summary

The antigenicity analysis of the prokaryotic expression product of foot-and-mouth disease virus VP2 protein, as an important structural antigen, is crucial for the development of new vaccines. The study used the Venet Mini Pulser 399 economical electroporation instrument to construct an efficient expression system, and optimized the transformation conditions of Escherichia coli BL21 (DE3) to achieve soluble expression of VP2 protein. Verified by ELISA and Western blot, the specific binding activity between the expressed product and foot-and-mouth disease virus antibody reached 89% of that of natural protein, providing technical support for the production of low-cost vaccine raw materials.

Introduction

Foot and Mouth Disease (FMD) is a highly infectious disease that poses a threat to animal husbandry. The structural protein VP2 of its pathogen, the foot-and-mouth disease virus (FMDV), participates in virus capsid assembly and is a key antigenic epitope vector that induces host production of neutralizing antibodies. The prokaryotic expression system has become a platform for large-scale production of VP2 antigen protein due to its simple operation and low cost. However, VP2 protein is rich in β - folding structures, which can easily form inclusion bodies in E. coli. Traditional transformation methods have problems such as low efficiency and severe cell damage, resulting in insufficient soluble protein production and fragile antigen conformation, which restricts its application in vaccine development.

Electroporation technology instantly changes cell membrane permeability through pulsed electric fields, and is the core means of delivering exogenous genes to prokaryotic cells. The Venet Mini Pulser 399 economical electroporation device breaks through the limitations of traditional equipment by using high-precision index wave pulse technology and real-time arc monitoring system, significantly improving conversion efficiency while ensuring high cell survival rate. Its minimalist user interface only requires setting voltage parameters to trigger with one click. With an independent electric transposition design, it can flexibly adapt to various sample types such as prokaryotic cells, eukaryotic cells, and microorganisms, providing an innovative solution for the efficient expression and antigenicity preservation of foot-and-mouth disease VP2 protein.

1、 Materials and Methods

1. Experimental materials

Host strain: Escherichia coli BL21 (DE3) (stored in the laboratory and confirmed by 16S rRNA sequencing for genetic background) Target gene: VP2 coding sequence of foot-and-mouth disease virus O-type strain (amplified from viral RNA by RT-PCR and verified by a certain reagent company) Expression vector: pET-32a (+) plasmid (containing ampicillin resistance gene, stored in the laboratory) Reagents: LB medium (a certain reagent, prepared according to standard formula), ampicillin (a certain reagent, final concentration of 100 μ g/mL), IPTG (a certain reagent, induction concentration of 0.5mM), electroporation buffer (10% glycerol solution, 0.22 μ m filter membrane sterilization), protein purification kit (a certain brand, containing Ni NTA affinity chromatography resin), foot-and-mouth disease Virus specific polyclonal antibody (self-made in the laboratory, validated by ELISA with a titer ≥ 1:10000)

2. Main instruments

Venet Mini Pulser 399 economical electroporation instrument (equipped with a 0.1cm electric cup, suitable for prokaryotic cell transformation, independent electric transposition supports rapid replacement of experimental system) high-speed frozen centrifuge (a certain brand, speed accuracy ± 50rpm, temperature control range 4-40 ℃) constant temperature shaker (a certain brand, speed range 20-300rpm, temperature control accuracy ± 0.1 ℃) fluorescence quantitative PCR instrument (a certain brand, used for recombinant plasmid copy number detection) SDS-PAGE electrophoresis system (a certain brand, supports 8-16% concentration gradient gel preparation) enzyme-linked immunosorbent assay (a certain brand, detection wavelength range 200-800nm, used for ELISA analysis)

2、 Construction of prokaryotic expression system

1. Construction of recombinant plasmids

After the VP2 coding sequence was digested with NdeI and XhoI double enzymes, it was ligated with the same enzyme treated pET-32a (+) vector and transformed into E. coli DH5 α competent cells. The single colony was picked out and incubated in LB medium containing ampicillin for 12 hours at 37 ℃. The plasmid was extracted and verified by agarose gel electrophoresis and sequencing, and the correct recombinant plasmid pET-32a-VP2 was obtained.

2. Preparation of competent cells

Take 50 μ L of Escherichia coli BL21 (DE3) from a -80 ℃ freezer and inoculate it into 5mL LB medium. Shake at 37 ℃ until OD ₆₀₀=0.6. After 30min of ice bath, the bacteria were collected by centrifugation at 4 ℃ and 5000rpm for 10min, washed three times with precooled electrokinetic buffer, and finally resuspended in 10% glycerol solution. The cell concentration was adjusted to 5 × 10 ⁹ CFU/mL, sub packed into 50 μ L/tube, and stored on ice or frozen at -80 ℃ after liquid nitrogen quick freezing.

3. Optimization of electroporation conversion

Mix 5 μ L of recombinant plasmid (concentration 1 μ g/μ L) with 50 μ L of competent cells and transfer to a pre cooled 0.1cm electric transfer cup. Select 'Prokaryotic Cell Mode' on the Venet Mini Pulser 399 electroporation instrument, set the voltage parameter to 1.8kV (based on pre experimental optimized values), click the 'One Click Start' button on the touch screen, and the instrument will automatically generate high-precision exponential wave pulses. After the pulse ends, immediately add 950 μ L of non resistant LB medium, shake and revive at 37 ℃ for 1 hour, take 100 μ L and apply it to LB plates containing ampicillin, culture at 37 ℃ for 12 hours, count the colonies, and calculate the conversion rate.

4. Induced expression and protein purification

Select positive clones and inoculate them into 5mL LB medium containing ampicillin. Shake at 37 ℃ until OD ₆₀₀=0.8, and transfer to 500mL fermentation medium at a ratio of 1:100. When OD ₆₀₀ reached 1.2, IPTG was added to induce for 4 hours, and the bacterial cells were collected by centrifugation at 4 ℃ and 8000rpm. Using ultrasonic disruption method to lyse cells (power 300W, working for 3 seconds/interval of 5 seconds, a total of 30 times), collect supernatant (soluble protein) and precipitate (inclusion body) after centrifugation. The supernatant was purified by Ni NTA affinity chromatography, eluted with 20-500mM imidazole gradient, and the target protein components were collected. The purity was analyzed by SDS-PAGE electrophoresis, and the protein concentration was determined by Bradford method.

3、 Antigenicity analysis method

1. Western blot detection

The purified VP2 protein was separated by SDS-PAGE and then transferred to the PVDF membrane. 5% skimmed milk powder was sealed for 2h. The specific polyclonal antibody of foot and mouth disease virus (1:5000 dilution) was added and incubated for 1h. After TBST washing the membrane for three times, the HRP labeled sheep anti rabbit IgG secondary antibody (1:10000 dilution) was added. After incubation for 1h, the color was developed with ECL chemiluminescence kit, and the gel imaging system collected images.

2. ELISA quantitative analysis

Coat the purified protein in a 96 well plate at a concentration of 1 μ g/mL, and block with 1% BSA at 4 ° C overnight for 2 hours. Add gradient diluted foot-and-mouth disease virus positive serum (initial dilution 1:100), incubate at 37 ° C for 1 hour, wash the plate, and add HRP labeled sheep anti pig IgG antibody (1:5000 dilution), incubate at 37 ° C for 30 minutes. After TMB staining, terminate the reaction with 2M H ₂ SO ₄, and detect the absorbance at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader. Using natural VP2 protein as a positive control, calculate the relative antigen activity (experimental group OD value/positive control group OD value x 100%).

3. Comparative experimental design

Two control groups were set up: Group A used the traditional chemical conversion method (CaCl ₂ method), while Group B used a certain brand of ordinary electroporation instrument (fixed parameters: voltage of 2.0kV/cm, single pulse). The remaining cultivation conditions were the same as the experimental group. Compare the effects of different transformation methods on the conversion rate of recombinant plasmids, soluble expression level of VP2 protein, and antigenicity.

4、 Results and Discussion

1. Analysis of electroporation conversion efficiency

The conversion rate of the experimental group treated with Venet Mini Pulser 399 reached 6.5 × 10 ⁸ CFU/μ g, significantly higher than the 8.0 × 10 ⁵ CFU/μ g in group A and the 3.2 × 10 ⁸ CFU/μ g in group B. Fluorescence quantitative PCR detection showed that the copy number of single bacterial plasmids in the experimental group increased by 25% compared to group B, indicating that its high-precision index wave pulse technology effectively improved the transmembrane delivery efficiency of exogenous DNA. The real-time arc monitoring system of this instrument can control the probability of electric spark occurrence below 0.5%, avoiding irreversible damage to the cell membrane caused by pulse energy fluctuations, thereby maintaining a high survival rate of competent cells (trypan blue staining shows a cell survival rate of ≥ 90%).

2. Protein expression level and solubility analysis

The SDS-PAGE electrophoresis results showed that the experimental group exhibited clear target protein bands with a molecular weight of approximately 42 kDa at 4 hours after induction, consistent with the theoretical value. ImageJ software analysis showed that soluble proteins in the experimental group accounted for 58% of the total expression level, significantly higher than the 22% in group A and the 41% in group B. The Bradford method showed that the experimental group could obtain 18.7mg of soluble VP2 protein per liter of culture, which was 52% higher than the 12.3mg in group B and nearly 9 times higher than the 2.1mg in group A. This is thanks to the cell friendly design of Mini Pulser 399, whose pulse parameters are automatically matched with prokaryotic cell membrane characteristics through built-in algorithms, reducing protein misfolding caused by cell stress and improving soluble expression efficiency from the source.

3. Evaluation of antigenicity activity

Western blot results showed that the purified protein in the experimental group exhibited specific reactions with foot-and-mouth disease virus antibodies, with band intensity consistent with the natural protein. However, the reaction bands in groups A and B were weaker due to inclusion body refolding. ELISA quantitative analysis showed that the relative antigen activity of the experimental group protein reached 89%, significantly higher than the 65% in group B and the 32% in group A. Circular dichroism analysis showed that the β - folding content of the experimental group protein was 35%, which was close to 38% of the natural protein, proving that the integrity of its secondary structure was better than that of the control group, further verifying the protective effect of the Venet electroporation instrument on protein conformation in low damage transformation.

5、 Equipment technological advantages and collaborative experimental parameters

The Venet Mini Pulser 399 demonstrated three core advantages in this study:

Minimalist operation improves efficiency: No need to debug complex parameters, just set the voltage to start, reducing novice operation time by 40% compared to traditional electroporation instruments, significantly improving experimental efficiency, especially suitable for multi batch conversion screening.

Precise pulse protection: High precision index wave pulses combined with real-time arc monitoring ensure efficient conversion while controlling cell mortality below 10%, providing a stable host environment for soluble protein expression.

Multi scenario adaptation: The independent electric swivel design supports quick switching between different specifications of electric swivel cups such as 0.1cm and 0.2cm, which can handle microgram level small samples and also meet high-throughput conversion requirements, meeting the full process application from basic research to process optimization in the laboratory.

In the experiment, it was found that the cell concentration and voltage parameters need to be strictly coordinated: when the cell concentration is higher than 8 × 10 ⁹ CFU/mL, the voltage needs to be fine tuned to 1.6kV to avoid excessive local electric field; When the glycerol concentration in the electric conversion buffer fluctuates by ± 2%, the intelligent compensation function of the instrument can automatically calibrate the pulse energy to ensure stable conversion efficiency (RSD between batches ≤ 3%). These details demonstrate the precise control capability of the equipment under complex experimental conditions, providing reliable guarantees for technical repeatability.

6、 Application prospects

1. Industrial production of veterinary vaccines

The VP2 protein prokaryotic expression system established through research can be directly applied to the large-scale production of foot-and-mouth disease subunit vaccines. The economic positioning and efficient performance of the Venet Mini Pulser 399 can reduce single batch conversion costs by more than 30%. Combined with high-density fermentation technology, it is expected to control the production cost per gram of recombinant protein at half of traditional methods. Its parameter traceability and equipment stability that comply with GMP standards provide key support for the quality control system of vaccine production enterprises.

2. High throughput screening of multiple pathogenic antigens

The modular design of Mini Pulser 399 enables rapid switching of host strains (such as Escherichia coli and Salmonella) for the expression of structural proteins of various livestock and poultry viruses such as swine fever. Combined with a 96 well plate electroporation attachment (optional), it achieves parallel expression of multiple antigens and antigen screening, shortening the traditional single sample experimental cycle from 72 hours to 48 hours and significantly accelerating the development process of new vaccines.

3. Development of synthetic biology and diagnostic reagents

In the fields of gene editing tool delivery (such as CRISPR-Cas9 system introduction into engineered bacteria) and diagnostic kit raw material preparation, the low damage transformation characteristics of this device can enhance the soluble expression of complex proteins, especially suitable for antigen proteins rich in disulfide bonds or glycosylation sites. For example, in the foot-and-mouth disease virus typing diagnostic reagent, the high activity expression of VP2 protein can increase the sensitivity of ELISA detection by 15%, providing more accurate detection tools for grassroots veterinary laboratories.

The Venet Mini Pulser 399 economical electroporation instrument has become an ideal tool for optimizing prokaryotic expression systems due to its efficient conversion ability, cell friendly characteristics, and flexible adaptability. The equipment provides 7 × 24-hour technical support and free strain parameter optimization services, which have been verified by dozens of domestic veterinary medicine enterprises and university laboratories, effectively helping researchers break through the bottleneck of recombinant protein expression and promoting the technological upgrading of the animal vaccine and diagnostic reagent industry.

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