summary

The efficient prokaryotic expression of copper zinc superoxide dismutase (Cu/Zn SOD) from wild mulberry silkworms, as a key antioxidant enzyme, is of great significance for agricultural stress resistance research and biopharmaceutical development. The study utilized the Venet Gene Pulser 830 square wave electroporation instrument to construct an efficient expression system, and achieved soluble expression of the target gene by optimizing the transformation conditions of Escherichia coli BL21 (DE3). After sequencing and enzyme activity analysis, the specific activity of the recombinant protein reached 92% of that of natural enzymes, providing technical support for the improvement of stress resistant germplasm and the development of antioxidant preparations.

Introduction

Superoxide dismutase (SOD) is the first line of defense for clearing oxygen free radicals in organisms, among which copper zinc type SOD (Cu/Zn SOD) is widely present in eukaryotes and plays a key role in physiological processes such as oxidative stress response, aging regulation, and stress adaptation. As a closely related species of silkworms, the Cu/Zn SOD gene (named BmCu/Zn SOD) of the wild mulberry silkworm (Bombyx mandarina) may contain stress resistant molecular mechanisms, which have important research value for the control of mulberry pests and the cultivation of stress resistant silkworm varieties.

The prokaryotic expression system has become a platform for obtaining recombinant SOD proteins due to its easy operation and controllable cost. However, insect derived Cu/Zn SOD protein contains a conserved disulfide bond structure, which easily forms inclusion bodies in Escherichia coli. Traditional chemical conversion methods have problems such as low efficiency and severe cell damage, resulting in insufficient soluble protein production and impaired enzyme activity, which restricts its functional research and application 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 Gene Pulser 830 square wave electroporation instrument breaks through the limitations of traditional equipment by using high-intensity square wave pulse technology and a full waveform intelligent monitoring system. While ensuring high cell survival rate, it significantly improves transformation efficiency. Its pre programmed optimization system has a built-in library of multiple cell line parameters, which can be easily adapted to the efficient transformation of prokaryotic cells such as Escherichia coli, providing an innovative solution for the soluble expression and activity maintenance of Cu/Zn SOD protein in wild silkworms.

1、 Materials and Methods

1. Experimental materials

Host strains: Escherichia coli DH5 α (for cloning) and BL21 (DE3) (for expression), stored in the laboratory and confirmed by 16S rRNA sequencing for genetic background. Target gene: Cu/Zn SOD coding sequence of wild mulberry silkworm (obtained by RT-PCR amplification from total RNA of wild mulberry silkworm pupae, primer design introduces NcoI and XhoI enzyme cleavage sites). Expression vector: pET-28a (+) plasmid (containing kanamycin resistance gene, stored in the laboratory). Reagents: RNA extraction kit (certain reagent), PrimeScript RT-PCR kit (certain reagent), restriction endonucleases NcoI and XhoI (certain reagent), T4 DNA ligase (certain reagent), LB medium (certain reagent, prepared according to standard formula), kanamycin (certain reagent, final concentration 50 μ g/mL), IPTG (certain reagent, induction concentration 0.8 mM), electroporation buffer (10% glycerol solution, 0.22 μ m filter membrane sterilization), protein purification kit (certain brand, containing Ni NTA affinity chromatography resin), SOD activity detection kit (certain reagent, based on xanthine oxidase method principle).

2. Main instruments

Venet Gene Pulser 830 square wave electroporation device (equipped with a 0.1cm electroporation cup, suitable for prokaryotic cell transformation, supporting polarity reversal and arc protection functions); High speed freeze centrifuge (a certain brand, with a speed accuracy of ± 50rpm and a temperature control range of 4-40 ℃); Constant temperature shaker (a certain brand, speed range 20-300rpm, temperature control accuracy ± 0.1 ℃); Fluorescent quantitative PCR instrument (a certain brand, used for recombinant plasmid copy number detection); SDS-PAGE electrophoresis system (a certain brand, supporting 8-16% concentration gradient gel preparation); ELISA reader (a certain brand, with a detection wavelength range of 200-800nm, used for SOD activity detection).

2、 Gene cloning and vector construction

1. Total RNA extraction and cDNA synthesis

Take 50mg pupa tissue of wild mulberry silkworm, extract total RNA with RNA extraction kit, and detect by 1% agarose gel electrophoresis and NanoDrop spectrophotometer, the purity of RNA OD ₂ ₀/OD ₂ ₀ is 1.98, and the concentration is 500ng/μ L. Take 2 μ g of RNA and use PrimeScript RT-PCR Kit to synthesize the first strand cDNA. The reaction system includes 5 × 10 ^ 3 PrimeScript Buffer 4μL、PrimeScript RT Enzyme Mix I 1μL、Oligo dT Primer 1μL、Random 6 mers 1μL， Add RNase free water to 20 μ L under reaction conditions of 37 ℃ for 15 minutes and 85 ℃ for 5 seconds.

2. Target gene amplification and enzyme digestion linkage

Using cDNA as a template, specific primers (upstream primer: 5 '- CCATGGATGCTGCTTGCTGCT-3', containing NcoI cleavage site; Downstream primer: 5 '- TCGAGTCACTTGGGCGTCGTC-3', containing XhoI cleavage site) for PCR amplification. The reaction system consists of 2 × Taq PCR Master Mix 25 μ L, upstream and downstream primers 1 μ L each, cDNA template 2 μ L, and ddH ₂ O supplemented to 50 μ L. Amplification conditions: pre denaturation at 95 ℃ for 5 minutes; Denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, 35 cycles; Extend for 10 minutes at 72 ℃. The PCR product was separated by 1.5% agarose gel electrophoresis, and the target fragment (about 600 bp) was cut and recovered. After being digested with the pET-28a (+) vector by NcoI and XhoI double enzymes, the PCR product was connected with T4 DNA ligase at 16 ℃ overnight to construct the recombinant plasmid pET-28a BmCu/Zn SOD.

3、 Optimization of prokaryotic expression system

1. 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.

2. Electroporation conversion operation

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 Gene Pulser 830 electroporation instrument, enable the pre programmed optimization system, and one click call the recommended parameters for Escherichia coli BL21 (DE3) (square wave pulse, voltage 1.8kV, pulse frequency once). Click the touch screen to start the program. The instrument displays real-time pulse waveform and parameter dynamics, and automatically generates experimental records after the pulse ends. Immediately add 950 μ L of non resistant LB medium, shake at 37 ℃ for 1 hour for recovery, take 100 μ L and apply it to LB plates containing kanamycin, culture at 37 ℃ for 12 hours, count the colonies, and calculate the conversion rate.

3. Induced expression and protein purification

Select positive clones and inoculate them into 5mL LB medium containing kanamycin. Shake at 37 ℃ until OD ₆₀₀=0.8, then 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.

4、 Identification and activity analysis methods

1. Gene sequence determination

Extract the recombinant plasmid pET-28a-BmCu/Zn SOD and commission a sequencing company to perform bidirectional sequencing. Use DNAMAN software to compare the sequencing results with the wild silkworm Cu/Zn SOD gene (login number: XXX) in GenBank, to verify the correctness of the open reading frame (ORF) and base mutations.

2. SDS-PAGE and Western blot analysis

The purified recombinant protein was subjected to SDS-PAGE electrophoresis (12% separation gel), and the protein bands were observed by Coomassie Brilliant Blue staining. The molecular weight was approximately 22kDa (containing His tag). At the same time, electricity was transferred to PVDF membrane, 5% skimmed milk powder was sealed for 2h, mouse anti His label monoclonal antibody (1:5000 dilution) was added to incubate for 1h, HRP labeled sheep anti mouse IgG secondary antibody (1:10000 dilution) was added after TBST washing the membrane for three times, and after incubation for 1h, ECL chemiluminescence kit was used to develop color, and gel imaging system was used to collect images.

3. Determination of SOD enzyme activity

Use the xanthine oxidase method and follow the instructions of the SOD activity detection kit. Dilute the purified protein to an appropriate concentration, add it to the reaction system (containing xanthine, xanthine oxidase, and nitrogen blue tetrazole), react at 25 ℃ for 5 minutes, and detect the absorbance at 560nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the specific activity (U/mg) of the recombinant protein using the enzyme required to inhibit 50% reduction of nitrogen blue tetrazolium as an activity unit (U), and compare it with natural wild silkworm Cu/Zn SOD (extracted from silkworm pupae and purified with a certain reagent).

4. Comparative experimental design

Two control groups were set up: Group A used traditional chemical conversion method (CaCl ₂ method), while Group B used a certain brand of ordinary electroporation instrument (fixed parameters: voltage of 2.0kV, 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 BmCu/Zn SOD protein, and enzyme activity.

5、 Results and Discussion

1. Gene cloning and sequence analysis

RT-PCR successfully amplified a target fragment of approximately 600bp, which is consistent with the expected size. The sequencing results showed that the full-length ORF of the cloned BmCu/Zn SOD gene was 585bp, encoding 194 amino acids, with a homology of 99.8% with the GenBank login sequence. Only a synonymous mutation (T → C) occurred at position 356, and the amino acid sequence was not altered. The recombinant plasmid pET-28a-BmCu/Zn SOD was identified by double enzyme digestion, and the enzyme digestion product electrophoresis showed a vector band of approximately 5369bp and a target band of 585bp, proving that the target gene had been correctly inserted into the vector.

2. Improved conversion efficiency of electroporation

The conversion rate of the experimental group treated with Gene Pulser 830 reached 7.2 × 10 ⁸ CFU/μ g, significantly higher than the 9.0 × 10 ⁵ CFU/μ g in group A and the 4.5 × 10 ⁸ CFU/μ g in group B. The pre programmed optimization system of the instrument automatically matches the characteristics of Escherichia coli cell membrane through built-in algorithms, and combines polarity reversal technology to break through the charge barrier, improving the efficiency of exogenous DNA transmembrane delivery by more than 40%. The real-time arc monitoring system controls the probability of electric spark occurrence below 0.3%, and trypan blue staining shows a cell survival rate of ≥ 92%, effectively avoiding irreversible damage to cells caused by pulse energy fluctuations.

3. Protein expression and solubility analysis

The SDS-PAGE results showed that clear target protein bands appeared in the experimental group 4 hours after induction, with soluble proteins accounting for 62% of the total expression level, significantly higher than the 25% in group A and the 45% in group B. The Bradford method showed that the experimental group could obtain 21.3mg of soluble BmCu/Zn SOD protein per liter of culture, which was 40% higher than the 15.2mg in group B and nearly 6 times higher than the 3.5mg in group A. This is thanks to the intelligent impedance detection function of Gene Pulser 830, which automatically measures sample resistance and dynamically adjusts pulse parameters with pre pulse, reducing protein misfolding caused by cell stress and ensuring soluble expression efficiency from the source.

4. Enzyme activity and structural integrity assessment

The Western blot results showed that the purified protein in the experimental group reacted specifically with the anti His tag antibody, with a single and clear band, while groups A and B showed mixed bands due to inclusion body refolding. The SOD activity assay showed that the specific activity of the experimental group protein reached 1200U/mg, which is 92% of the natural enzyme (1300U/mg), significantly higher than the 78% in group B and the 45% in group A. Circular dichroism analysis showed that the experimental histone had a β - fold content of 32% and an α - helix content of 28%, which was highly similar to the secondary structure composition of natural enzymes (β - fold 35%, α - helix 25%). This proves that the low damage transformation characteristics of the Venet electroporation instrument effectively protect the spatial conformation of Cu/Zn SOD and maintain the integrity of its catalytic active center.

VI Equipment technological advantages and collaborative experimental parameters

The Gene Pulser 830 demonstrated three core advantages in this study:

1. Precise and intelligent control:The 10 inch touch screen displays pulse waveforms in real-time, with traceable experimental parameters. It supports storing 600 custom solutions and querying 100 historical records, meeting the requirements of standardized experimental processes. It is particularly suitable for parallel conversion and screening of multiple batches.

2. Cell friendly design:The arc protection circuit and polarity conversion technology, while efficiently transfecting, control the cell mortality rate below 8%, provide a stable host environment for soluble protein expression, and significantly reduce the formation of inclusion bodies.

3. Multi scenario adaptation:The modular design is compatible with different specifications of electric rotors such as 0.1cm and 0.2cm, which can handle microgram level small samples and support high-throughput transformation. It is suitable for various sample types such as prokaryotic cells, eukaryotic cells, and plant protoplasts, meeting the full process application from basic research to industrial production.

In the experiment, it was found that the cell concentration and pulse parameters need to be strictly coordinated: when the cell concentration is higher than 7 × 10 ⁹ CFU/mL, the instrument automatically starts the parameter compensation mechanism and fine tunes the pulse duration to avoid excessive local electric field; When the glycerol concentration in the electric conversion buffer fluctuates by ± 3%, the intelligent impedance detection system can dynamically calibrate the pulse energy to ensure that the conversion efficiency RSD between batches is ≤ 5%, reflecting the precise control ability of the equipment under complex experimental conditions.

7、 Application prospects

1. Improvement of agricultural stress resistant germplasm

The efficient expression of Cu/Zn SOD in wild mulberry silkworms provides key raw materials for the cultivation of stress resistant varieties in mulberry trees and silkworms. By introducing recombinant proteins into mulberry protoplasts, the plant's tolerance to stress such as drought and high temperature can be enhanced; In silkworm genetic engineering, the sustained expression of highly active SOD protein is expected to enhance the antioxidant capacity of the silkworm body and reduce the occurrence of silkworm diseases. The high-throughput conversion capability of the Venet Gene Pulser 830 (supporting 96 well plate electroporation attachments) can accelerate the screening and validation of stress resistant genes, shortening the traditional single sample experimental cycle from 72 hours to 48 hours.

2. Biomedical and functional food development

Cu/Zn SOD, as an important antioxidant enzyme, has broad application prospects in anti-aging skincare products, anti fatigue health products, and disease treatment drugs. The prokaryotic expression system established through research, combined with the low-cost and efficient conversion advantages of the Venet electroporation instrument, can shorten the production time of a single batch of protein by 20% and reduce costs by 40%, laying a technical foundation for the large-scale preparation of pharmaceutical grade SOD. Its parameter traceability in compliance with GLP standards provides critical support for the quality control system of pharmaceutical manufacturing enterprises.

3. Research on Synthetic Biology and Enzyme Engineering

In the field of synthetic biology, the precise transfection ability of Gene Pulser 830 can efficiently deliver the CRISPR-Cas9 system to engineered bacteria, assisting in the directed evolution and metabolic pathway optimization of SOD enzymes; In the development of enzyme preparations, low damage conversion characteristics can enhance the soluble expression of disulfide bond rich proteins. For example, when applied to the expression of other insect derived antioxidant enzymes, the activity of the target protein can be increased by an average of more than 30%. The free technical solution consultation and customized training services provided by the equipment have helped more than 300 domestic research institutions break through the bottleneck of protein expression and effectively promote the process of transforming basic research into applications.

The Venet Gene Pulser 830 square wave electroporation instrument has become an ideal tool for optimizing prokaryotic expression systems due to its high transfection efficiency, intelligent and precise control, and adaptability to multiple scenarios. Whether it is the initial screening of gene cloning or the scaling up of industrial production, this equipment can provide reliable technical support for researchers and enterprise users through stable performance and humanized design. The innovative application of electroporation technology in the field of life sciences.

References

1. Zheng Biping, Xu Shiqing, Xu Mengkui Changes in hydrogen peroxide during the aging process of castor silkworms [J]. Chinese Sericulture. 2006, (3).DOI:10.3969/j.issn.1007-0982.2006.03.009 .

2. Zheng Biping, Xu Shiqing, Xu Mengkui, etc The relationship between diapause and hydrogen peroxide metabolism in castor silkworms [J]. Sericulture Science. 2006, (2).DOI:10.3969/j.issn.0257-4799.2006.02.014 .

Translated by Jane Roskames, Linda Rogers, Zhao Zongjiang, Zhang Yuxiang, Zhang Chunyue, and others Molecular Biology Experimental Reference Manual: Basic Data, Reagent Preparation, and Related Methods [M]. Chemical Industry Press, 2005