Prokaryotic expression and purification method of rumen butyric acid vibrio RnfC protein

Abstract

The invention belongs to the technical field of protein separation and purification, and particularly relates to a prokaryotic expression and purification method of rumen butyric acid vibrio RnfC protein. The method comprises the following steps: transferring a recombinant expression vector for coding rumen butyric acid vibrio RnfC protein into BL21 (DE3) competent cells to carry out protein induced expression; the recombinant expression vector for coding the rumen butyric acid vibrio RnfC protein comprises a basic vector and an RnfC protein coding gene inserted into the basic vector, the amino acid sequence of the RnfC protein is as shown in SEQ ID NO: 3. According to the method, conditions such as inducer concentration, induction time, induction temperature and the like are limited, a bacterial solution subjected to induced expression is collected, a supernatant is collected after ultrasonication treatment, and protein magnetic bead purification is performed, so that prokaryotic in-vitro expression and purification of rumen butyric acid vibrio RnfC protein can be realized, a large amount of rumen butyric acid vibrio RnfC can be obtained within a short time (2-3 days), and large-scale production is easy.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation and purification, and specifically relates to a prokaryotic expression and purification method for rumen butyrivibrio RnfC protein. Background Art

[0002] Ruminal Butyrivibrio ( Butyrivibrio ) is a kind of cellulose-degrading anaerobic bacteria widely found in the rumen of ruminants, which plays an important role in the normal fermentation and metabolism of the rumen. Studies have found that feeding guanidine acetate significantly increases the concentration of adenosine triphosphate (ATP) in the rumen fluid of beef cattle and the number of Butyrivibrio spp. ( Butyrivibrio ) abundance, and there was a significant positive correlation between the two. The results of metagenomic sequencing analysis showed that guanidine acetate significantly upregulated R The expression of genes mainly promotes the synthesis of ATP by rumen butyrivibrio through the (Electron transport phosphorylation, ETP) pathway. R The proteins encoded by the genes are particularly important for clarifying the mechanism of ATP synthesis in rumen microorganisms.

[0003] Protein prokaryotic expression and purification is to express recombinant proteins in vitro through E. coli, which is currently the most mature method for producing foreign proteins. However, the expression efficiency of foreign genes in E. coli is usually affected by factors such as inducer concentration, induction time, and induction temperature, which also results in different expression conditions for different foreign genes in E. coli. R A record of prokaryotic expression of gene-encoded proteins. Summary of the invention

[0004] The purpose of the present invention is to make up for the deficiencies of the prior art and provide a prokaryotic expression and purification method for ruminal butyrivibrio RnfC protein, so as to achieve prokaryotic in vitro expression of ruminal butyrivibrio RnfC protein, obtain a large amount of ruminal butyrivibrio RnfC in a short time, and facilitate large-scale production.

[0005] The present invention provides a prokaryotic expression and purification method of rumen butyrivibrio RnfC protein, comprising the following steps: The recombinant expression vector encoding the RnfC protein of ruminal butyrivibrio was transferred into BL21(DE3) competent cells for protein induction expression, and the bacterial liquid was collected; The bacterial solution is subjected to ultrasonic disruption and the supernatant is collected, and the supernatant is purified by protein magnetic beads to obtain the purified rumen butyrivibrio RnfC protein; The protein induction expression method includes: using isopropyl-β-D-thiogalactoside with a final concentration of 1mM to induce culture of BL21 (DE3) competent cells containing a recombinant expression vector encoding the rumen butyrivibrio RnfC protein; The induction culture time is 24 hours and the temperature is 16°C; The recombinant expression vector encoding the rumen butyrivibrio RnfC protein comprises a basic vector and a RnfC protein encoding gene inserted into the basic vector; the amino acid sequence of the RnfC protein is shown in SEQ ID NO:3.

[0006] Preferably, the rotation speed of the induction culture is 180 rpm.

[0007] Preferably, the OD of the BL21 (DE3) competent cells containing the recombinant expression vector encoding the rumen Butyrivibrio RnfC protein is 600 The value is 0.6.

[0008] Preferably, the nucleotide sequence of the RnfC protein encoding gene is shown in SEQ ID NO:1.

[0009] Preferably, the method for preparing the recombinant expression vector encoding the ruminal Butyrivibrio RnfC protein comprises: amplifying the genomic DNA of Butyrivibrio fibrinolyticus using RnfC protein encoding gene amplification primers to obtain an RnfC amplification product; Amplifying the basic vector using basic vector amplification primers to obtain a basic vector amplification product; The RnfC amplification product and the basic vector amplification product are connected by homologous recombination, and positive recombination products are screened to obtain the recombinant expression vector encoding the rumen butyrivibrio RnfC protein.

[0010] Preferably, the RnfC protein encoding gene amplification primers include an upstream primer RnfC-F and a downstream primer RnfC-R; The nucleotide sequence of the upstream primer RnfC-F is shown in SEQ ID NO:4; The nucleotide sequence of the downstream primer RnfC-R is shown in SEQ ID NO:5.

[0011] Preferably, the basic vector comprises pET28a plasmid.

[0012] Preferably, the primers for amplifying the pET28a plasmid include an upstream primer pET28a-F and a downstream primer pET28a-R; The nucleotide sequence of the upstream primer pET28a-F is shown in SEQ ID NO:6; The nucleotide sequence of the downstream primer pET28a-R is shown in SEQ ID NO:7.

[0013] Preferably, the nucleotide sequence of the recombinant expression vector encoding the ruminal Butyrivibrio RnfC protein is shown in SEQ ID NO:2.

[0014] Preferably, before transferring the recombinant expression vector encoding the RnfC protein of ruminal butyrivibrio into BL21 (DE3) competent cells, the method further comprises: transferring the recombinant expression vector encoding the RnfC protein of ruminal butyrivibrio into trans1-T1 competent cells for plasmid amplification.

[0015] Beneficial effects: The present invention transfers a recombinant expression vector encoding the rumen butyrivibrio RnfC protein into BL21 (DE3) competent cells for protein induction expression, collects bacterial liquid; collects supernatant after ultrasonic disruption of the bacterial liquid, purifies the supernatant with protein magnetic beads, and obtains purified rumen butyrivibrio RnfC protein; the recombinant expression vector encoding the rumen butyrivibrio RnfC protein includes a basic vector and a RnfC protein encoding gene inserted into the basic vector; the amino acid sequence of the RnfC protein is as described in SEQ ID NO: 3. The present invention utilizes BL21 (DE3) competent cells, and by limiting conditions such as inducer concentration, induction time, and induction temperature, the prokaryotic in vitro expression of the rumen butyrivibrio RnfC protein can be achieved, and a large amount of rumen butyrivibrio RnfC can be obtained within 2 to 3 days, which is easy for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 The graphs are the absorbance results of the recombinant bacteria at different culture times; Figure 2 The graph shows the results of protein concentration detection at different IPTG concentrations and temperatures; different lowercase letters indicate significant differences between the groups; Figure 3 This is a diagram of the SDS-PAGE polyacrylamide gel electrophoresis detection results. DETAILED DESCRIPTION

[0018] The present invention provides a prokaryotic expression and purification method of rumen butyrivibrio RnfC protein, comprising the following steps: The recombinant expression vector encoding the RnfC protein of ruminal butyrivibrio was transferred into BL21(DE3) competent cells for protein induction expression, and the bacterial liquid was collected; The bacterial solution is subjected to ultrasonic disruption and the supernatant is collected, and the supernatant is purified by protein magnetic beads to obtain the purified rumen butyrivibrio RnfC protein; The protein induction expression method includes: using isopropyl-β-D-thiogalactoside with a final concentration of 1mM to induce culture of BL21 (DE3) competent cells containing a recombinant expression vector encoding the rumen butyrivibrio RnfC protein; The induction culture time is 24 hours and the temperature is 16°C; The recombinant expression vector encoding the rumen butyrivibrio RnfC protein comprises a basic vector and a RnfC protein encoding gene inserted into the basic vector; the amino acid sequence of the RnfC protein is shown in SEQ ID NO:3.

[0019] As an embodiment, the present invention constructs a recombinant expression vector encoding the RnfC protein of ruminal butyrivibrio. As an embodiment, the present invention uses RnfC protein encoding gene amplification primers to amplify the genomic DNA of Butyrivibrio fibrinolyticus to obtain the RnfC amplification product. As an embodiment, the nucleotide sequence of the RnfC protein encoding gene is shown in SEQ ID NO:1. The present invention optimizes the nucleotide sequence according to the codon preference of ruminal butyrivibrio and the characteristics of the basic vector, and can efficiently express the RnfC protein without changing the encoded amino acid sequence. As an embodiment, the RnfC protein encoding gene amplification primers include an upstream primer RnfC-F and a downstream primer RnfC-R; the nucleotide sequence of the upstream primer RnfC-F is shown in SEQ ID NO:4; the nucleotide sequence of the downstream primer RnfC-R is shown in SEQ ID NO:5. As an embodiment, the ruminal butyrivibrio RnfC protein is ruminal butyrivibrio ( Butyrivibrio fibrisolvens ) RnfC protein As an embodiment, the present invention uses a basic vector amplification primer to amplify the basic vector to obtain a basic vector amplification product. As an embodiment, the basic vector of the present invention includes a pET28a plasmid. As an embodiment, the primers for amplifying the pET28a plasmid include an upstream primer pET28a-F and a downstream primer pET28a-R; the nucleotide sequence of the upstream primer pET28a-F is shown in SEQ ID NO:6; the nucleotide sequence of the downstream primer pET28a-R is shown in SEQ ID NO:7. The present invention uses the pET28a plasmid as the basic vector. Compared with other pET series vectors and pGEX series vectors, from the acquisition of the target gene, the construction of the recombinant plasmid to the prokaryotic expression of the protein until the final protein separation and purification, the expression cycle of BL21 (DE3) is shorter, and all links can be completed in 2 to 3 days to obtain a large amount of rumen butyrivibrio RnfC.

[0020] As an embodiment, after obtaining the RnfC amplification product and the basic vector amplification product, the present invention uses homologous recombination to connect the RnfC amplification product and the basic vector amplification product, screens positive recombination products, and obtains a recombinant expression vector encoding the rumen butyrivibrio RnfC protein. The present invention has no strict requirements on the homologous recombination method, and the operation can be performed according to the instructions of the homologous recombination kit. As an embodiment, the nucleotide sequence of the recombinant expression vector encoding the rumen butyrivibrio RnfC protein is shown in SEQ ID NO:2.

[0021] After obtaining the recombinant expression vector encoding the rumen butyrivibrio RnfC protein, the present invention transfers the recombinant expression vector encoding the rumen butyrivibrio RnfC protein into BL21 (DE3) competent cells for protein induction expression, and collects the bacterial liquid. The present invention has no strict requirements on the method of transferring the recombinant expression vector encoding the rumen butyrivibrio RnfC protein into the BL21 (DE3) competent cells, and conventional steps in the art can be used. The present invention selects BL21 (DE3) competent cells. Compared with other Escherichia coli competent cells, BL21 (DE3) contains a T7 RNA polymerase gene regulated by the lacUV5 promoter, which can efficiently transcribe the target gene, and lacks certain proteases. The expressed protein is more stable and not easy to be degraded. At the same time, after adding IPTG induction, the transcription and expression of the target gene can be quickly started, and the regulation is relatively simple; compared with other Escherichia coli competent cells, BL21 (DE3) has a higher expression level and is suitable for large-scale protein production. As an embodiment, the method of inducing the expression of the protein of the present invention includes: using isopropyl-β-D-thiogalactoside to induce the culture of BL21 (DE3) competent cells containing a recombinant expression vector encoding the rumen butyrivibrio RnfC protein. As an embodiment, the final concentration of the isopropyl-β-D-thiogalactoside of the present invention is 0.5~1mM; as another embodiment, the final concentration of the isopropyl-β-D-thiogalactoside of the present invention is 1mM. As an embodiment, the time of the induction culture of the present invention is 24h. As an embodiment, the temperature of the induction culture of the present invention is 16°C. As an embodiment, the rotation speed of the induction culture of the present invention is 180rpm.

[0022] After obtaining the bacterial liquid, the present invention performs ultrasonic crushing on the bacterial liquid and collects the supernatant, and performs protein magnetic bead purification on the supernatant to obtain the purified rumen butyrivibrio RnfC protein. As an embodiment, the ultrasonic power of the ultrasonic crushing treatment of the present invention is 200W. As an embodiment, the ultrasonic crushing time of the present invention is 15min, ultrasonic 2s, and interval 1s. The present invention has no strict requirements on the method of protein magnetic bead purification, and conventional steps in the art can be used, for example, according to the instructions for use of beaver histidine tag protein purification magnetic beads.

[0023] To further illustrate the present invention, a prokaryotic expression and purification method of rumen Butyrivibrio RnfC protein provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1 1. Experimental consumables trans1-T1 competent cells: purchased from Beijing Quanshijin Biotechnology Co., Ltd.; BL21(DE3) competent cells: purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Homologous recombination kit: purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Plasmid extraction kit: purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. (CB101-01); Beaver histidine-tagged protein purification magnetic beads: purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.; pET28a plasmid: purchased from Wuhan Miaoling Biotechnology Co., Ltd.; Upstream primer RnfC-F: 5′-GTGGTGGTGGTGGTGTTTCTTCTTGCGCTCAGCA-3′ (SEQ ID NO: 4), synthesized by Beijing Qingke Biotechnology Co., Ltd.; Downstream primer RnfC-R: 5′-TAAGAAGGAGATATACCATGGCGCGACTAACGTTCGTAGG-3′ (SEQ ID NO: 5), synthesized by Beijing Qingke Biotechnology Co., Ltd.; Upstream primer pET28a-F: 5′-CGTTAGTCGCGCCATGGTATATCTCCTTCTTAAAGTTA-3′ (SEQ ID NO: 6), synthesized by Beijing Qingke Biotechnology Co., Ltd.; Downstream primer pET28a-R: 5′-GCAAGAAGAAACACCACCACCACCACCACTGAGATCCG-3′ (SEQ ID NO: 7), synthesized by Beijing Qingke Biotechnology Co., Ltd.; PCR high-fidelity enzyme premix: purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; PCR product purification kit: purchased from Nanjing Novozymes Biotech Co., Ltd.

[0025] 2. Cloning of RnfC protein gene

[0026] 3. Construction of recombinant expression vector pET28a- R (1) PCR amplification of plasmid pET28a was performed using upstream primer pET28a-F and downstream primer pET28a-R. After DNA gel electrophoresis, the plasmid pET28a was purified using a PCR product purification kit to obtain a linear fragment of the plasmid pET28a. The linear fragment of the plasmid pET28a and the RnfC protein encoding gene obtained in step 2 were homologously recombined using a homologous recombination kit to obtain a recombinant product. The reaction system was: 2×Basic Assembly Mix 5μL, pET28a plasmid linear fragment 1μL, RnfC protein encoding gene 2μL, Nuclease-free Water was added to 10μL, gently mixed, and reacted at 50℃ for 15min. After the reaction was completed, the centrifuge tube was placed on ice for a few seconds to obtain a recombinant product.

[0027] (2) Take 50 μL of Trans1-T1 competent cells melted on ice, add 2 μL of the recombinant product obtained in step (1), gently flick the wall of the centrifuge tube to mix (no vortexing), place on ice for 30 minutes, heat shock in a 42°C water bath for 30 seconds, then immediately transfer to ice to cool for 2 minutes, add 450 μL of room temperature LB culture medium, then culture at 200 rpm in a 37°C shaker for 1 hour, and preheat the kanamycin LB plate at 37°C. Take 100 μL of cells and spread them evenly on the plate, then culture in a 37°C incubator overnight. Pick the positive colonies and add them to the liquid LB culture medium containing kanamycin for shaking culture at 37°C. When the culture medium becomes turbid, use a plasmid extraction kit to extract the plasmid and perform PCR verification. If the verification result is positive, it is the recombinant expression vector pET28a- R

[0028] Example 2 1. Protein induction expression (1) Take 50 μL of BL21 (DE3) competent cells melted on ice and add 2 μL of the recombinant expression vector pET28a- R , gently flick the wall of the centrifuge tube to mix (no vortexing), place on ice for 30 minutes, heat shock in a 42°C water bath for 30 seconds, then immediately transfer to ice to cool for 2 minutes, add 450μL of room temperature LB medium, and then culture at 200rpm in a 37°C shaker for 1 hour, and preheat the kanamycin LB plate at 37°C. Take 100μL of cells and spread them evenly on the plate, then culture in a 37°C incubator overnight, and verify the colonies by PCR. Pick the colonies that are positive for PCR in step 1 for bacterial expansion culture, and add 50μg / mL kanamycin to the culture medium for screening. Culture in a constant temperature shaker at 37°C, 180rpm, and detect the OD at different culture times. 600 Value, the result is Figure 1 As shown. 600 The value can reflect the number and density of bacteria in the culture medium. Figure 1 It can be seen that when the culture time is between 12 and 24 hours, OD 600 It grows rapidly and is in the exponential growth period. After 24 hours, the growth rate slows down and enters the slow growth period of bacterial life.

[0029] (2) Step (1) Incubate in a constant temperature shaker at 37°C, 180 rpm until OD 600 When the pH value reached 0.6, IPTG solution was added to the culture medium to make the final IPTG concentration of 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.2 mM and 1.4 mM, and the culture was transferred to an appropriate temperature (16°C, 25°C, 30°C and 37°C) and cultured at 180 rpm overnight.

[0030] 2. Protein extraction and concentration detection The bacteria induced and cultured overnight were centrifuged at 8000rpm for 5min, and the supernatant was discarded to retain the bacterial precipitate. PB solution (phosphate buffer) protein binding buffer was added to resuspend the cells in a cell disruptor to disrupt the cells. Before disruption, phenylmethylsulfonyl fluoride (PMSF) was added to the centrifuge tube to prevent the protein from dissolving under the action of the enzyme, so that the final concentration of PMSF was 1mM. After cell disruption, the cells were centrifuged at 8000rpm for 5min, and the supernatant and precipitate were separated and divided into different centrifuge tubes for later use.

[0031] Refer to the steps of determining protein concentration by Coomassie Brilliant Blue method to detect protein concentration at different IPTG induction concentrations and different induction temperatures. The results are as follows: Figure 2 And as shown in Table 1.

[0032] Table 1 Protein concentration test results

[0033] According to Table 1 and Figure 2 It can be seen that when the final IPTG concentration was 1.0 mM, the protein concentration was the highest when cultured overnight at 16°C and 180 rpm, which was significantly higher than other treatment conditions.

[0034] 3. Protein Purification Referring to the instructions for beaver histidine tag protein purification magnetic beads, the bacteria induced and cultured at 16°C with a final IPTG concentration of 1.0 mM were subjected to ultrasonic disruption and centrifugation, and the separated protein supernatant was purified by magnetic beads to obtain the purified target protein. The protein size was verified by SDS-PAGE polyacrylamide gel electrophoresis. The results are as follows: Figure 3 As shown; wherein, lanes 1 and 2 are protein purification solutions, and lane 3 is the filtrate. Figure 3 It can be seen that the protein purification solution contains the target protein with a size of 47.84Kda.

[0035] 4. Protein Identification According to the conclusion of step 3, the correct protein band after purification ( Figure 3 The amino acid sequence of the gelatin protein was identified by PCR (red frame part), and its amino acid sequence was MARLTFVGGVHPYEGKELTMDKPIKSVLPKGDLVYPLSQHIGAPASPIVAVGDHVLTGQKIAEASGFVSAPIYATVSGTVKAIEPRRLATGGMCNSIIVENDGLYEEVEWPEVKPWEEMTSEEIVAAVREAGVVGMGGAGFPTAVKLSPKEPDKIEYVIANCSECEPYLTSDYRRMIEEPELIIGGLKIAVSLFPNARGILAVEDNK PEAIKKLRELVKDEPKLEVKQLQTKYPQGAERMLIYACTGRSINSSMLPADAGCIVDNDTLCAIYRAVTEGRPLMERIVTITGDAIKEPCNYKVRIGTSYNELLEDAGGFYKTPQKI ISGGPMMGFAVWDTEVPTTKTASALTCLTEDAVSAMEPTACINCARCVEACPEQLIPKNLADAVEHGQTERFLKEYGMECCECGCCSFICPAHRHLTQLIKGMRKIQLAERKKK (SEQ ID NO:3).

[0036] It can be seen from the above content that the scheme provided by the present invention can achieve the prokaryotic in vitro expression of ruminal butyrivibrio RnfC protein, obtain a large amount of ruminal butyrivibrio RnfC in a short time, and facilitate large-scale production.

[0037] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A prokaryotic expression and purification method for rumen Butyrivibrio RnfC protein, characterized in that: The steps include: The recombinant expression vector encoding the RnfC protein of ruminal butyrivibrio was transferred into BL21(DE3) competent cells for protein induction expression, and the bacterial liquid was collected; The bacterial solution is subjected to ultrasonic disruption and the supernatant is collected, and the supernatant is purified by protein magnetic beads to obtain the purified rumen butyrivibrio RnfC protein; The protein induction expression method includes: using isopropyl-β-D-thiogalactoside with a final concentration of 0.5-1 mM to induce culture of BL21 (DE3) competent cells containing a recombinant expression vector encoding the rumen butyrivibrio RnfC protein; The induction culture time is 24 hours and the temperature is 16°C; The recombinant expression vector encoding the rumen butyrivibrio RnfC protein comprises a basic vector and a RnfC protein encoding gene inserted into the basic vector; the amino acid sequence of the RnfC protein is shown in SEQ ID NO:

3.

2. The prokaryotic expression purification method according to claim 1, characterized in that: The rotation speed of the induction culture is 180 rpm.

3. The prokaryotic expression purification method according to claim 1, characterized in that: The OD of the BL21 (DE3) competent cells containing the recombinant expression vector encoding the rumen butyrivibrio RnfC protein is 600 The value is 0.

6.

4. The prokaryotic expression purification method according to claim 1, characterized in that: The nucleotide sequence of the RnfC protein encoding gene is shown in SEQ ID NO:

1.

5. The prokaryotic expression purification method according to claim 1, characterized in that: The method for preparing the recombinant expression vector encoding the rumen Butyrivibrio RnfC protein comprises: amplifying the Butyrivibrio fibrinolyticus genomic DNA using RnfC protein encoding gene amplification primers to obtain the RnfC amplification product; Amplifying the basic vector using basic vector amplification primers to obtain a basic vector amplification product; The RnfC amplification product and the basic vector amplification product are connected by homologous recombination, and positive recombination products are screened to obtain the recombinant expression vector encoding the rumen butyrivibrio RnfC protein.

6. The prokaryotic expression purification method according to claim 5, characterized in that: The RnfC protein encoding gene amplification primers include an upstream primer RnfC-F and a downstream primer RnfC-R; The nucleotide sequence of the upstream primer RnfC-F is shown in SEQ ID NO:4; The nucleotide sequence of the downstream primer RnfC-R is shown in SEQ ID NO:

5.

7. The prokaryotic expression purification method according to claim 1, characterized in that: The basic vector includes the pET28a plasmid.

8. The prokaryotic expression purification method according to claim 7, characterized in that: The primers for amplifying the pET28a plasmid include an upstream primer pET28a-F and a downstream primer pET28a-R; The nucleotide sequence of the upstream primer pET28a-F is shown in SEQ ID NO:6; The nucleotide sequence of the downstream primer pET28a-R is shown in SEQ ID NO:

7.

9. The prokaryotic expression purification method according to claim 7, characterized in that: The nucleotide sequence of the recombinant expression vector encoding the rumen Butyrivibrio RnfC protein is shown in SEQ ID NO:

2.

10. The prokaryotic expression purification method according to any one of claims 1 to 9, characterized in that: Before transferring the recombinant expression vector encoding the RnfC protein of rumen butyrivibrio into BL21 (DE3) competent cells, the method further comprises: transferring the recombinant expression vector encoding the RnfC protein of rumen butyrivibrio into trans1-T1 competent cells for plasmid amplification.

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