Bovine herpesvirus 4 type gb protein recombinant expression method and application
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-03-17
Smart Images
Figure CN121673376A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of molecular biology and immunology, specifically to a method for recombinant expression of bovine herpesvirus type 4 gb protein and its application. Background Technology
[0002] Recombinant protein expression and antiserum preparation are important directions in modern molecular biology and immunology research. By recombinantly expressing specific antigen protein fragments and immunizing experimental animals, specific antibodies can be obtained. These antibodies are widely used in disease diagnosis, vaccine development, and molecular detection.
[0003] Currently, common methods for recombinant protein expression and antiserum preparation in related technical fields mainly include: expressing proteins using full-length genes or random fragments, purifying proteins using conventional methods, and immunizing experimental animals to obtain antiserum. However, existing technologies have many defects and shortcomings: In terms of protein expression, when using full-length genes or random fragments for expression, there are often problems such as poor protein solubility and low expression levels; moreover, the strict induction conditions lead to unstable protein expression, which further affects the efficiency of subsequent experiments. In terms of protein purification, the purification methods are unstable, making it difficult to obtain high-purity antigen proteins, which in turn leads to poor immunization effects. Regarding the preparation of antiserum, the titer of serum obtained after animal immunization is not high, and there are large batch-to-batch variations, with a lack of a unified and reliable preparation method; In terms of specificity, there is currently a lack of mature recombinant expression of bovine herpesvirus type 4 gb protein and methods for preparing antiserum. In terms of cost and efficiency, existing technologies require repeated experiments, which are time-consuming and material-intensive, resulting in high costs and low efficiency.
[0004] To address the shortcomings of the existing technologies, there is an urgent need for a technical solution that can efficiently express and purify the target protein, reduce preparation costs, optimize the process, and improve serum specificity and stability. In particular, it is necessary to solve the key technical problem of how to efficiently express and purify bovine herpesvirus type 4 gb protein fragments (104–384aa) to obtain high-titer mouse polyclonal antibodies. Summary of the Invention
[0005] The purpose of this invention is to provide a method for recombinant expression of bovine herpesvirus type 4 gb protein and its application. Based on the above method, this invention obtains a high-titer mouse polyclonal antibody.
[0006] The objective of this invention is achieved through the following technical solution: This invention provides a method for recombinant expression of bovine herpesvirus type 4 gb protein, comprising the following steps: (1) The 104-384aa region of bovine herpesvirus type 4 gb protein was selected as the expression fragment, and the amino acid sequence of the 104-384aa region is shown in SEQ ID NO.2; (2) The expression fragment is constructed into the E. coli expression vector pet22b or pet32a to obtain the recombinant plasmid that is correctly identified by sequencing; (3) The recombinant plasmid is transformed into the expression host, and bacterial cells containing recombinant gb protein are obtained after induction culture; (4) The bacterial cells are broken and separated to obtain a sample containing recombinant gb protein; (5) The sample was purified using Ni-NTA resin to obtain recombinant bovine herpesvirus type 4 gb protein.
[0007] Furthermore, in step (3), the induction culture operation includes: For small-scale expression validation: host bacteria containing the recombinant plasmid are cultured to OD200. 600 After reaching approximately 0.6, add IPTG to a final concentration of 0.5 mM, and incubate at 37°C with shaking for 3 hours. For high-level expression: Culture the host bacteria containing the recombinant plasmid to OD. 600 After reaching approximately 0.6, the temperature was lowered to 30°C, and IPTG was added to a final concentration of 0.5 mM. The mixture was then incubated at 30°C with shaking for 3 hours.
[0008] Furthermore, in step (4), the bacterial cells are broken down by ultrasonic disruption under ice bath conditions. The disruption parameters are: power 300W, ultrasonic for 4s, pause for 4s, and a total of 90 ultrasonic disruptions. After disruption, the cells are centrifuged at 20000g and 4℃ for 30min, and the supernatant is taken as the sample containing the recombinant gb protein.
[0009] Furthermore, the specific operation of purification using Ni-NTA resin is as follows: (1) Equilibrate the chromatography column packed with Ni-NTA resin with 10 times the column bed volume of NTA-0 Buffer; (2) The sample containing recombinant gb protein was added to the chromatography column at a flow rate of 0.5 ml / min; (3) Wash the chromatography column with 10 times the column bed volume of NTA-0 Buffer at a flow rate of 1 ml / min; (4) Perform gradient elution sequentially with 10 column bed volumes of NTA-20 Buffer, NTA-60 Buffer, NTA-200 Buffer, and NTA-500 Buffer at a flow rate of 1 ml / min, and collect the elution peak components; (5) The elution peak components were dialyzed with 1×PBS buffer and concentrated by ultrafiltration to obtain high-purity recombinant bovine herpesvirus type 4 gb protein.
[0010] The present invention also provides a recombinant bovine herpesvirus type 4 gb protein, which is prepared by the recombinant expression method described above. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.2, and the molecular weight is 48.5 kDa.
[0011] The present invention also provides the application of the recombinant bovine herpesvirus type 4 gb protein in the preparation of mouse antiserum.
[0012] Furthermore, the step of preparing mouse antiserum includes: (1) Using the recombinant bovine herpesvirus type 4 gb protein as the antigen, a polystyrene 96-well reaction plate was coated at a concentration of 1 μg / ml and placed at 4°C overnight; (2) After washing and sealing, add serially diluted immune mouse antiserum and incubate at 37°C for 1-2 hours; (3) After washing, add diluted goat anti-mouse IgG-HRP and incubate at 37°C for 1 hour; (4) After washing, the color is developed and the reaction is terminated. The absorbance at a wavelength of 450 nm is measured to determine the antiserum titer.
[0013] Furthermore, in step (2), the starting ratio of the serial dilution is 1:1000, and the dilution ratio of the goat anti-mouse IgG-HRP is 1:10000.
[0014] Furthermore, the titer of the mouse antiserum is not less than 1:128000.
[0015] Furthermore, the purification procedure for the mouse antiserum is as follows: (1) Equilibrate the chromatography column with PBS or TBS buffer, mix the antiserum supernatant with an equal volume of 2×PBS buffer and then load the sample; (2) Wash the chromatography column with PBS buffer until no protein is detected in the effluent, and then elute with 0.1M citric acid or 0.1M glycine as the elution buffer; (3) After adding 2 / 5 volume of 1M Tris to neutralize the elution buffer, concentrate it and switch to 2×PBS buffer containing 0.02% NaN3 and 1mM EDTA, and store it at -20℃.
[0016] Beneficial effects
[0017] This invention precisely selects the 104-384aa region of bovine herpesvirus type 4 gb protein as the expression fragment, combines it with the construction of the pet22b / pet32a E. coli expression vector, and adopts an optimized induction (30℃ + 0.5mM IPTG), disruption (controllable ultrasonic parameters), and Ni-NTA gradient elution purification process. This effectively solves the problems of poor solubility and low expression levels in traditional protein expression. The recombinant gb protein obtained is of uniform molecular weight (approximately 48.5kDa), with stable purity meeting the standards. The operation process is highly controllable, avoiding the consumption of consumables and time in repeated experiments, and significantly improving the efficiency and cost-effectiveness of protein preparation.
[0018] Meanwhile, the invention has significant application value: when the prepared recombinant gb protein is used to prepare mouse antiserum, the resulting antiserum titer is no less than 1:128000, exhibiting high specificity and minimal batch-to-batch variation; the purified antibody purity meets experimental requirements and can stably recognize the target antigen. This recombinant protein and antiserum overcome the shortcomings of existing bovine herpesvirus type 4 gb protein-related reagents, which suffer from insufficient titer and poor specificity, providing a reliable tool for molecular detection, immunoassay, and other research and applications of this virus, thus possessing strong practical value. Attached Figure Description
[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a graph showing the SDS-PAGE detection results of the purified protein in this invention. Figure 2 This is a graph showing the antibody purity results after purification and concentration in this invention. Detailed Implementation
[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] Unless otherwise specified, all chemical reagents, biochemical reagents and materials used in this invention are commercially available.
[0027] The present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the invention can be more easily understood by those skilled in the art, thereby providing a clearer and more definite definition of the scope of protection of the present invention. However, this does not limit the present invention to the scope of the described embodiments. The reagents and raw materials used in the following embodiments are all commercially available, and the test methods without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.
[0028] Example 1: Recombinant Expression of Bovine Herpesvirus Type 4 gb Protein and Preparation of Mouse Antiserum 1.1 Experimental Materials 1.1.1 Main Instruments and Equipment The main instruments and equipment are shown in Table 1 below: Table 1 Main Instruments and Equipment
[0029] 1.1.2 Key Reagents The main reagents are shown in Table 2 below: Table 2 Key Reagents
[0030] All other reagents were prepared in the laboratory.
[0031] 1.2 Antigen Analysis and Expression Vector Construction 1.2.1 Antigen sequence analysis The complete amino acid sequence of bovine herpesvirus type 4 gb protein is shown in SEQ ID NO. 1.
[0032] 1.2.2 Selection of Expression Segments and Construction of Vector The 104-384aa region of bovine herpesvirus type 4 gb protein was selected as the expression fragment. The amino acid sequence of this fragment is shown in SEQ ID NO.2. The above expression fragment was constructed into the pet22b / pet32a Escherichia coli expression vector. After construction, the correctness of the recombinant plasmid was identified by sequencing.
[0033] 1.3 Protein Preparation 1.3.1 Small-scale expression verification (1) Transform the recombinant plasmid that has been correctly identified by sequencing into the expression host; (2) Pick a single colony containing the recombinant plasmid and inoculate it into 3 ml of LB medium containing the corresponding antibiotic, and incubate overnight at 37°C with shaking. (3) Take 30 μl of overnight culture and add it to 3 ml of LB medium containing the corresponding antibiotic. Incubate at 37°C with shaking until OD. 600 ≈0.6; Add glycerol to the remaining overnight culture medium to a volume fraction of 20%, store at -80℃, and use as working seed for later use; (4) Take a portion of the above-mentioned culture liquid as the uninduced control group, and add IPTG inducer to the remaining culture liquid to a final concentration of 0.5mM as the experimental group. Both groups continue to be cultured at 37℃ with shaking for 3h. (5) Take 1 ml of bacterial culture from the experimental group and the control group respectively, centrifuge at 12000g for 30s to collect the bacterial precipitate; (6) Resuspend the bacterial precipitate in 100 μl of 1% SDS solution, mix thoroughly, and heat in a water bath at 100°C for 10 min. (7) Centrifuge at 12000g for 10min, and take the supernatant for SDS-PAGE detection and analysis of protein expression.
[0034] 1.3.2 High-level expression and bacterial rupture detection (1) Take 20 μl of working seed stored at -80℃ and transfer it into 20 ml of liquid LB medium containing the corresponding antibiotic, and culture overnight at 37℃ with shaking. (2) Take 2 ml of the above overnight culture solution and add it to 2000 ml of LB medium containing the corresponding antibiotic. Shake and culture at 37°C until OD600≈0.6, then lower the culture temperature to 30°C. (3) Add IPTG inducer to the culture medium to a final concentration of 0.5 mM, and continue to culture at 30°C with shaking for 3 h; (4) Collect the fermentation broth, centrifuge at 6000g for 10min, and collect the cell precipitate; (5) Resuspend the bacterial precipitate in 40 ml of pre-cooled NTA-0 Buffer and mix thoroughly; (6) Bacteria were disrupted by ultrasound under ice bath conditions. The disruption parameters were: power 300W, ultrasound for 4s, pause for 4s, and a total of 90 ultrasounds. (7) After crushing, centrifuge at 20000g and 4℃ for 30min, and collect the supernatant and precipitate respectively; (8) Take a small amount of supernatant and precipitate sample for SDS-PAGE detection; place the remaining supernatant and precipitate in an environment of 0-7℃ for later use.
[0035] 1.3.3 Protein purification (1) Pack Ni-NTA resin into a chromatography column of appropriate specifications, wash the chromatography column with 10 times the column bed volume of NTA-O Buffer, and perform equilibration treatment; (2) Slowly add the collected supernatant sample to the chromatography column, control the sample loading rate to 0.5 ml / min, and collect the breakthrough fraction at the same time; (3) After loading the sample, rinse the chromatography column with 10 times the column bed volume of NTA-0 Buffer, and control the rinsing flow rate to 1 ml / min; (4) Protein elution was performed using a gradient elution method: 10 column bed volumes of NTA-20 Buffer, NTA-60 Buffer, NTA-200 Buffer and NTA-500 Buffer were used for elution, and the elution flow rate was controlled at 1 ml / min. The eluent corresponding to each elution peak was collected. (5) SDS-PAGE analysis was performed on each elution peak sample (the results are shown in the table below). Figure 1 ), and screen for components that meet the required purity; (6) Pack the qualified components into a dialysis bag and dialyze them with 1×PBS buffer at 4°C, changing the dialysate twice during the process; (7) After dialysis, the dialysis product was concentrated by ultrafiltration at 4°C to obtain high-purity recombinant gb protein (the molecular weight of the pet32a vector expression product was about 48.5 kDa, and it was obtained by Ni column purification under 16°C induction conditions).
[0036] 1.4 Detection of mouse immune and antiserum titers 1.4.1 Antigen Coating Using the purified recombinant gb protein as the antigen, 100 μL of antigen solution was added to each well of a polystyrene 96-well plate at a coating concentration of 1 μg / ml, and the plate was incubated overnight at 4°C.
[0037] 1.4.2 Washing The next day, the coating solution in the wells of the 96-well reaction plate was poured out, and the plate was washed three times with washing solution, discarding the residual liquid after each wash.
[0038] 1.4.3 Closure Add 100 μL of blocking solution to each well and let stand at room temperature for 0.5 h.
[0039] 1.4.4 Washing After sealing, wash three times with detergent, discarding any residual liquid after each wash.
[0040] 1.4.5 Add the sample to be tested (primary antibody) The antiserum from immunized mice was serially diluted with PBS buffer in another 96-well plate (initial dilution ratio 1:1000). Then, 100 μL of the diluted antiserum sample was added to each well of the pre-coated and sealed 96-well plate, with two replicates for each dilution. A negative control (PBS buffer or blank culture medium) and a positive control (known positive sample) were also set up. After capping, the plate was incubated at 37°C for 1-2 hours.
[0041] 1.4.6 Washing After incubation, wash three times with detergent, discarding any residual liquid after each wash.
[0042] 1.4.7 Add enzyme-labeled anti-antibody Goat anti-mouse IgG-HRP was diluted with blocking buffer at a ratio of 1:10000. 100 μL of the diluted enzyme-labeled anti-antibody was added to each well, and the well was capped and incubated in a 37°C incubator for 1 hour.
[0043] 1.4.8 Washing After incubation, wash 5 times with washing solution, then wash 2 times with distilled water, discarding any residual liquid after each wash.
[0044] 1.4.9 Colorimetric properties Add 100 μL of freshly prepared substrate solution to each well and incubate in the dark at room temperature for 5–30 min. Stop the color development process when the reaction solution turns blue.
[0045] 1.4.10 Termination of the reaction and colorimetric analysis Add 50 μL of stop solution to each well; the reaction solution turns yellow. Measure the absorbance of each well at 450 nm using a microplate reader. The highest dilution with a positive reaction is taken as the titer of the antiserum to be tested. The absorbance data for different dilutions of antiserum are shown in Table 3 below (left and right are parallel replicates; blank is the negative control): Table 3 Absorbance data of antiserum at different dilutions
[0046] 1.5 Antibody Purification (1) Wash the column with 10 column bed volumes of PBS buffer (or TBS buffer) to equilibrate it; (2) Centrifuge the antibody-containing antiserum at high speed, take the supernatant and mix it with an equal volume of 2×PBS buffer. After adjusting the pH and ion concentration of the mixture, slowly add it to the equilibrated chromatography column. (3) Wash the chromatography column with PBS buffer at least 10 times the column bed volume until no protein is detected in the eluent; (4) Add 2 column volumes of 0.1M citric acid (pH 2.7) as eluent, clamp the column effluent, let stand for 5 minutes, and collect the eluent. Repeat this elution operation three times. During this process, the OD can be measured. 280 Antibody concentration can be estimated using a elution buffer, or 0.1M glycine (pH 3.0) can be used as the elution buffer. (5) Add 2 / 5 volume of 1M Tris (pH 8.0) solution to the collected eluent for neutralization; (6) Use a Millipore protein concentrate tube to switch the neutralized antibody solution to the required buffer (usually 2×PBS buffer containing 0.02% NaN3 and 1mM EDTA). (7) Concentrate the antibody solution to the required volume and detect the antibody purity by SDS-PAGE (see results below). Figure 2 The molecular weight standards of the protein markers used for detection are 116 kDa, 66.2 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, and 14.4 kDa. (8) Store the purified antibody at -20°C and avoid repeated freezing.
[0047] 1.6 Result Validation (1) SDS-PAGE results showed that the molecular weight of bovine herpesvirus type 4 gb protein (104-384aa) obtained by expression with pet22b / pet32a vector and purification with Ni-NTA column was about 48.5kDa, and the purity met the experimental requirements. (2) The ELISA titer test results showed that the prepared mouse antiserum still had a significant positive signal when diluted to 1:128000, indicating that the antiserum titer was high; (3) After purification, the antibody was tested by SDS-PAGE and the purity met the requirements for subsequent application. It can specifically recognize recombinant gb antigen.
[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A recombinant expression method of bovine herpes virus 4 type gb protein, characterized by, The method comprises the following steps: (1) selecting a 104-384 aa segment of a bovine herpes virus 4 gb protein as an expression fragment, wherein the amino acid sequence of the 104-384 aa segment is shown as SEQ ID NO. 2; (2) constructing the expression fragment into a pet22b or pet32a E. coli expression vector to obtain a recombinant plasmid which is identified correct by sequencing; (3) transforming the recombinant plasmid into an expression host, and obtaining a bacterial body containing the recombinant gb protein after induced culture; (4) crushing and separating the bacterial body to obtain a sample containing the recombinant gb protein; (5) purifying the sample by using Ni-NTA resin to obtain the recombinant bovine herpes virus 4 gb protein.
2. The recombinant expression method of claim 1, wherein, In step (3), the operation of the induced culture comprises: Small-scale expression verification: the host bacteria containing recombinant plasmid were cultured to OD 600 ≈0.6, then IPTG was added to a final concentration of 0.5 mM, and cultured at 37 °C for 3 h with shaking; For large scale expression: the host bacteria containing the recombinant plasmid was cultured to OD 600 After the OD≈0.6, the temperature was decreased to 30℃, IPTG was added to the final concentration of 0.5mM, and the culture was shaken at 30℃ for 3h.
3. The recombinant expression method of claim 1, wherein, In step (4), the bacterial body is crushed by ultrasonic wave under ice bath condition, and the crushing parameters are as follows: power 300 W, ultrasonic 4 s, pause 4 s, and cumulative ultrasonic 90 times; after crushing, the sample is centrifuged at 20000 g and 4 ℃ for 30 min, and the supernatant is taken as the sample containing the recombinant gb protein.
4. The recombinant expression method of claim 1, wherein, The specific operation of purifying by using Ni-NTA resin comprises: (1) equilibrating a chromatography column filled with Ni-NTA resin by using 10 times column bed volume of NTA-0 Buffer; (2) adding the sample containing the recombinant gb protein to the chromatography column at a sample flow rate of 0.5 ml / min; (3) flushing the chromatography column by using 10 times column bed volume of NTA-0 Buffer at a flow rate of 1 ml / min; (4) sequentially performing gradient elution by using 10 times column bed volume of NTA-20 Buffer, NTA-60 Buffer, NTA-200 Buffer and NTA-500 Buffer at a flow rate of 1 ml / min, and collecting elution peak components; (5) dialyzing and ultrafiltration-concentrating the elution peak components by using 1xPBS buffer to obtain the recombinant bovine herpes virus 4 gb protein with high purity.
5. A recombinant bovine herpes virus type 4 gb protein, characterized in that, The recombinant bovine herpes virus 4 gb protein is prepared by the recombinant expression method of any one of claims 1-4, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 2, and the molecular weight is 48.5 kDa.
6. Application of the recombinant bovine herpes virus 4 gb protein of claim 5 in preparing mouse antisera.
7. Use according to claim 6, characterized in that, The steps of preparing the mouse antisera comprise: (1) coating a polystyrene 96-hole reaction plate with the recombinant bovine herpes virus 4 gb protein as an antigen at a concentration of 1 μg / ml, and placing the plate at 4 ℃ overnight; (2) after washing and blocking, adding serially diluted immune mouse antisera, and incubating at 37 ℃ for 1-2 h; (3) after washing, adding diluted goat anti-mouse IgG-HRP, and incubating at 37 ℃ for 1 h; (4) after washing, developing and terminating the reaction, and determining the absorbance value at 450 nm to determine the titer of the antisera.
8. Use according to claim 7, characterized in that, In step (2), the initial ratio of the serially diluted immune mouse antisera is 1:1000, and the dilution ratio of the goat anti-mouse IgG-HRP is 1:10000.
9. Use according to claim 6, characterized in that, The titer of the mouse antiserum is not less than 1:128000.
10. Use according to claim 6, characterized in that, The purification operation of the mouse antiserum is as follows: (1) Equilibrate the chromatography column with PBS or TBS buffer, mix the antiserum supernatant with equal volume of 2×PBS buffer, and then load; (2) Wash the chromatography column with PBS buffer until no protein is detected in the effluent, and then elute with 0.1M citric acid or 0.1M glycine as eluent; (3) After adding 2 / 5 volume of 1M Tris to neutralize the eluent, concentrate and switch to 2×PBS buffer containing 0.02% NaN3 and 1mM EDTA, and store at -20℃.