CN109485704B - Expression system of meningococcal fHbp protein - Google Patents

Abstract

The invention provides an expression system of meningococcal fHbp protein, which comprises an fHbp antigen gene and a vector pCAMBIA1301, wherein the expression system is pCAMBIA-fHbp, and the promoter is a beta-phaseolin promoter. The full length of the nucleotide sequence of the codon preference of fHbp Arabidopsis thaliana is synthesized through design, the fHbp Arabidopsis thaliana codon preference nucleotide sequence is subcloned into a plant expression vector with a kidney bean seed specific promoter, and the fHbp gene is integrated into an Arabidopsis thaliana genome through agrobacterium mediation. The fHbp is expressed in seeds, the production process is simplified, the production cost is reduced, and the possibility of producing the oral vaccine fHbp in large quantities by plants is realized.

Description

An expression system of meningococcal fHbp protein

technical field

The invention belongs to the technical field of genetic engineering, in particular to an expression system of meningococcal fHbp protein.

Background technique

Meningococcal disease is a serious global public health problem due to its high morbidity and mortality. Almost every year there are more than 500,000 cases of invasive meningitis disease worldwide, of which 60,000 suffer from severe sequelae. 2% of children born in developing countries die of meningitis before the age of 5. Almost all meningococcal cases in humans are caused by serotypes A, B, C, W135 and Y. The tetravalent capsular polysaccharide vaccine developed by Sanofi-Pasteur for A, C, W135 and Y proved to be effective against these four serotypes. The capsular polysaccharide of group B is homologous to the neuronal cell adhesion molecule in a few mature tissues of humans. Therefore, immunity cannot be induced. However, a vaccine developed by Novartis using "reverse vaccinology" containing four outer membrane protein antigens to prevent meningococcal group B has entered Phase III clinical trials, and it has been proved that the immunogenicity of fHbp is better than other outer membrane proteins. At present, the genetically engineered vaccine of fHbp is still expressed by prokaryotic expression system. However, the high cost and the problem of easily containing heat sources in the products cannot meet the needs of society, and a new expression system is urgently needed.

The rise of plant bioreactors has shown great technical advantages in low-cost large-scale production of genetically engineered vaccines, simplified post-purification processes, and products that do not contain pyrogens, and have broad application prospects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an expression system of meningococcal fHbp protein.

The present invention is specifically realized through the following technical solutions:

An expression system of meningococcal fHbp protein, said expression system comprising fHbp antigen gene and carrier pCAMBIA1301, said expression system is pCAMBIA-fHbp.

The expression system includes a promoter capable of efficiently expressing the fHbp antigen gene in plants, and the promoter is a β-phaseolin promoter.

The nucleotide sequence of the β-phaseolin promoter is shown in SEQ ID NO.1.

The expression system includes a selectable marker, and the selectable marker is Bar gene.

The application of the above-mentioned β-phaseolin promoter of the present invention in improving the high-efficiency expression of the fHbp antigen gene in plants also falls within the protection scope of the present invention.

The application of the described Bar gene in screening the expression system pCAMBIA-fHbp also falls within the protection scope of the present invention.

Another method, the present invention also provides a method for preparing the above-mentioned expression system, which specifically includes the following content: subcloning the antigenic protein gene comprising fHbp of the present invention into a vector that can efficiently express the gene, which will increase the expression level. The seed-specific promoter of , and a gene that can screen out transformed plants are introduced into the expression system.

The promoter introduced by the expression system is β-phaseolin promoter, the selection marker is Bar gene, and the vector is pCAMBIA1301.

In a specific embodiment of the present invention, the expression cassette of the fHbp gene and the expression cassette of the Bar gene of the above construct can be randomly integrated into the plant genome by the Agrobacterium infection method, so that the plant can produce the fHbp antigen protein.

In a specific embodiment of the present invention, Bar gene-specific primers are designed to perform PCR detection on plants that are positive for herbicide screening, and "false positive" plants that are negative in PCR detection but resistant to herbicides are removed.

Preferably, PCR detection of the target gene of fHbp is performed on the "Bar gene positive plants" screened by PCR detection of Bar gene. Plants in which only the Bar gene was integrated into the plant genome and the fHbp was not integrated into the plant genome during the transformation were removed. Through two rounds of PCR detection, it was confirmed that all the screened Arabidopsis thaliana contained the fHbp target gene and the Bar selection marker gene.

Since there is currently no commercially available fHbp antibody for detecting the expression level of fHbp protein, the integration mechanism of Agrobacterium is to integrate the T-DNA region containing the target gene and the expression cassette of the selection marker into the plant genome. Therefore, in the present invention, the expression level of the target gene fHbp gene is indirectly determined by detecting the expression level of the screening marker Bar gene. In the plants screened by PCR in the present invention, the expression level of the Bar gene is detected by the Bar gene colloidal gold test strip. The concentration of the sample was diluted 10 times, and the test strip was tested. The result was still positive, which was determined to be Arabidopsis thaliana with high Bar gene expression, which indirectly proved the high expression of the target gene fHbp.

The integration of foreign genes mediated by Agrobacterium is multi-site and random integration, and the genetic factors will be separated when plants are inherited. Since the foreign genes are not single copies in plants, the foreign genes will not be stably inherited with the genome. In the present invention, the copy of the exogenous gene is detected by Southern-Blot to screen out the high-expression plants that can stably inherit.

The beneficial effects of the present invention are:

The invention first makes a breakthrough in the expression vector, replacing the constitutive promoter with the seed-specific promoter, so that the protein is specifically expressed in the seed and the expression amount of the protein is increased. The Agrobacterium-mediated Arabidopsis infection method is a unique technology platform. This shows great advantages in many aspects, such as reducing production costs, simplifying production processes and improving product safety.

Description of drawings

Fig. 1 is the restriction enzyme digestion identification of expression vector p1301-phas-fHbp;

Figure 2 shows the PCR detection results of the transformed Arabidopsis Bar gene: M: DL100 DNA marker (Trans Gen); M: p1301-phas-fHbp; WT: untransformed Arabidopsis genomic DNA; 1-21: transformed Arabidopsis;

Figure 3 is the PCR detection result of transformed Arabidopsis fHbp; M: DL2000 DNA marker (TAKARA); P: p1301-phas-fHbp; WT: untransformed Arabidopsis genomic DNA; 1-23: transformed Arabidopsis;

Figure 4 is the result of the test strip detecting Bar gene expression; Left: Bar test strip detection result; Right: Bar test strip detection result after diluting 10 times;

Figure 5 is the result of SDS-PAGE electrophoresis of the target protein fHbp; M: protein low marker (TAKARA); WT: untransformed Arabidopsis; 1-8: transformed Arabidopsis;

Figure 6 shows the results of Southern-Blot detection of plants with high expression of target protein fHbp; M: Trans 15K DNA marker; +: p1301-phas-fHbp; -: untransformed Arabidopsis; 01-06: plants with high fHbp expression;

Figure 7 shows the results of SDS-PAGE after purification of fHbp high-expressing plants; M: Protein Molecular WeightMarker (Low); 1: untransformed Arabidopsis total protein; 2: transformed Arabidopsis total protein; 3: purified fHbp protein;

Figure 8 is the titer of serum IgG detected by ELISA: the control group is PBS buffer solution; the experimental group is fHbp/Freund's adjuvant preparation.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Cloning of fHbp antigen gene and construction of plant expression vector

The MenB standard strain MC58 was streaked on chocolate solid medium. Incubate for 36-48hr at 37°C, 5% _CO2 . A single colony with a diameter of 1.0-1.5 mm was picked and resuspended in PBS, and the whole genome of MC58 was extracted using Universal Genomic DNA Extraction Kit. According to the known fHbp sequence, Primer Premier5.0 was used to design two specific primers upstream and downstream of fHbp and introduced NcoI and HindIII restriction sites at the 5' and 3'. The primer sequences are:

Upstream primer: 5'ATCGT CCATGG GACATCATCATCATCATC 3';

Downstream primer: 5' AAGCTT AATTAGCCATAGAAATAGCAGC 3'.

The fHbp fragment was amplified and ligated into pEASY-T1. The vector was named pEASY-T1-fHbp.

The pEASY-T1-fHbp and p1301-phas were digested by Nco I and Hind III enzymes, respectively, and the digestion effect was detected by 1% agarose gel electrophoresis, and the small fragment of pUC57-fHbp and the large fragment of p1301-phas were recovered respectively. The recovered product was ligated with T4 DNA ligase at ₁₆ °C overnight. The ligation product was transformed into E. coli and plated on LB solid medium containing ampicillin (100 μg/mL). The resistant monoclonal colonies were picked on solid medium for PCR and identification by Nco I and Hind III digestion. The digestion results are shown in Figure 1.

PCR reaction system:

Enzyme cleavage reaction system:

Ligation reaction system:

Example 2 Transformation of Agrobacterium with plant expression vector

1) Preparation of Agrobacterium EHA105 competent cells

A single colony of Agrobacterium EHA105 was inoculated into 5 mL YEP liquid medium, and cultured overnight at 28°C and 220 rpm. Transfer 2 mL of overnight cultured bacterial solution into 50 mL YEP liquid medium, cultivate at 28 °C at 220 rpm until OD ₆₀₀ is about 0.5, ice bath for 30 min, centrifuge at 5000 rpm at 4 °C for 5 min, discard the supernatant, add 700 μL 50 mmol/L CaCl ₂ and 300 μL 80 Resuspend the cells in % glycerol, aliquot 100 μL per tube, and store at -80°C.

2) Transform Agrobacterium

Take 20ng of purified plasmid pCAMBIA-fHbp, add it to 100μL of Agrobacterium competent, mix well, ice bath for 30min, transfer to liquid nitrogen for 5min, quickly incubate at 37°C for 5min, add 800μL YEP liquid medium, 28°C 220rpm Incubate for 4-5h. The bacterial liquid was transferred to solid YEP medium containing 50 mg/L kanamycin, spread on the entire plate, and cultured at 28°C for 1-2 days.

3) Screening and identification of transformed Agrobacterium

The resistant colonies were screened in 5 mL of YEP liquid medium containing 50 mg/L kanamycin, cultured at 28°C and 220 rpm for 1 day, centrifuged, and the plasmid was extracted, and the size of the fragment was detected by enzyme digestion and electrophoresis.

Example 3 Agrobacterium-mediated transformation and screening of transformed plants for herbicide resistance

1) Preparation of Agrobacterium engineering bacteria

Take the Agrobacterium strain with the expression vector, and add it to the YEP liquid medium containing 50 mg/L kanamycin and 25 mg/L rifampicin at a ratio of 1:200 to the medium at 28°C. Cultivated at 220 rpm until OD ₆₀₀ was 0.5-0.6, and centrifuged at 5000 rpm for 5 min to collect bacterial cells. The collected bacteria were resuspended in the infection solution, adjusted to OD ₆₀₀ of 1.0-1.2, and used for later use.

2) Transformation of Arabidopsis

A. Soak the whole plant of Arabidopsis thaliana that has been bolted and contain flower buds in Agrobacterium solution for 5 minutes, take out the Arabidopsis thaliana, and place it in the dark for 24 hours;

B. After 2-3 weeks of infection, collect yellow seed pods;

C. Evenly sow the collected seeds into flowerpots, when two true leaves grow, spray 1% glufosinate for screening, and spray 3 times in total, with an interval of 5 days each time.

Example 4 Molecular biological detection of transformed plants

1) PCR detection of fHbp gene and Bar gene of transformed plants

Take 0.05g Arabidopsis leaves and grind them in liquid nitrogen, add 1mL CTAB extraction buffer (75mM 1M Tris-HCl (pH8.0), 100mM 0.5M EDTA (pH8.0), 1.05M NaCl, 0.75% 40K PVP, Add water to volume to 1L), incubate at 65°C for 30min, centrifuge at 12000rpm for 10min, transfer the supernatant to a new centrifuge tube, add an equal volume of phenol/chloroform/isoamyl alcohol (volume ratio 25:24:1) and mix well , 12000rpm centrifugation for 10min. The supernatant was transferred to a new centrifuge tube, an equal volume of chloroform was added to mix well, and centrifugation was performed at 12,000 rpm for 10 min. Transfer the supernatant to a new centrifuge tube, add twice the volume of absolute ethanol, and precipitate DNA at -20°C. After 30 minutes, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, dry the precipitate, and add ddH ₂ O to redissolve for later use. Using the total DNA of the resistant plants as a template, the total DNA of the transformed Arabidopsis thaliana was used as a negative control, and the pUC57-fHbp plasmid was used as a positive control, and specific primers were designed according to fHbp. The two primers are:

Upstream primer: 5'ATCGTCCATGGGACATCATCATCATCAT 3';

Downstream primer: 5'AAGCTTAATTAGCCATAGAAATAGCAG 3'.

Specific primers are designed according to the Bar gene, and the two primers are:

Upstream primer: 5'TCAAATCTCGGTGACGGGCAGGA 3';

Downstream primer: 5'ATGAGCCCAGAACGACGCCCGGC 3'.

Amplification products were detected by 1% agarose gel electrophoresis. Amplification of fHbp gene reaction program: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s; annealing at 56°C for 30s; extension at 72°C for 1min20s; 30 cycles; extension at 72°C for 7min; termination of the reaction at 4°C. The test results are shown in Figure 2.

PCR reaction system:

Bar gene PCR reaction program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30s; annealing at 56°C for 30s; extension at 72°C for 50s; 30 cycles; extension at 72°C for 7 min; termination at 4°C. The test results are shown in Figure 3.

PCR reaction system:

2) Detection of plants with high expression of Bar gene

Use Bar test strips to detect the plants that are positive for the target gene by PCR, and detect the expression level of Bar gene according to the instructions of the Bar gene detection kit. After diluting the positive result of the Bar gene test strip by 10 times, recheck with the Bar test strip. Plants that were still positive were retained. The test results are shown in Figure 4.

3) Expression of antigenic fHbp protein

Weigh 0.1 g of seed pods and extract total protein with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ (pH 7.4), add water to volume to 1 L), and the solid-liquid ratio is 1 : 3. The extraction temperature was 4°C, 10000rpm, and centrifugation for 20min. The supernatant is the electrophoresis total protein sample.

Recipe for 12% separating gel (5mL):

Recipe for 5% Stacking Gel (2mL):

Prepare the separating gel and stacking gel according to the above recipe, mix the protein with loading buffer 5:1, take a boiling water bath for 10 min, and take 10 μL for SDS-PAGE detection of protein fHbp. The results are shown in Figure 5.

4) Detection of Southern-Blot of transformed plants

Take 1 g of sample into a suitable centrifuge tube and triturate in liquid nitrogen. Add 5mL of CTAB extraction buffer (75mM 1M Tris-HCl (pH8.0), 100mM 0.5M EDTA (pH8.0), 1.05M NaCl, 0.75% 40K PVP, add water to volume to 1L), incubate at 65°C for 45min, during Mix well 2-3 times. Take out from the water bath and place at room temperature, centrifuge at 13,000 rpm for 10 min, and transfer the supernatant to a new centrifuge tube. Add an equal volume of chloroform, invert and mix for 2-3 min. Centrifuge at 13000rpm for 10min. Repeat the above operation until the supernatant is colorless. Add 2 volumes of absolute ethanol to precipitate DNA. Centrifuge at 13000rpm for 5-7min. The supernatant was discarded and the pellet was dried. Add 100 μL of ddH ₂ O to redissolve the pellet. Select appropriate restriction enzymes to digest the extracted plant DNA. Enzyme cleavage system:

After enzyme digestion, invert and mix, add 1/10 volume of 3M NaAc (pH 5.2) and 2 times volume of absolute ethanol, and invert and mix. Centrifuge at 13,000 rpm for 2 min, discard the supernatant, and dry the precipitated DNA. Add 40 μL of ddH ₂ O to redissolve the pellet. The positive control was pCAMBIA1301-fHbp, and the negative control was non-transformed Arabidopsis DNA. DNA was separated in a 0.8% agarose gel at 50V. Detected according to Southern-Blot kit. The results are shown in Figure 6.

Example 5 Separation, purification and content determination of fHbp

1) Extraction of total protein from Arabidopsis seed pods

The seeds were placed in a mortar, ground into powder with liquid nitrogen, added with 1 volume of PBS extract, shaken to homogenize, placed on ice for 30 min, and mixed by inversion every 5 min. The mixture was centrifuged at 10,000 g at 4°C for 40 min in a centrifuge. Aspirate the supernatant and repeat the centrifugation once. Aspirate the supernatant. The OD ₅₉₅ value was measured according to the method above, and the concentration of total protein was calculated.

2) Ni-NTA purification of fHbp

The extracted total plant protein was filtered through a 0.22 μm filter. 10 mL of total plant protein was taken each time and poured onto a Ni-NTA affinity chromatography column equilibrated with Binding Buffer (300 mM NaCl, 50 mM NaH ₂ PO ₄ , 10 mM imidazole, 10 mM Tris base, pH 8.0). The flow rate of the effluent was controlled at 1 mL·min ^-1 by a peristaltic pump, and the effluent was collected. Wash with WashBuffer (20mmol·L ^-1 NaH ₂ P0 ₄ , 500mmol·L ^-1 NaCl, pH7.4, 20mmol·L imidazole) for about 5 column volumes, and then use Elution Buffer (20mmol·L ^-1 NaH ₂ P0 ₄ , 500 mmol·L ^-1 NaCl, pH 7.4, 100 mmol·L (imidazole) eluted fHbp from the column. The results are shown in Figure 7.

3) Determination of purified fHbp concentration

Take 10 μL of protein standard (BSA 5 mg·mL ^-1 ) and add PBS to dilute to 100 μL, that is, the concentration is 0.5 mg·mL ^-1 . The diluted standards were added to 96-well plates at 0, 2, 4, 6, 8, 12, 16, and 20 μL, and PBS was added to make up all the standards to 20 μL. Add purified protein to 96-well plate. Add 200 μL of Bradford staining solution to each well, gently mix with a pipette, place at room temperature for 3-5 min, and measure the absorbance at A ₅₉₅ with a microplate reader. The protein concentration was calculated according to the standard curve, and the protein concentration was about 3.437 mg·mL ^-1 .

Example 6 Identification of the immunological activity of fHbp subunit vaccines

1) Preparation of immune preparations

Take the purified fHbp protein, add PBS to adjust its concentration to 1.000 μg·μL ^-1 , and prepare an emulsifier with 1:1 Freund's complete adjuvant and incomplete Freund's adjuvant for later use.

2) Animal immunity test

Twenty 6-week-old female BALB/C mice were randomly divided into 2 groups, namely the experimental group and the control group (10 mice in each group), and the prepared immune preparations were injected by intraperitoneal injection at 0, 15, and 30 days, respectively. The immunization injection was fHbp/Freund's complete adjuvant, and the booster immunization injection was fHbp/Freund's incomplete adjuvant. Each animal in the experimental group was injected with 0.3 mL (containing 20 μg of fHbp protein), and each animal in the control group was injected with the same amount of PBS solution.

3) Obtaining animal serum

Before immunization, and 10 days after primary immunization, 25 days and 40 days after booster immunization, mice were tail-docked and bled, and serum was separated and stored frozen at -80°C.

4) ELISA detection of fHbp antiserum

A 96-well microtiter plate was coated with purified fHbp at a concentration of 20 μg·mL ^-1 . Wash the plate 3 times. The cells were blocked with PBST containing 3% BSA for 2 hr, and the blocking solution was removed. The serum obtained at 10, 25, and 40 days was added to set a negative control, namely, the total protein of non-transformed Arabidopsis thaliana seeds, and the reaction was performed at 37°C for 2 hr, and the plate was washed three times. Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG diluted 5000 times in the blocking solution was added to remove the antibody solution. Add 100 μL of the substrate o-phenylenediamine (OPD) to develop color in the dark. The reaction was stopped by adding 50 μL of 2M sulfuric acid to each well. The absorbance of the sample at 492 nm with a microplate reader. The absorbance value of the serum sample was more than 2 times that of the negative control, and it was judged as a positive result. The antibody titer was expressed as the reciprocal of the highest dilution to obtain a positive result, and the average of the A ₄₉₂ absorbance values of 10 mouse serum samples expressed the antibody titer at that time. The results are shown in Figure 8.

5) Preparation of MenB (strain number 29315) strain suspension

MenB was inoculated on chocolate solid medium, 37°C, 5% CO ₂ , and cultured for 36-48 hr. Pick a single colony. The cells were cultured on chocolate liquid medium to an OD ₆₀₀ of 0.5, and the cells were collected and resuspended in PBS buffer to a final concentration of 5× ^{10 3} cfu·mL ^-1 .

6) Immune effect protection of fHbp on MenB (strain number 29315) strain

A total of 20 mice (10 experimental groups, 10 control groups) after 40 days of intraperitoneal immunization with fHbp and PBS were selected, injected with 100 μL of bacterial solution, and the mortality of the mice was observed 72 hours later. The following formula was used to calculate the immune protection rate (RPS): RPS=100×(1-death percentage of mice in immunization group)/(death percentage of mice in control group). Therefore, the recombinant fHbp subunit vaccine can protect mice against MenB infection by 80%.

Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principle and spirit of the invention Variations, the scope of the invention is defined by the appended claims and their equivalents.

sequence listing

<110> Wenzhou University

<120> An expression system of meningococcal fHbp protein

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Claims (3)

1. An expression system of meningococcal fHbp protein is characterized by comprising an fHbp antigen gene, a vector pCAMBIA1301 and a promoter capable of enabling the fHbp antigen gene to be efficiently expressed in a plant, wherein the promoter is a beta-phaseolin promoter, the expression system is pCAMBIA-fHbp, and the nucleotide sequence of the beta-phaseolin promoter is shown in SEQ ID No. 1;

by streaking MenB Standard strain MC58 on chocolate solid Medium at 37 deg.C, 5% CO₂Culturing for 36-48hr, picking single colony with diameter of 1.0-1.5mm, re-suspending with PBS, extracting MC58 whole genome with Universal genomic DNA Extraction Kit, designing two specific primers at the upstream and downstream of fHbp according to known fHbp sequence and introducing NcoI and HindIII enzyme cutting sites at 5 'and 3', respectively:

an upstream primer: 5'ATCGTCCATGGGACATCATCATCATCATC 3';

a downstream primer: 5'AAGCTTAATTAGCCATAGAAATAGCAGC 3' of the formula I,

amplifying a fHbp fragment and connecting the fHbp fragment into pEASY-T1, wherein the carrier is named as pEASY-T1-fHbp, and pEASY-T1-fHbp and p1301-phas are subjected to double enzyme digestion, recovering a small fragment of pUC57-fHbp and a large fragment of p1301-phas, and connecting to obtain the plasmid pCAMBIA-fHbp.

2. The meningococcal fHbp protein expression system of claim 1, comprising a selection marker, wherein the selection marker is the Bar gene.

3. Use of the promoter of claim 1 for increasing the high expression of the fHbp antigen gene in plants.

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