CN104152469A - Lactobacillus plantarum nitrite reductase gene, protein encoded by lactobacillus plantarum nitrite reductase gene and preparation method of protein - Google Patents

Abstract

The invention discloses a plant lactobacillus nitrite reductase gene and its encoded protein and a preparation method. The nucleotide sequence of the plant lactobacillus nitrite reductase gene is shown in SEQ ID NO.1. The gene sequence shown as SEQ ID NO.1 was cloned into the prokaryotic expression vector pET-32a(+), induced and expressed in recombinant Escherichia coli, and purified by affinity chromatography to obtain a high-purity recombinant protein. The high-purity protein purified by the method of the present invention can effectively degrade nitrite, can be produced into an enzyme preparation, and can effectively degrade nitrite in food; it can also be used as an antigen for Lactobacillus plantarum nitrite reductase Antibody preparation, and can be used for research on its structure and properties.

Description

A plant Lactobacillus nitrite reductase gene and its encoded protein and preparation method

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector containing the gene of Lactobacillus plantarum nitrite reductase, a host transformed with the vector, and a large amount of expression of nitrite reductase and nitrite reduction by using the transformant High-efficiency affinity chromatography purification method for enzymes.

Background technique

Nitrite is a potential carcinogen, which is easy to accumulate in the process of vegetable fermentation, bringing potential food safety problems to the product, and excessive intake of nitrite can induce methemoglobinemia, so strict control of nitrite in food The salt content is very important. Many studies have shown that nitrite is the precursor of nitrosamines, which are strong carcinogens that can induce various cancers in the digestive system, such as gastric cancer, intestinal cancer and liver cancer. In view of the potential food safety problems of excessive nitrite pollution in food and nitrite in meat products, it is imperative to find effective ways to control or degrade nitrite. In addition to adding edible safe lactic acid bacteria, nitrite can also be used to Nitrate reductase (Nir) carries out the biodegradation of nitrite. However, there is no good method for how to obtain a large amount of edible safe nitrite reductase at present. The main reason is that the production cost is too high due to the difficulty in the separation and purification of enzyme proteins, and industrialization cannot be realized. Compared with conventional protein separation techniques, Immobilized metal ion affinity chromatography (IMAC) is used for enzyme proteins. The separation has high selectivity and can realize separation and purification with high throughput and high adsorption. The principle of separation is mainly to use histidine, tryptophan, cysteine, etc. on the surface of the protein to have affinity with immobilized metal ions to achieve the separation of enzyme proteins. Therefore, design polyhistidine, tryptophan, cysteine, arginine, etc. as affinity tags to fuse with the target enzyme protein and then use IMAC for purification, which can achieve the expected separation effect.

At home and abroad, there are few reports on the extraction of nitrite reductase from lactic acid bacteria and large-scale expression. The Chinese invention patent application number 201110207974.3 discloses "a production method of lactic acid bacteria nitrite reductase", which uses simple mechanical crushing and The crude enzyme is collected by centrifugation. Since the crude enzyme solution contains a large amount of foreign proteins, the subsequent separation and purification work is complicated, and the target product nitrite reductase cannot be obtained in large quantities. In addition, the medium used for enzyme production in this method The ingredient contains tomato juice. Due to the red color of the tomato, red pigment is inevitably contained in the final nitrite reductase, which affects the subsequent purification work. The Chinese invention patent with the application number 201210037774.2 discloses "a nitrite reductase gene of Lactobacillus plantarum and its encoded protein and its application", in which the main basis for verifying the successful expression of nitrite reductase is the whole The results of protein SDS-PAGE electrophoresis did not go further into the separation and purification, enzyme activity and application of the recombinant protein, because the microbial engineering bacteria E.coli BL 21, E.coli DE 3 and JM 105 can all express nitrite reduction before transformation Enzymes, by the method of the present invention, it is not possible to well identify whether the expression of nitrite reductase is successful.

Contents of the invention

In order to solve the deficiencies in the mass expression, extraction and purification of existing nitrite reductase, the present invention provides a method for recombining the gene encoding nitrite reductase, and provides a mass expression of nitrite recombination protein and purification methods, mainly using the prokaryotic expression vector pET-32a(+) containing histidine tag (HIS tag) to recombine the nitrite reductase gene, then transform the recombinant vector into the host, and use the transformant A large amount of nitrite reductase was expressed, and the nitrite protein was purified by affinity chromatography.

In order to achieve the above object, the present invention adopts the following technical solutions:

A plant lactobacillus nitrite reductase gene, its nucleotide sequence is shown in SEQ ID NO.1.

The recombinant plasmid containing the nitrite reductase gene of Lactobacillus plantarum according to claim 1, which contains a histidine tag.

The protein encoded by the above-mentioned Lactobacillus plantarum nitrite reductase gene.

The preparation method of the protein encoded by the above-mentioned reductase gene comprises the following steps:

(1) Using the nitrite reductase coding gene of Lactobacillus plantarum in NCBI as a template, the upstream and downstream primers were designed, and the designed enzyme cutting sites were EcoRI and XhoI;

(2) Collect the bacterial liquid of Lactobacillus plantarum (Lactobacillus plantarum) DMDL 9010 cultured at 37°C for 16 hours, extract its DNA, and use this as a template to amplify the gene fragment of Lactobacillus plantarum nitrite reductase ( As shown in SEQ ID NO.1); the preservation number of the Lactobacillus plantarum DMDL is CGMCC NO.5172;

(3) Use EcoRI and XhoI tool enzymes to cut the amplified fragment and plasmid pET-32a(+) into fragments with two cohesive ends respectively at 4°C; use a recovery kit to recover the above two fragments, and use T4DNA ligase Connected, then transferred into recombinant Escherichia coli, and screened with the antibiotic ampicillin to obtain positive engineering bacteria;

(4) The above-mentioned engineered bacteria are induced and expressed, and then purified by affinity chromatography to obtain the recombinant nitrite reductase.

The recombinant Escherichia coli is E.coli 5α or E.coli BL 21.

The upstream primer described in step (1) is 5'-CCGGAATTCATGAGTCAAAGCTTATGGCA-3' (EcoRI), and the downstream primer is 5'-CCGCTCGAGTTAATTCCGTACACTGTTTG-3' (Xhol).

The induced expression is induced by adding an inducer IPTG.

The concentration of the IPTG is 1 mmol/L.

The affinity chromatography is Ni sepharose affinity chromatography.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The present invention verifies that engineering bacteria can induce the expression of nitrite reductase (Nir) recombinant protein through affinity chromatography.

(2) The present invention can induce and express a large amount of nitrite reductase through IPTG, and can be applied to industrial production of nitrite reductase in large quantities.

(3) The present invention uses affinity chromatography to purify and obtain Nir recombinant protein with higher purity, which solves the difficult problem of isolating nitrite reductase with higher purity from Lactobacillus plantarum.

(4) The recombinant protein expressed by the engineering bacteria obtained by the present invention can effectively degrade nitrite, can be produced into enzyme preparations, and can effectively degrade nitrite in food; it can also be used as an antigen for Lactobacillus plantarum nitrite reduction Preparation of enzyme antibodies, and can be used to study its structure and properties.

Preservation information of Lactobacillus plantarum DMDL 9010: Lactobacillus plantarum DMDL 9010 was preserved on August 19, 2011 in the General Microbiology Center of China Committee for the Collection of Microorganisms, referred to as: CGMCC, address: Datun Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, deposit number: CGMCC NO.5172; the deposit certificate has been submitted in Chinese patent CN103060229A.

Description of drawings

Fig. 1 is that utilizes Lactobacillus plantarum DMDL 9010 genomic DNA as template to carry out PCR amplification product identification figure in embodiment 1, and wherein M is DNA molecular weight standard; 1,2 is negative control; 3,4 is PCR amplification product.

Figure 2 is a diagram of double enzyme digestion identification of the recombinant plasmid constructed in Example 2, wherein M is a DNA molecular weight standard; 1, 2 are double enzyme digestion recombinant plasmids; 3, 4 are negative controls.

Fig. 3 is the PCR product identification diagram of the recombinant plasmid constructed in Example 3, wherein M is the DNA molecular weight standard; 1 is the negative control; 2 is the PCR amplification product.

Fig. 4 is the crude enzyme solution (recombination group) obtained after inducing the expression of E.coli DH5α containing recombinant plasmid pET-32a (+)-Nir in Example 5, the E.coli of blank plasmid pET-32a (+) The protein content of the crude enzyme solution obtained after DH5α induced expression (empty plasmid group) and the crude enzyme solution obtained after the induced expression of E.coli DH5α without plasmid (wild type group) through the affinity chromatography column Trend.

Figure 5 is the SDS-PAGE electrophoresis image of the purified Nir protein, wherein M is the protein molecular weight standard; 1 is the purified protein; 2 is the negative control.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.

Example 1

The preparation of the genomic DNA of plant lactobacillus nitrite reductase comprises the steps:

1. PCR primer design

by PCR primers are designed from the DNA in: the upstream primer is 5'-CCGGAATTCATGAGTCAAAGCTTTATGGCA-3' (EcoRI), such as SEQ ID NO.2, and the downstream primer is 5'-CCGCTCGAGTTAATTCCGTACACTGTTTG-3' (Xhol), such as SEQ ID NO.3, And artificially synthesize this pair of upstream and downstream primers;

2. Genomic DNA extraction of Lactobacillus plantarum nitrite reductase (bacterial genomic DNA small purification kit TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0, a product of Takara Biotechnology Company, purchased from Guangzhou Ruizhen Biotechnology Co., Ltd. Company), wherein the solution is the solution in the kit, the specific steps are: (1) 3-4 mL of Lactobacillus plantarum DMDL 9010 culture is centrifuged at 12000rp for 1 min to discard the supernatant, and the bacterial cell pellet is placed in 1.5 mL Add 0.6mL of 10% lysozyme solution to the EP, mix by inverting for 5-10 times, then keep it at 37°C for at least 30min;

(2) Centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant carefully.

(3) Add 150 μL SP Buffer (containing RNase A1) to fully suspend the bacterial pellet.

(4) Add 20 μL of Lysozyme solution, mix evenly and let stand at room temperature for 5 minutes.

(5) Add 30 μL of EDTA Buffer, mix evenly and let stand at room temperature for 5 minutes.

(6) Add 200 μL of Solution A, shake vigorously and keep warm at 65°C for 10 minutes.

(7) Add 400μL of Solution B and shake vigorously for 15s.

(8) Add 650 μL of Solution C and mix evenly by inverting up and down.

(9) Centrifuge at 12,000 rpm for 1 min.

(10) First discard the upper organic phase, then transfer the aqueous phase solution (colorless lower layer) into a Filter Cup pre-placed on a 1.5mL centrifuge tube, and centrifuge at 12,000rpm for 1min.

(11) Discard the Filter Cup, add 450 μL of DB Buffer to the filtrate, and mix well.

(12) Place the Spin Column in the kit on the Collection Tube.

(13) Transfer the mixture of the above (11) to the Spin Column, centrifuge at 12,000rpm for 1min, and discard the filtrate.

(14) Add 500 μL of Rinse A to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

(15) Add 700 μL of Rinse B to the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

(16) Repeat operation step (15).

(17) Place the Spin Column on the Collection Tube and centrifuge at 12,000rpm for 1min.

(18) Place the Spin Column on a new 1.5mL centrifuge tube, add 60μL of sterilized distilled water to the center of the Spin Column membrane, and let stand at room temperature for 1min.

(19) Centrifuge at 12,000 rpm for 1 min to elute DNA.

3. Amplification of the target gene

Using the genomic DNA of Lactobacillus plantarum extracted in step 2 (13) as a template, use the PCR method to amplify the target gene fragment. The PCR reaction conditions are: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30s, annealing at 56°C for 30s, and extension at 72°C 2min, a total of 30 cycles, to obtain the genomic DNA of Lactobacillus plantarum nitrite reductase extended by PCR; meanwhile, a negative control was set up, and the above operations were performed using sterile water as a template.

Use 1% agarose electrophoresis to detect the PCR product of the target gene; the electrophoresis detection result is shown in Figure 1, and there is an obvious DNA band near 1600KD, which is the Lactobacillus plantarum Nir genomic DNA obtained above.

Example 2

Containing the construction of the recombinant expression vector of the genomic DNA of the PCR-expanded Lactobacillus plantarum Nir of the gained of embodiment 1, the steps are as follows:

The amplified DNA of the PCR-extended Lactobacillus plantarum nitrite reductase obtained in Example 1 and the plasmid pET-32a (+) (a product of Novagen, USA, purchased from Shanghai Jiran Biotechnology Co., Ltd.) were subjected to EcoRI respectively. , XhoI enzyme digestion, and the reaction conditions were overnight at 37°C for 14-16 hours, and then the fragment DNA and plasmid DNA were recovered respectively.

Ligate the above two fragments and react overnight in a refrigerator at 4°C for 14-16 hours to obtain the ligation product. The ligation reaction system is: recover 19 μL of pET-32a(+), recover 2.5 μL of the target gene fragment, and T4 DNA ligation Enzyme 1 μL, T4 DNA ligase buffer 2.5 μL;

Then the above ligation product was transformed into E.coli DH5α cells to obtain the recombinant plasmid pET-32a(+)-Nir;

The obtained recombinant plasmid pET-32a(+)-Nir was identified by PCR and double enzyme digestion method, the negative control group was double enzyme digestion of blank plasmid pET-32a(+), pET-32a(+)-Nir recombinant vector and The double enzyme digestion identification of the blank plasmid pET-32a(+) is shown in Figure 2. It can be clearly seen from the figure that the recombinant plasmid has two bands, while the blank plasmid has only one band, indicating that the constructed vector was passed through EcoRI and XhoI respectively. Two fragments were cut into two fragments at the sites of GAATTC and CTCGAG, indicating that the recombinant plasmid pET-32a(+)-Nir was successfully constructed;

During the PCR of the recombinant plasmid pET-32a(+)-Nir, the negative control group was the blank plasmid pET-32a(+) as a template for PCR, and the identification results were shown in Figure 3. It can be seen from the figure that the recombinant plasmid pET- 32a(+)-Nir was used as a template to amplify DNA fragments, but the blank plasmid pET-32a(+) could not amplify DNA fragments, again indicating that the recombinant plasmid pET-32a(+)-Nir was successfully constructed;

The sequence of the recombinant plasmid was determined by a conventional method with a DNA sequencer, and the sequencing results showed that the recombinant plasmid was successfully constructed and named pET-32a(+)-Nir;

The recombinant plasmid pET-32a(+)-Nir was cut at the 5'-GAATTC-3' and 5'-CTCGAG-3' sites by double enzymes EcoRI and XhoI, respectively, to obtain a 5.9kb vector fragment and a size consistent with the target fragment As shown in Figure 2, it can be seen from the figure that the recombinant plasmid pET-32a(+)-Nir was successfully constructed.

Example 3

The expression vector pET-32a(+)-Nir plasmid constructed in embodiment 2 is transferred in E.coli DH5α competent cells to obtain recombinant Escherichia coli, and its specific steps are as follows:

(1) Add 5 μL of the ligation product to 200 μL of E.coli DH5α competent cell suspension, mix gently, and place on ice for 30 minutes;

(2) Heat-shock in a water bath at 42°C for 90s, quickly remove and cool on ice for 30min;

(3) Add 400 μL of LB liquid medium, mix well, and then bathe in water at 37°C for 1 hour to restore the bacteria to normal growth state;

(4) Centrifuge at 4000rpm for 10 minutes, discard 400 μL of supernatant, take the remaining 200 μL of bacterial solution and evenly spread it on LB solid medium, place it face-up at 37°C for 30 minutes, and culture it upside down for 8-12 hours after the bacterial solution is completely absorbed by the medium;

(5) After picking a single clone and culturing it, it was identified by PCR. The positive clone culture was the successfully constructed recombinant E. coli E. coli DH5α containing the pET-32a(+)-Nir plasmid transformed with the Nir gene.

Inoculate successfully recombined E.coli DH5α into LB liquid medium (containing 50 mg/mL ampicillin and 50 μg/mL NaNO ₂ ) (recombinant group), culture at 37°C and 180 rpm on a shaker for 12 hours, and take 1 mL of bacterial liquid After centrifuging at 12000rpm for 1min, take 100μL of the supernatant and measure the _NaNO2 content. The _NaNO2 determination method is: take 100μL of the sample in a 25mL colorimetric tube, add 20mL of distilled water, then add 2mL of sulfanilic acid, mix well and avoid Light reaction for 3 minutes, then add 1 mL of naphthalene ethylenediamine hydrochloride, add water to make up to 25 mL, mix well, and react in the dark for 15 minutes, then measure the absorbance at 540 nm.

The control group was: E.coli DH5α without plasmid was inoculated in LB liquid medium (50 μg/mL NaNO ₂ ) (no plasmid group) and LB liquid medium (50 μg/mL NaNO ₂ ) without inoculation ( In the group without bacteria), after culturing on a shaking table at 37°C and 180rpm for 12h, take 1mL of the bacterial solution and centrifuge at 12,000rpm for 1min, then take 100μL of the supernatant, and measure the _NaNO2 content according to the above method. The absorbance values are shown in Table 1. It can be seen that _the NaNO that has added the recombinant engineering bacteria (recombination group) has been degraded by 90%, while in the two groups of control groups without plasmid group and bacteria-free group, _the NaNO content has no degradation.

Table 1 detects _undegraded NaNO Absorbance value results at 540nm

1, 2, and 3 in Table 1 refer to three groups of parallel experiments.

The LB liquid medium is as follows: Weigh 10 g of peptone, 5 g of yeast extract, and 10 g of NaCl according to the following formula, then dilute to 1 L with distilled water, sterilize at 121° C. for 20 minutes and cool before use.

The LB solid medium refers to adding 15 g of agar to the LB liquid medium, then distilling the volume to 1 L with distilled water, sterilizing at 121° C. for 20 minutes and cooling down.

Example 4

Utilize the recombinant E.coli DH5α of embodiment 3 gained to carry out the induced expression of plantaractobacillus nitrite reductase, specifically as follows:

Spread the positive clone strains obtained in Example 3 on the LB solid medium (containing 50 mg/mL ampicillin), cultivate overnight in a 37° C. Pick a single colony and inoculate it into a liquid medium (containing 50 mg/mL ampicillin), and cultivate overnight at 37°C and 200 rpm; Cultivate at 200 rpm to logarithmic growth phase (OD ₆₀₀ = 0.5), add IPTG with a final concentration of 1 mmol/L for induction, and induce culture at 37 °C and 200 rpm for 4 hours to obtain a culture solution, and collect the bacteria in the culture solution by centrifugation Body, extract Nir, its specific steps are as follows:

Centrifuge the culture medium at 8000rpm to collect the bacteria, wash twice with sterile water, add 10mL of buffer A pre-cooled at 4°C for every 100mL of culture medium, break the bacteria by ultrasonication in an ice bath for 10min, stop for 2s every 2s, after ultrasonication Take it out, put it in a refrigerator at 4°C for 12 hours, centrifuge at 4°C and 9000 rpm for 15 minutes, take the supernatant as Nir crude protein solution, and store it at 4°C for later use.

The buffer A preparation method: 0.2mol/L Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer preparation, 0.2mol/L Na ₂ HPO ₄ preparation, weigh 71.6g Na ₂ HPO ₄ .12H ₂ O solution In 1000mL water; 0.2mol/L NaH ₂ PO ₄ preparation, weigh 31.2g NaH ₂ PO ₄ .2H ₂ O dissolved in 1000mL water; take 0.2mol/L Na ₂ HPO ₄ solution 81mL and 0.2mol/L 19mL of NaH ₂ PO ₄ solution, mixed evenly to make 0.2mol/L Na ₂ HPO ₄ -NaH ₂ PO ₄ solution. Take the above 0.2mol/L Na ₂ HPO ₄ -NaH ₂ PO ₄ solution 100mL, add 29.22g of NaCl, 480g of urea to it, adjust the pH to 7.4, and set the volume to 1000mL, which is buffer A.

Set up control group simultaneously, be about to non-recombinant vector pET-32a (+) according to the method for embodiment 3, transfer in the cell of E.coli DH5α to obtain control group bacterial strain (empty plasmid group), press the induced expression process of the present embodiment simultaneously Induce expression. The cell protein of the empty plasmid group and the recombinant bacteria was subjected to ultrasonic disruption and centrifugation under the same conditions to collect the crude enzyme solution.

Example 5

Utilize the crude protein solution induced and expressed by the recombinant Escherichia coli obtained in Example 4 to purify to obtain high-purity nitrite reductase, specifically as follows:

First use the affinity chromatography column Ni Sepharose 6Fast Flow (GE Company) to purify the protein, the operation is as follows:

(1) Pack the column and wash it with 5 times the column volume of distilled water at a speed of about 2mL/min.

(2) Equilibrate the column with 5-10 column volumes of binding buffer B at a speed of about 2 mL/min.

(3) Add crude enzyme solution, rinse with binding buffer B at a speed of about 1mL/min, collect the rinsed solution in a 2mL centrifuge tube, 1.5mL per tube; at the same time, use a microplate reader to detect the absorbance of each tube solution at 280nm value until the absorbance value reaches the baseline.

(4) Elute with elution buffer C at a speed of about 1mL/min, collect the solution after washing with a 2mL centrifuge tube, 1.5mL in each tube; at the same time, use a microplate reader to detect the absorbance value of each tube solution at 280nm until Absorbance reaches baseline.

Wherein, the preparation method of binding buffer B: take the above-mentioned 0.2mol/L Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution 100mL, add 29.22g of NaCl, 480g of urea, and 1.36g of imidazole to it, adjust the pH to 7.4, Dilute to 1000mL, as binding buffer B.

The preparation method of elution buffer C: take 100 mL of the above-mentioned 0.2 mol/L Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer solution, add 29.22 g of NaCl, 480 g of urea, and 34 g of imidazole to adjust the pH to 7.4, Dilute to 1000mL as elution buffer C.

The control group is respectively the E.coli DH5α (empty plasmid group) of the blank plasmid pET-32a (+) and the E.coliDH5α (wild type group) without the plasmid, which are induced and expressed according to the induction expression method of Example 4, and carried out protein purification. The protein content change table obtained by the elution of these three groups of experiments is shown in Figure 4. It can be seen from the figure that the eluate of E.coli DH5α (wild type group) without plasmid does not contain protein, indicating that E.coli The protein expressed by DH5α cannot adhere to the affinity chromatography column NiSepharose 6Fast Flow, while the E.coli DH5α (recombinant group) containing the recombinant plasmid pET-32a(+)-Nir and the E.coli DH5α containing the blank plasmid pET-32a(+) E.coli DH5α (wild-type group) can elute the protein, indicating that the plasmid can be successfully expressed in E.coli DH5α, that is, the engineered bacteria E.coli DH5α can successfully express the recombinant protein, and the protein can be adsorbed to the affinity chromatography On the column Ni Sepharose 6Fast Flow, and can be eluted with imidazole solution. The purified recombinant protein was detected by SDS-PAGE of 12.5% separating gel, and its electrophoresis diagram is shown in Figure 5. It can be seen from the figure that the target protein Lactobacillus plantarum nitrite reductase in the recombinant bacteria with higher purity was obtained.

Claims (10)

1. a plant lactobacillus nitrite reductase gene, its nucleotide sequence is as shown in SEQ ID NO.1.

2. contain the recombinant plasmid of plant lactobacillus nitrite reductase gene described in claim 1.

3. recombinant plasmid according to claim 2, is characterized in that, contains histidine-tagged in this recombinant plasmid.

4. the protein of plant lactobacillus nitrite reductase genes encoding described in claim 1.

5. the preparation method of the protein of reductase gene coding described in claim 4, is characterized in that, comprises the steps:

(1) utilize EcoRI, XhoI toolenzyme that the gene fragment as shown in SEQ ID NO.1 and plasmid pET-32a (+) are cut into respectively to the fragment with two sticky ends at 4 DEG C; Adopt and reclaim test kit and reclaim above two fragments, and with the connection of T4DNA ligase enzyme, then proceed in recombination bacillus coli, utilize microbiotic penbritin to screen and can obtain positive engineering bacteria;

(2) by above-mentioned engineering bacteria through abduction delivering, adopt affinity chromatography purifying, obtain plant lactobacillus nitrite reductase.

6. method according to claim 5, is characterized in that, described recombination bacillus coli is E.coli5 α or E.coli BL21.

7. according to the method described in claim 5 or 6, it is characterized in that, described abduction delivering is for adding inductor IPTG to induce.

8. method according to claim 7, is characterized in that, the concentration of described IPTG is 1mmol/L.

9. according to the method described in claim 5 or 6, it is characterized in that, described affinity chromatography is Ni sepharose affinity chromatography.

10. method according to claim 7, is characterized in that, described affinity chromatography is Ni sepharose affinity chromatography.