people pay more attention to the DNA vaccine.

The DNA vaccine based on the Brucella L7/L12 gene can produce good protection effect. GroES is an accessory protein of the hsp60 family of heat shock proteins, can activate the immune response of a host, and simultaneously participate in antigen presentation to enhance the immune response level of the host.

Therefore, the problem to be solved by the technical personnel in the field is to provide a construction method and application of Brucella L7/L12 and GroES eukaryotic expression vectors.

Disclosure of Invention

In view of the above, the invention provides a construction method and application of Brucella L7/L12 and GroES eukaryotic expression vectors.

In order to achieve the purpose, the invention adopts the following technical scheme:

a construction method of Brucella L7/L12 and GroES eukaryotic expression vectors comprises the following steps:

(1) abortus 2308 genomic DNA was extracted;

(2) PCR amplifying L7/L12 gene by using the primers LG-1 and LG-2, and PCR amplifying GroES gene by using LG-3 and LG-4; after purification of the amplification product 1:1, uniformly mixing the mixture as a template of overlapped PCR, amplifying by using primers LG1 and LG4, and connecting two genes of L7/L12 and GroES to obtain L7/L12-GroES;

wherein the primer sequences of LG-1 and LG-2, and LG-3 and LG-4 are as follows:

LG-1：5’-GCGGATCCACCATGGTGGCTGATCTCGCAAAGATCGTT-3’；SEQ ID NO.1；

LG-2：5’-GCGCTCGGGCAGTATCATCATTCCTCCCTTGAGTTCAACCTTGGCGCCAGCA-3’；SEQ ID NO.2；

LG-3：5’-GGCGCCAAGGTTGAACTCAAGGGAGGAATGATGATACTGCCCGAGCGCCT-3’；SEQ ID NO.3；

LG-4：5’-GCCTCGAGTCAGACAAGCGCATGTAGAGAAGCA-3’；SEQ ID NO.4；

(3) carrying out double enzyme digestion, connection and transformation on the purified L7/L12-GroES and plasmid pcDNA3.1, and coating an Amp plate for overnight culture; selecting a single colony for PCR identification to obtain positive clone, extracting plasmid, and obtaining the eukaryotic expression vector pcDNA3.1-LG through sequencing verification.

Further, the reaction system of the PCR and the overlap PCR in the step (2) is as follows: 1 mul of each upstream and downstream primer, 1 mul of DNA template, 25 mul of PCR mix and 50 mul of distilled water; the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 60s at 94 ℃, 30s at 50 ℃, 60s at 72 ℃ for extension, 32 cycles; final extension at 72 ℃ for 8 min.

Further, the Brucella L7/L12 and GroES eukaryotic expression vector are applied to preparation of Brucella vaccines.

According to the technical scheme, compared with the prior art, the invention discloses a construction method and application of Brucella L7/L12 and GroES eukaryotic expression vectors, and by constructing the fusion expression eukaryotic plasmid of L7/L12 and GroES, an organism generates good body fluid and cell immunity after a mouse is immunized, so that an important foundation is laid for the development of Brucella nucleic acid vaccines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram showing the PCR amplification and overlap PCR of L7/L12 and GroES gene according to the present invention;

wherein, M: DL2000 Plus DNA Marker; 1: L7/L12; 2: GroES; 3: L7/L12-GroES;

FIG. 2 is a SDS-PAGE analysis of IPTG-induced recombinant protein expression of the invention;

wherein, M: pre-dyeing a Marker with protein; 1: the recombinant plasmid pET32a-LG is transformed into escherichia coli competence BL 21; 2: the empty vector pET32a is transformed into escherichia coli competence BL 21;

FIG. 3 is a Western blot analysis of the IPTG induced recombinant protein expression of the invention;

wherein, 1: the recombinant plasmid pET32a-LG is transformed into escherichia coli competence BL 21; 2: the empty vector pET32a is transformed into escherichia coli competence BL 21;

FIG. 4 is an SDS-PAGE analysis of the expression form of the recombinant protein of the invention;

wherein, M: pre-dyeing a Marker with protein; 1: performing whole bacteria; 2: precipitating; 3: supernatant fluid;

FIG. 5 is an SDS-PAGE analysis of recombinant proteins of the invention before and after purification;

wherein, M: pre-dyeing a Marker with protein; 1: inclusion bodies; 2: purifying inclusion bodies;

FIG. 6 is a Western blot assay of the polyclonal antibody of the present invention;

wherein, 1: pET32 a-LG; 2: pET32 a;

FIG. 7 is a diagram showing the indirect immunofluorescence of the present invention to verify the expression of recombinant proteins in 293T cells;

FIG. 8 is a diagram showing the expression of recombinant proteins in 293T cells verified by Western blot;

wherein, 1: pcDNA3.1-LG; 2: pcDNA3.1;

FIG. 9 is a graph showing the measurement of serum antibody levels in immunized mice by ELISA method according to the present invention; *: p < 0.05; **: p < 0.01; ***: p < 0.001;

FIG. 10 is a graph showing the determination of the cellular immunity level (MTT method) in the lymphocyte transformation assay according to the present invention; **: p < 0.01; ***: p < 0.001.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The Ni-agarose gel, the His label mouse-derived monoclonal antibody, the HRP-goat anti-mouse IgG and endotoxin-free plasmid extraction kit is purchased from Beijing kang, a century Biotech Co., Ltd; ConA, DMSO, MTT, erythrocyte lysate, SDS-PAGE, and Western blot related reagents were purchased from Solebao Biotech, Inc., Beijing.

EXAMPLE 1 construction of recombinant expression vectors and expression strains

Abortus 2308 genomic DNA was extracted and amplified by PCR to obtain L7/L12 and GroES fusion gene fragments. The primer information is shown in Table 1, wherein the primers LG-1 and LG-2 are used for amplifying L7/L12 gene, LG-3 and LG-4 are used for amplifying GroES gene, and the amplification product is purified 1:1, uniformly mixing the two genes to be used as a template of overlapped PCR, amplifying by using primers LG1 and LG4, and connecting two genes of L7/L12 and GroES together to obtain L7/L12-GroES; is used for constructing prokaryotic or eukaryotic expression vectors.

The nucleotides of L7/L12-GroES are listed below:

atggctgatctcgcaaagatcgttgaagacctttcggccctgaccgttctggaagccgctgagctgtccaagcttctcgaagagaagtggggcgtttcggctgctgctccggtcgctgttgctgctgccggtggcgctgcccctgctgctgccgcagaagaaaagaccgaattcgacgtcgttctcgctgacggcggcgctaacaagatcaacgtgatcaaggaagtgcgcgcactcaccggtctcggcctcaaggaagccaaggacctggtcgaaggcgctccgaaggctgtcaaggaaggcgcctcgaaggacgaagctgagaagatcaaggcacagctcgaagctgctggcgccaaggttgaactcaaggaagaaatgatgatactgcccgagcgcctcgacgatctgacggaccgatatgatgcgatcttttgcgacgtctggggcgttgtacacaacggcgaaacgtcctttgcgccggccatcgccgccttgcagcgcgctcgcgccaagggtgtgacaatcattctcgtgaccaattctccacgcccgcatccgggcgttgtcgcgcagatgtcgcttctgggtgtgcccgaagatgcctatgaccgtgttgtcacttccggtgatgttacgcgcgatcttatcgccgaagggccgcgccggattttccacatcggctgcgagcgcgagcttgccatttatgatgggcttgacgtggagctggtcgaggaatttgaggcggcaggcgtggtttgtaccgggctttatgacgatgaagtcgaaacgccagaagactacagggaattgctccagcgtctgcgcgcgcgcaacctgcctttcatttgcgccaatcctgatatcatggtggaacgcgggcctcgcctcatctggtgcgcgggcgcgcttgcacgcgaatacggccagcttggagggcgcaccctgattgcaggcaagccccatcgcccgatctatgaggcggcgcttcgcgcggtggaaagcattcgcggtggaagcgtggacaagagccgcattcttggcattggcgacggcgttttgaccgatgtgaaaggggctgcggattttggcctcgatgtgctttatatttcgggcggcgttcatgccgccgattatgcggtgaacggggatctcgatatggcgaagatggaggcttttctggaaaagcatggccatcgccccattgcttctctacatgcgcttgtctga；SEQ ID NO.9。

both PCR and overlap PCR reaction systems are: mu.L of each of the upstream and downstream primers, 1. mu.L of DNA template, 25. mu.L of PCR mix, and 50. mu.L of distilled water. The PCR reaction conditions were all as follows: pre-denaturation at 94 ℃ for 5 min; 60s at 94 ℃, 30s at 50 ℃, 60s at 72 ℃ for extension, 32 cycles; final extension at 72 ℃ for 8 min.

The amplification results are shown in FIG. 1, which shows that L7/L12 and GroES genes are successfully fused by the Overlap PCR method by using brucella bovis 2308 genomic DNA as a template.

And carrying out double enzyme digestion on the purified amplification product, the plasmid pET32a and the pcDNA3.1, respectively connecting and transforming the amplification product subjected to enzyme digestion purification with the plasmid pET32a and the pcDNA3.1 subjected to enzyme digestion purification, and coating an Amp plate for overnight culture. A single colony is picked and PCR identification is carried out by using corresponding primers (T7 and S-tag primer, pcDNA3.1-F and pcDNA3.1-R, see table 1), and positive strains are extracted with plasmid, subjected to double enzyme digestion identification and subjected to sequencing. The identified error-free recombinant plasmids were designated as pET32a-LG and pcDNA3.1-LG, respectively.

TABLE 1 primer information

Example 2 inducible expression, validation and purification of recombinant proteins

The recombinant plasmid pET32a-LG is transformed into escherichia coli competence BL21, and after the expression is induced for 4 hours at 37 ℃ by IPTG with the final concentration of 1mmol/L, SDS-PAGE and Western blot analysis are carried out to verify the expression.

The SDS-PAGE result is shown in FIG. 2, and the size of the band is about 65kDa, which is expected. In order to further verify the expression of the secretory protein, Western blot identification is carried out on the fusion protein aiming at the expression tag His, and the result is shown in figure 3, thereby further proving that the target protein is expressed.

After induction expression, adding a part of thalli into a protein loading buffer solution for boiling to serve as whole bacteria loading; ultrasonic cracking of a part of the thalli, centrifugal separation of supernatant and precipitation, SDS-PAGE respectively, analysis of expression product form, and the result is shown in figure 4, and the recombinant protein is mainly expressed by inclusion body. The expression product was purified by Ni column and verified by SDS-PAGE, and the result is shown in FIG. 5, and a fusion expression protein (recombinant protein) with high purity was obtained.

EXAMPLE 3 preparation and characterization of murine polyclonal antibodies to recombinant proteins

Emulsifying the recombinant protein by Freund complete adjuvant for the first immunization, and injecting the immunized mice into the back of the mice at multiple subcutaneous points with the dose of 100 mu Ag/Ag; two weeks later, two immunizations were given with 50 μ Ag/Ag emulsified protein in Freund's incomplete adjuvant; three-time immunization after one week, and the same two-time immunization; one week later, the immunization was boosted, without adjuvant, at a dose of 100/ip, i.p. injection; after one week, fasting is performed for one day, blood is taken the next day, and serum is separated. Taking the recombinant protein to carry out SDS-PAGE and membrane transfer, using the prepared mouse serum as a primary antibody, using an HRP-labeled goat anti-mouse polyclonal antibody as a secondary antibody, and using LG prokaryotic protein as an antigen to carry out Western blot analysis, wherein the result is shown in figure 6, which indicates that the preparation of the polyclonal antibody is successful.

The serum was diluted in multiples with 100ng protein concentration per well of coated plate and the serum antibody titer was determined using a 1:5000 dilution of HRP-goat anti-mouse IgG for the secondary antibody.

The serum was determined to have an antibody titer of 15000 by ELISA since the P/N value was still greater than 2.1 at a serum dilution of 1: 15000.

Example 4 transient transfection of recombinant plasmid pcDNA3.1-LG, Indirect immunofluorescence assay and Western blot assay of eukaryotic expression products

Plasmid pcDNA3.1-LG was extracted using an endotoxin-free plasmid extraction kit. 293T cells were plated in 6-well plates at a concentration of 2X 10⁵CFU/well. The next day, dissolve 2 μ Ag in 200 μ Lbuffer, vortex oscillation for 10 seconds; then add 4. mu.Lreagent, vortex shaking for 10 seconds, and incubating for 10min at room temperature; then removing the culture medium in the cell culture hole, adding a serum-free culture medium, gently adding the transfection mixture into the culture hole, fully and uniformly mixing, placing the mixture into a cell culture box for culture for 4 hours, removing the culture medium by suction, changing the culture medium into a culture medium containing 10% of serum, continuously culturing for 24 hours, and taking one 6-hole plate for indirect immunofluorescence; after another 6-well plate is cultured for 48h, 293T cells are collected for Western blot detection.

(1) Indirect immunofluorescence detection of expression of pcDNA3.1-LG in 293T cells

Taking the transfected 293T cells, discarding the culture medium, and washing for 3 times by using PBS; fixing 2% paraformaldehyde at room temperature for 15min, and washing with PBS for 3 times; 1% Triton X-100, reacting at room temperature for 15min, and washing with PBS 3 times; 2% BSA, blocked overnight at 4 ℃ and washed 3 times with PBS; using LG antiserum prepared by immunizing a mouse with prokaryotic expression purified protein, incubating for 1h at 37 ℃, and washing for 3 times by PBS; adding a goat anti-mouse secondary antibody labeled by FITC, and incubating for 1h at 37 ℃ in the dark; PBS was washed 6 times, added to PBST 1mL, observed under a fluorescence microscope, and stored by photographing, and the result is shown in FIG. 7. FIG. 7 shows that in 293T cells transfected with pcDNA3.1-LG recombinant plasmid, specific green fluorescence was observed, and it can be seen that pcDNA3.1-LG recombinant plasmid can be expressed in 293T cells.

(2) Western blot analysis of expression of pcDNA3.1-LG in 293T cells

The transfected 293T cells were removed and placed on ice, washed 3 times with ice-cold PBS, added with protein loading buffer, harvested into 1.5mL EP tubes, boiled for 15 min; centrifuging at 14000rpm/min for 10min at 4 deg.C, collecting supernatant as protein sample, and analyzing expression with western blot, and finding the result shown in FIG. 8. Western blot results show that a protein band with the size of about 45kDa can be detected in 293T cells transfected with the recombinant plasmid pcDNA3.1-LG; in 293T cells transfected with the empty vector plasmid, no corresponding band of interest was detected.

Example 5 evaluation of immune Effect

(1) Animal immunization

Dividing BALB/c mice into 3 groups, each group comprises 5 mice, the first two groups respectively immunize recombinant plasmid pcDNA3.1-LG and empty plasmid pcDNA3.1, the dose is 100 mug/mouse, and the immunization mode is leg muscle injection; the third group was a control group, which was intramuscularly infused with an equal volume of PBS. Following the first immunization, the immunization was boosted at one and two weeks, with the same dose and method as the first immunization. Randomly taking 3 mice for blood sampling at intervals of 7 days after the first immunization, collecting serum, preserving at minus 20 ℃, and collecting blood for 3 times. After each blood sampling, mice were sacrificed, splenocytes isolated, and lymphocyte transformation experiments performed.

(2) Serum antibody titer detection

Detecting serum antibody titer by indirect ELISA method, coating with purified LG recombinant protein as antigen (200 ng/well), mouse serum as primary antibody (dilution multiple of 1:50), HRP-labeled goat anti-mouse IgG as secondary antibody (dilution multiple of 1:5000), and determining OD₄₅₀。

Serum antibody detection was performed on the immunized mice by ELISA, with the results shown in figure 9; FIG. 9 shows the results that the antibody level after immunizing the recombinant plasmid pcDNA3.1-LG was higher than that of the empty plasmid group and that of the PBS group.

(3) Lymphocyte transformation assay

Spleens of sacrificed mice were taken and spleen cell suspensions were prepared, and erythrocytes were lysed using erythrocyte lysate, and after washing with PBS, cells were resuspended using cell culture medium. Cells with 10 percent⁵Laying CFU/well on a 96-well plate, laying spleen cells of each mouse on 6 wells, and adding 11 mu L of purified LG recombinant protein (5 mu g/mL) into 1-3 wells to serve as a test group; adding 11 mu L1640 culture medium into 4-6 holes to serve as an unstimulated group; adding 16 to 10-12 holes40 medium as cell-free control group. After 72h of action, adding MTT, continuing to culture for 4h, then adding Formazan dissolving solution into the hole, uniformly mixing, culturing for 4h, and determining OD₅₇₀. Calculated stimulation index ═ (test group OD_570nmmean-OD of cell-free control group_570nmmean)/(OD of unstimulated group_570nmmean-OD of cell-free control group_570nmMean value).

The stimulation index of the test group/the stimulation index of the non-stimulated group are used as the ordinate, the time is used as the abscissa for plotting, and the result is shown in a graph 10; FIG. 10 shows that the stimulation index of the recombinant plasmid pcDNA3.1-LG immunization group was higher than that of the PBS group and the empty plasmid group.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Hubei institute of science and technology

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gctgctgccg gtggcgctgc ccctgctgct gccgcagaag aaaagaccga attcgacgtc 180

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ggtctcggcc tcaaggaagc caaggacctg gtcgaaggcg ctccgaaggc tgtcaaggaa 300

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ccggccatcg ccgccttgca gcgcgctcgc gccaagggtg tgacaatcat tctcgtgacc 540

aattctccac gcccgcatcc gggcgttgtc gcgcagatgt cgcttctggg tgtgcccgaa 600

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gtggacaaga gccgcattct tggcattggc gacggcgttt tgaccgatgt gaaaggggct 1080

gcggattttg gcctcgatgt gctttatatt tcgggcggcg ttcatgccgc cgattatgcg 1140

gtgaacgggg atctcgatat ggcgaagatg gaggcttttc tggaaaagca tggccatcgc 1200

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