CN117126881A - Preparation method of rabies virus antigen - Google Patents

Abstract

The invention discloses a preparation method of rabies virus antigen, which comprises the steps of transferring rabies virus antigen Pcl genes into tobacco, screening to obtain transgenic tobacco capable of expressing the rabies virus antigen, and obtaining rabies virus antigen proteins from the transgenic tobacco, wherein the nucleotide sequence of the rabies virus antigen Pcl genes is shown as SEQ ID NO. 1; research results show that the transgenic tobacco plant obtained by the invention can synthesize rabies virus antigen, can reduce the production cost of the rabies virus antigen, improves the safety of antigen production, has the advantages of high efficiency, environmental protection and the like, and the synthesized rabies virus antigen can stimulate the mice to produce rabies antibodies in vivo, thereby providing a new way for obtaining the rabies virus antigen.

Description

Preparation method of rabies virus antigen

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for heterologously synthesizing rabies virus antigen in tobacco.

Background

Rabies (Rabies) is a nervous system infectious disease caused by Rabies virus (Rabies), and the mortality rate of the infectious disease is almost 100% after the infectious disease is caused, which is a serious threat to human life safety. There is currently no therapeutic method and drug for rabies, and the most effective method for preventing rabies occurrence and transmission is still to vaccinate rabies. The rabies vaccines currently used are mainly of three types: an inactivated vaccine cultured by animal brain tissue, an inactivated vaccine cultured by duck embryo and chicken embryo cells, and an inactivated vaccine cultured by primary cells of the kidney of a mice and passage cells of Vero. Although the traditional methods for preparing rabies vaccine are dominant in rabies prevention and control, the production cost is high, the immunization period is short, and the risks of virus escape and the like in the vaccine preparation process exist. Therefore, it is of great importance to find new methods for stable, safe and efficient vaccine production. Hitherto, antigens of rabies virus have been expressed in different hosts, such as E.coli, silkworm, etc., respectively, but these expression hosts have respective disadvantages, such as antigen expressed by silkworm, possibly due to the presence of some proteins with higher homology between the silkworm body and the immunized animal, which may generate some nonspecific immune responses.

In recent years, with rapid development of plant genetic engineering techniques, a method for synthesizing an exogenous target product using a plant as a host has become possible. Plant expression systems are more biosafety than microbial or animal expression systems. The plant can correctly express genes which are not derived from plants, the activity of the produced protein is high, compared with the post-modification capability of the protein is relatively weak when microorganisms such as yeast, escherichia coli and the like express exogenous proteins, and the activity of the protein can be influenced.

In the invention, tobacco is adopted as an expression host of rabies virus antigen, and the method has a plurality of advantages: the genetic background of the tobacco is clear, and a mature genetic operation system is provided; the tobacco can carry out photosynthesis, the biomass is large, and the expression cost of the exogenous protein can be reduced; because humans and livestock are not habitually used to consume tobacco, the risk of transfer of transgenic crops in the food chain is greatly reduced. At present, the synthesis of rabies virus antigen by using a plant bioreactor has not been reported in the literature.

Disclosure of Invention

The invention provides a preparation method of rabies virus antigen, which comprises the steps of transferring rabies virus antigen Pcl genes into tobacco, screening to obtain transgenic tobacco capable of expressing the rabies virus antigen, and obtaining rabies virus antigen protein from the transgenic tobacco, wherein the nucleotide sequence of the rabies virus antigen Pcl genes is shown as SEQ ID NO. 1.

The invention is realized by the following technical scheme:

1. connecting rabies glycoprotein Pcl genes to pCAMBIA2300s plant expression vectors, transforming agrobacterium, and screening positive monoclonal colonies by PCR;

2. the tobacco leaves are impregnated with positive agrobacterium containing the target gene, and the transgenic tobacco plants containing the Pcl gene are obtained through plant regeneration;

3. extracting total RNA of positive transgenic tobacco screened from DNA level, reverse transcribing the total RNA into cDNA, and carrying out RT-PCR to determine whether the Pcl gene is expressed;

4. extracting total protein of transgenic tobacco, performing Western-blot detection, and determining whether positive transgenic tobacco plants can synthesize PCL protein;

5. mice were divided into a blank group, which was intraperitoneally injected with physiological saline, and an experimental group, which was intraperitoneally injected with PCL protein extracted and purified from tobacco; the blank group and the experiment group are respectively injected with normal saline and PCL protein three times at intervals of one week;

6. and extracting serum proteins of mice in the blank group and the experimental group, and carrying out Western-blot detection to determine whether corresponding rabies virus antibodies are generated in the mice.

The invention has the advantages and technical effects that:

based on the characteristics of easy propagation and quick growth of tobacco, the rabies virus antigen Pcl gene is transferred into the tobacco to obtain the transgenic tobacco capable of expressing the rabies virus antigen, and research results show that the transgenic tobacco obtained by the invention can synthesize the rabies virus antigen with immune activity, thereby providing a new thought and a new way for preparing rabies virus antigen vaccine.

Drawings

FIG. 1 shows the result of PCR product electrophoresis detection of Pcl gene, wherein M is DNA marker DL2502;

FIG. 2 is a PCR electrophoresis of pCAMBIA2300s-Pcl vector transferred into Agrobacterium LBA4404, wherein 1-6: positive agrobacterium strain, M: DNA marker DL2502, +: positive control, -: a negative control;

FIG. 3 is a DNA level PCR electrophoretogram in transgenic tobacco, wherein M: DNA marker DL2502, WT wild tobacco, T1-T6 transgenic tobacco, "+" cDNA template;

FIG. 4 is a PCR electrophoretogram of RNA level in transgenic tobacco, wherein M: DNAmaroker DL2502, WT wild tobacco, T1-T6 transgenic tobacco, "+" cDNA template;

FIG. 5 is a diagram showing the detection of protein WB in transgenic tobacco, wherein 6 is wild-type tobacco and 1-5 are transgenic tobacco.

FIG. 6 is a diagram showing the WB detection of rabies virus antibodies in mouse serum, wherein 1-5 are experimental group mice.

Detailed Description

The present invention will be described in further detail by way of examples, but the scope of the invention is not limited to the above description, and the methods in the examples are all conventional methods unless otherwise specified, and the reagents used are all conventional commercial reagents or reagents prepared by conventional methods unless otherwise specified.

Example 1: construction of Pcl Gene overexpression vector

According to the nucleic acid sequence of the Pcl gene obtained by previous separation in the subject group, the primer with the homology arm is designed by using CE Design software in combination with the insertion position of the Pcl gene in a plant expression vector pCAMBIA2300 s; the upstream primer of Pcl is a catalystattacgaattcgagctcATGTGGAGGCTAATGACAGCCA, the downstream primer is cttgcatgcctgcaggtcgacTCAGACGCTTTTAGGTGGTAATCG; extracting total RNA from mouse (C57) serum, synthesizing mouse cDNA by reverse transcription, cloning Pcl gene by using cDNA first chain as template and high-fidelity enzyme, and PCR reaction system of 50 μL including Prime STAR MAX Premix μL, upstream primer 1 μL, downstream primer 1 μ L, cDNA 1 μL, ddH ₂ O22 μl; the PCR reaction conditions were: 98 ℃ for 3min;98℃for 10s,60℃for 15s,72℃for 15s,35cycles;72 ℃ for 5min; after the PCR was completed, the result of agarose gel electrophoresis was shown in FIG. 1, and after confirming that the amplification was successful, the target band was recovered, using the SanPrep column type DNA gel recovery kit (Shanghai Biotechnology); taking 1 mu L of recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 ℃ for standby.

Linearizing plant expression vector pCAMBIA2300s with restriction enzyme in 15 μL pCAMBIA2300s plasmid, 7.5 μL 10 xT buffer, 7.5 μL BSA, 2.5 μL NdeI, 2.5 μL SmaI, 15 μL ddH ₂ O, after mixing evenly, centrifuging for a short time, and placing the mixture at 37 ℃ for enzyme digestion for 3.5 hours; the enzyme-digested products are spotted on agarose gel for electrophoresis, then the linearized pCAMBIA2300s carrier fragment is subjected to gel recovery, and a SanPrep column type DNA gel recovery kit (Shanghai) is used in the whole process; taking 1 mu L of recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 ℃ for standby. In the case of homologous recombination, clonExpressMultiS One Step Cloning Kit was used for assembly, and in the case of homologous recombination, assembly was performed according to the instructions of homologous recombination, and then the amounts of the respective components were calculated according to the instructions of recombination based on the concentrations of the insert and the vector. Finally, adding each component into a PCR reaction tube on ice, placing the PCR reaction tube at 37 ℃ for reaction for 0.5h, after assembling, transforming the recombinant expression plasmid into host strain DH5 alpha competent cells, coating a plate, wherein the plate is an LB solid medium added with kanamycin, culturing in a dark way for 12-15h in a 37 ℃ incubator, then selecting single bacterial colony on the plate, performing bacterial liquid PCR amplification, detecting positive clone, sending to a sequencing company for detection, further finally confirming, and performing seed preservation after the assembling is successful, thus obtaining the large intestine containing Pcl geneAnd (3) bacillus.

Example 2: transformation of Agrobacterium cells

The pCAMBIA2300s-Pcl plasmid in E.coli of example 1 was extracted and purified using a plasmid extraction kit; competent cells of Agrobacterium LBA4404 strain were prepared and packed in 1.5mL centrifuge tubes, 50. Mu.L per tube, snap frozen with liquid nitrogen and stored at-80℃for further use. Transferring the constructed plant expression vector pCAMBIA2300s-Pcl plasmid into the prepared agrobacterium LBA4404 competent cells by adopting a liquid nitrogen freeze thawing method; the operation steps are as follows: adding 3 μg of pCAMBIA2300s-Pcl plasmid into a centrifuge tube containing 50 μl of competent cells, mixing, ice-bathing for 30min, quick-freezing in liquid nitrogen for 5min, and water-bathing at 37deg.C for 5min, and ice-bathing for 2min; the transformed Agrobacterium competent cells were transferred to LB liquid medium (without any antibiotics) and cultured with shaking at 28℃and 200rpm for 4 hours to resuscitate the cells, which were then plated on LB solid medium containing 50mg/L kanamycin and 25mg/L rifampicin and cultured upside down in an incubator at 28℃for about 48 hours. And (3) selecting monoclonal, shaking, performing PCR detection on bacterial liquid, and screening agrobacterium containing recombinant vector pCAMBIA2300s-Pcl plasmid, wherein the detection result is shown in figure 2.

Example 3: agrobacterium tumefaciens mediated genetic transformation of tobacco plants

The transgenic acceptor in this experiment was tobacco, tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water and then with 0.1% HgCl ₂ Soaking for 8min, then washing with sterile water for several times, seeding on 1/2MS culture medium, dark culturing at 28deg.C for 6d, transferring to illumination incubator (25deg.C, 16h/d illumination), and subculturing with 1/2MS culture medium once per month. Taking out the stored agrobacterium LBA4404 strain containing pCAMBIA2300s-Pcl plasmid from a refrigerator at-80 ℃, inoculating the agrobacterium containing the target gene into LB liquid medium containing 50mg/L kanamycin and 20mg/L rifampicin, and culturing at 28 ℃ and 200rpm until the medium is turbid; sucking 1mL of turbid mixed bacterial liquid onto LB solid medium containing 50mg/L kanamycin and 20mg/L rifampicin, and culturing at 28 ℃ for 48h; the agrobacterium on the LB solid medium is scraped and inoculated into MGL liquid medium added with 20mg/L acetosyringone, and the agrobacterium is activated by shaking culture for 2-3 hours at 28 ℃. Will be 1cm ² Putting tobacco leaves with the size into MGL liquid culture medium containing target gene agrobacterium tumefaciens, and culturing at 120rpm and 28 ℃ for 20min; after the dip dyeing is finished, the bacterial liquid of the extracted tobacco leaves is sucked by sterile filter paper, and then transferred into an MS solid culture medium containing 40mg/L acetosyringone, and the tobacco leaves are subjected to dark culture at 25 ℃ for 48 hours; transferring tobacco leaves after co-culture to a screening culture medium (MS+0.5 mg/L6-BA+0.1 mg/L NAA+50mg/L kanamycin+300 mg/L cephalosporin) for selective culture, transferring a culture flask to an illumination incubator for culture (25 ℃,16h/d illumination and 8h/d darkness) during screening culture, and carrying out secondary culture on tobacco leaves after differentiation and budding by using an MS culture medium containing 50mg/L kanamycin and 300mg/L cephalosporin, wherein the regeneration plants are required to be further screened due to higher callus differentiation rate of the tobacco, transferring tobacco regeneration seedlings to the MS culture medium containing 50mg/L kanamycin for rooting, and finally selecting the regeneration seedlings with better rooting for further detection.

Example 4: detection of Pcl gene in transgenic tobacco plants

(1) Transgenic tobacco plant genomic DNA level detection

Extracting DNA of transgenic tobacco leaves by adopting a CTAB method, taking genomic DNA of transgenic plants as a template, and carrying out PCR by using specific primers for amplifying genes Pcl; the PCR reaction system was 2. Mu.L of DNA, 0.4. Mu.L of upstream primer (10. Mu.M), 0.4. Mu.L of downstream primer (10. Mu.M), 10. Mu.L of PCR Mix, 7.2. Mu.L of ddH ₂ O; PCR reaction conditions: 94 ℃ for 5min;94℃30s,60℃90s,72℃1min,32 cycles; 7min at 72 ℃; after the PCR is finished, 10 mu L of the product is used for agarose gel electrophoresis to detect positive transgenic plants, the amplification result of part of tobacco transgenic plants is shown as figure 3, 6 positive plants are obtained by detection on the DNA level, and the result shows that all 6 transgenic tobacco plants are introduced with exogenous DNA and integrated into genome DNA for stable inheritance, and the Pcl transgenic tobacco plant line is obtained by preliminary determination.

(2) Transgenic tobacco plant genomic RNA level detection

Extracting total RNA from young leaves of positive transgenic individual plant and non-transgenic tobacco (wild type) at DNA level, grinding tobacco leaf into powder with liquid nitrogen, transferring into centrifuge tube, collectingExtracting total RNA by guanidine isothiocyanate method, and synthesizing cDNA first strand by using reverse transcriptase M-MLV (promega) and using total RNA as template, wherein the reaction system and operation process are as follows: mu.g of Total RNA was taken and 50ng of oligo (dT), 2. Mu.L of dNTP (2.5 mM each) and DEPC water were added in this order to a reaction volume of 14.5. Mu.L; after mixing, heating and denaturing for 5min at 70 ℃, placing on ice and cooling for 5min, then sequentially adding 4 mu L of 5 xfirst-stand buffer, 0.5 mu L of RNasin (200U) and 1 mu L M-MLV (200U), mixing uniformly and centrifuging for a short time, carrying out warm bath for 1.5h at 42 ℃, taking out, heating for 10min at 70 ℃, and stopping the reaction; the cDNA first strand is synthesized and then stored at-20 ℃ for standby. Using the synthesized first strand cDNA as a template, and carrying out PCR by using a specific primer for amplifying the Pcl gene; the PCR reaction system was 1.5. Mu.L cDNA, 0.4. Mu.L upstream primer (10. Mu.M), 0.4. Mu.L downstream primer (10. Mu.M), 10. Mu.L PCR Mix, 7.7. Mu.L ddH ₂ O; PCR reaction conditions: 94 ℃ for 5min;94℃30s,60℃30s,72℃90s,32 cycles; 7min at 72 ℃; after the PCR was completed, 10. Mu.L was used for agarose gel electrophoresis, and the expression of the transcription level of the Pcl gene in each transgenic individual was analyzed based on the result of the PCR, and after the PCR was completed, 10. Mu.L was used for agarose gel electrophoresis, and the detection results of some individuals were shown in FIG. 4, and the large amount of the expression of the Pcl gene at the transcription level in the T1-T6 transgenic individual was co-detected.

(3) Transgenic tobacco plant protein level detection

A clean mortar was prepared and pre-cooled with liquid nitrogen. The tobacco leaves were ground to a white powder. The powder was transferred to a pre-chilled 2mL centrifuge tube, and pre-chilled protein lysis buffer (prepared according to kit, as-is) was added and lysed at 4 ℃ for 20min. Centrifuging at 4deg.C for 20min at 15000g, and collecting supernatant as tissue total protein extract. The method for determining the protein concentration according to the kit can be used for western blot analysis after determining the protein concentration, and comprises the following specific operation steps: protein denaturation, adding a certain amount of Loading buffer and 5 XSDS-PAGE into each group of protein liquid to dilute the protein, and ensuring that the concentration of each group of protein after dilution is the same. Loading and electrophoresis, adding 1 XSDS-PAGE in an electrophoresis tank, adding protein markers and various histones in gel holes (ensuring that the loading amount is 30-50 mug), and concentrating gel at 80V; the protein bands were separated by running the separation gel at 120V. Transferring to membrane, transferring the gel with protein to polyvinylidene fluoride (PVDF) membrane (soaked and activated in methanol in advance), placing the membrane between clamping plates according to the positive and negative electrodes of the filter paper, adding membrane transferring solution, and transferring to membrane on ice for 120min. Sealing, namely placing the PVDF film into an incubation box, adding sealing liquid, sealing at 80r/min for 2 hours at room temperature. The primary antibody is incubated, the blocking solution is discarded, the primary antibody is added, and the primary antibody is incubated at 4 ℃ and 80r/min overnight. After the secondary antibody is incubated and the primary antibody is recovered, TBST is added, the washing is carried out for 3 times at room temperature of 80r/min, the secondary antibody is added, and the incubation is carried out for 2 hours at room temperature of 80 r/min. Development by a chemiluminescent gel imager, the results of which are shown in FIG. 5, were successfully screened at the protein level to obtain 5 positive tobacco plants.

Example 5: abdominal injection of mice with rabies glycoprotein produced by transgenic tobacco, and Western immunoblotting analysis of mouse serum for rabies antibody production

(1) Purification of rabies glycoprotein

Collecting the whole protein extract of the transgenic tobacco in the example 4, and purifying the whole protein extract to obtain rabies glycoprotein; and taking a Ni-NTA pre-packed column with corresponding specification according to the required load, and flowing out the storage buffer solution by gravity. The column was equilibrated with a Binding/Wash Buffer of twice the column volume and the Buffer was slowly drained off the resin using a flow rate of 0.5-1 mL/min. The protein extract was mixed with Binding/Wash Buffer 1:1 to prepare a sample solution, so that the total volume of the sample solution was twice the column volume. Sample fluid is added to the column and the flow through is collected into a centrifuge tube. If the redundant sample can be loaded again, the binding force between the sample and the filler can be improved by circulating again. The column was washed with twice the column volume Binding/Wash Buffer and the run-through was collected. This procedure was repeated using a new collection tube until the flow-through absorbance at 280nm was near baseline. The histidine-tagged proteins on the column were eluted with twice the column volume of the Elutation Buffer, and this procedure was repeated 2 times, each time the eluate was stored separately until the absorbance of the eluate at 280nm was close to baseline. Eluting the column with 5 volumes of Elution Buffer, balancing the column with 5 volumes of Binding/Wash Buffer, and finally with 5 volumes of ddH ₂ O cleaning the column material to obtain purified rabies glycoprotein, adding 20% ethanol protection solution into the rabies glycoprotein, and storing at 2-8 ℃.

(2) Animal experiment

Mice were divided into a blank group and an experimental group, 8 mice per group; the experimental group was intraperitoneally injected with 100. Mu.g/g PCL protein extracted and purified from tobacco, and the mice were intraperitoneally injected with an equal volume of physiological saline given in a blank group. In the experimental process, the blank group and the experimental group are respectively injected with physiological saline and PCL protein three times, and each time is separated by one week; mice were sacrificed and blood was collected after day 28 of dosing.

Detection of rabies antibody in mice: mouse blood was collected and its proteins were extracted. Conventionally separating serum, namely standing at normal temperature after taking blood, solidifying the blood, centrifuging at 3000rpm for 15min, and taking a supernatant; measuring the concentration of serum protein; diluting to the required concentration by Laemmli buffer; adding a proper amount of loading buffer, and boiling at 100 ℃ for 5min to finish the preparation of the sample. Western Blot detection is carried out, the prepared separating gel is slowly injected into the glass plate along one side of the glass plate, and ddH with proper dosage is added ₂ O or absolute ethyl alcohol sealing glue. And after the prepared separation gel is solidified, starting to load, adding 5-10 mu L of the Marker hole, and the total load of the rest proteins is 30-50 mu g. After the sample loading is completed, the sample is firstly subjected to constant voltage electrophoresis at 80V until the bromophenol blue indicator reaches the junction of the concentrated gel and the separation gel, the sample is changed into a linear sample, the sample is subjected to constant voltage electrophoresis at 120V until the bromophenol blue is electrophoresed to the bottom of the gel, and the time of the process is about 1.5 hours. The gel was removed, the target band was cut according to Marker, rinsed with distilled water, and PVDF membrane of the same size as the SDS-PAGE gel was cut, and the filter paper was of the same size as the fiber pad. Soaking PVDF membrane in methanol for 1min, and soaking in electric transfer buffer solution together with filter paper; sequentially placing a black plate, a fiber pad, filter paper, gel, PVDF membrane, filter paper, a fiber pad and a white plate, clamping the plates, and placing the plates into a wet-transfer electric transfer tank, wherein one side of the black plate faces to a black negative electrode; and filling the film transfer groove with electrotransfer liquid, and starting film transfer. PVDF membrane was soaked in TBST (blocking solution) containing 5% skimmed milk powder and blocked for 2h at room temperature with a shaker. The phosphorylated proteins were blocked with 5% BSA for 2h. The corresponding primary antibody was diluted with TBST containing 5% nonfat dry milk, and PVDF membranes were immersed in the primary antibody incubation solution and incubated overnight at 4 ℃. Diluting HRP-labeled secondary antibody with the same dilution of primary antibody system, soaking PVDF membrane in secondary antibody incubation solution, and incubating at room temperature for 1 hr to obtain the final productAs shown in FIG. 6, rabies virus antibodies were successfully detected in 5 mice.

Claims (2)

1. A preparation method of rabies virus antigen is characterized by comprising the following steps: transferring rabies virus antigen Pcl gene into tobacco, screening to obtain transgenic tobacco capable of expressing rabies virus antigen, and obtaining rabies virus antigen protein from transgenic tobacco, wherein the nucleotide sequence of rabies virus antigen Pcl gene is shown as SEQ ID NO. 1.

2. The method for preparing rabies virus antigen according to claim 1, characterized in that: firstly, constructing a plant expression vector of a rabies virus antigen Pcl gene, and transforming tobacco by using the plant expression vector of the rabies virus antigen Pcl gene.