CN113583926B - A kind of AKP heterologous expression engineering bacteria and its construction method and preparation method of high enzyme activity alkaline phosphatase - Google Patents

Abstract

The invention discloses an engineering bacterium for heterologous expression of AKP, which is obtained by taking Bacillus subtilis (B.subtilis) as a host and transferring a recombinant plasmid carrying the phoA gene of Escherichia coli (E.coli) into the host cell; The nucleotide sequence of the Escherichia coli phoA gene is shown in any one of SEQ ID NO.1‑3. Also disclosed are a construction method of the AKP heterologous expression engineering bacteria and a method for preparing high enzyme activity alkaline phosphatase using the AKP heterologous expression engineering bacteria. The concentrated enzyme protein fermented by the AKP heterologously expressed engineering bacteria is used as an enzyme preparation, which has good thermal stability, high enzyme activity, and can inhibit inflammation, which can enhance the immunity and disease resistance of piglets, and promote the growth and development of animals. Provide sustainable development momentum.

Description

A kind of AKP heterologous expression engineering bacteria and its construction method and preparation method of high enzyme activity alkaline phosphatase

technical field

The invention belongs to the field of biotechnology, and in particular relates to an AKP heterologous expression engineering bacterium, a construction method thereof, and a preparation method of alkaline phosphatase with high enzyme activity.

Background technique

In pig farming, weaning of piglets may increase the risk of infection with Gram-negative pathogens that can produce many virulence factors, including the endotoxin lipopolysaccharide (LPS), but the use of antibiotics is prohibited in animal husbandry . Because the non-standard use of antibiotics has led to serious problems, such as antibiotic resistance, serious adverse drug reactions, and drug residues in animal products, etc. Therefore, it is necessary to find effective measures to replace antibiotics to promote the high-quality development of animal husbandry. Under existing commercial conditions, alternatives to antibiotics can be used to improve animal productivity and help poultry and pigs reach their genetic potential, including probiotics, organic acids, auxins, prebiotics, synbiotics, enzymes, antimicrobial peptides, hyperimmune proteins Antibodies, clays and metals.

Alkaline phosphatase (AKP) is a non-specific phosphate monoesterase, which can catalyze the hydrolysis of phosphate monoesters to generate inorganic phosphate and corresponding alcohols, phenols or sugars, and can also catalyze the transfer reaction of phosphate groups . It can promote intestinal health in mammals, including prevention and reduction of intestinal inflammation and bacterial translocation, regulation of intestinal bacterial growth and local intestinal pH, local and systemic detoxification of intestinal endotoxin lipopolysaccharide and anti-inflammatory effects, etc. Function, AKP can be made into enzyme preparation as green feed additive. However, the current methods for producing AKP are difficult to obtain AKP with high enzyme activity, which limits the application of AKP in animal feed additives.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology, and provide a kind of AKP heterologous expression engineering bacteria and its construction method and the preparation method of high enzyme activity alkaline phosphatase.

In order to solve the problems of the technologies described above, the technical solution proposed by the present invention is:

A kind of AKP heterologous expression engineering bacterium, take Bacillus subtilis (B.subtilis) as host, obtain after being transformed into the recombinant plasmid that carries Escherichia coli (E.coli) phoA gene in this host cell; Said Escherichia coli The nucleotide sequence of the phoA gene is shown in any one of SEQ ID NO.1-3. Wherein, the nucleotide sequence of the untreated phoA gene is shown in SEQ ID NO.1, and the nucleotide sequence of the DelSigphoA gene obtained by deleting the signal peptide through Red/ET recombination technology is shown in SEQ ID NO.2. The nucleotide sequence of the DelSigD ₁₅₃ GD ₃₃₀ N gene obtained by ₁₅₃ GD ₃₃₀ N directed mutagenesis is shown in SEQ ID NO.3.

For the above-mentioned AKP heterologous expression engineering bacteria, preferably, the recombinant plasmid carrying the Escherichia coli phoA gene is obtained by inserting the Escherichia coli (E. coli) phoA gene into a shuttle vector, and the shuttle vector is a pP43NMK vector. More preferably, the mazE-mazF system is introduced into the recombinant plasmid, and the nucleotide sequence of the mazE-mazF plasmid stabilization system is shown in SEQ ID NO.9.

The construction method of the AKP heterologous expression engineering bacteria comprises the following steps:

(1) Insert the Escherichia coli (E.coli) phoA gene into the shuttle vector, and make a recombinant plasmid carrying the Escherichia coli phoA gene by alkaline lysis; the nucleotide sequence of the Escherichia coli phoA gene is as shown in SEQ ID NO.1 - Shown in any one of 3;

(2) Transform the recombinant plasmid obtained in the step (1) into Bacillus subtilis (B. subtilis) by means of competent transformation to obtain an engineering bacterium for heterologous expression of AKP.

In the above preparation method, preferably, the shuttle vector is a pP43NMK vector, or is prepared by the following method: Escherichia coli DH5α is screened and incubated with a Km antibody, and the genomic DNA of the pP43NMK plasmid is prepared by alkaline lysis extraction and ethanol precipitation, The homology was exposed by Pst I digestion, and the linear vector was recovered by electrophoresis running gel to obtain the shuttle vector.

Preferably, the preparation methods of the recombinant plasmids are as follows:

1) Method 1 specifically includes the following steps: using the primer pair P43-phoA-5/P43-phoA-3 to amplify the plasmid pGB-Cm-ccdA-Ptet-PhoA, and co-electroporating the obtained PCR product and the shuttle vector into L-arabinose-induced overexpression of DH5α cells, and direct cloning of the ETgA operon expressed under the control of the BAD promoter, the transformants were selected with kanamycin, the plasmid was extracted by alkaline lysis, and passed through Ava I/EcoR I and BamH I/Ava I further screen the correct restriction plasmid to the transformant, obtain the recombinant plasmid pP43NMK-PhoA-km; the nucleotide sequences of the primers to P43-phoA-5/P43-phoA-3 are respectively as SEQ Shown in ID NO.4, SEQ ID NO.5, the nucleotide sequence of described plasmid pGB-Cm-ccdA-Ptet-PhoA is shown in SEQ ID NO.7.

2) Method 2 specifically includes the following steps: using the primer pair P43-DelSig-5/P43-phoA-3 to amplify the plasmid pGB-Cm-ccdA-Ptet-PhoA, and co-electroporating the obtained PCR product and the shuttle vector into In the overexpression of DH5α cells induced by L-arabinose, transformants were selected after culturing on LB agar plates containing kanamycin, and the plasmids were extracted by alkaline lysis, and passed through BsrG I/Apal I, BamH I/Ava I and EcoR I/Pst I further screen the correct restriction plasmid for the transformant, obtain the recombinant plasmid pP43NMK-DelSigphoA-km; the nucleotide sequences of the primers to P43-DelSig-5/P43-phoA-3 are respectively as SEQ ID As shown in NO.6 and SEQ ID NO.5, the nucleotide sequence of the plasmid pGB-Cm-ccdA-Ptet-PhoA is shown in SEQ ID NO.7.

3) Method three specifically includes the following steps: the preparation method of the recombinant plasmid specifically includes the following steps: using the plasmid pGB-Cm-ccdA-Ptet-PhoA as a template, respectively using the primer pair A DelSigphoA-3/A DelSigphoA-5, BDelSigphoA -3/B DelSigphoA-5 and C DelSigphoA-3/C DelSigphoA-5 were amplified, the obtained PCR products were mixed with the shuttle vector, and co-transformed into pSC101-BAD-ETgA-tet in DH5G induced by Ara, Obtain recombinant plasmid pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km after recombination; The primer is to the nucleoside of A DelSigphoA-3/ADelSigphoA-5, B DelSigphoA-3/B DelSigphoA-5 and C DelSigphoA-3/C DelSigphoA-5 The acid sequences are respectively shown in SEQ ID NO.12-17.

4) Method 4 specifically includes the following steps: use the primer pair mazEF-5/mazEF-3 to amplify the mazE-mazF plasmid stable system, combine the obtained PCR product with the phoA gene group and co-electroporate the shuttle vector to L-arabinose Induced DH5α cell overexpression, ready to obtain; the nucleotide sequence of the mazE-mazF plasmid stabilization system is shown in SEQ ID NO.9, and the nucleotide sequences of the primer pair mazEF-5/mazEF-3 are respectively As shown in SEQ ID NO.10, SEQ ID NO.11. Genomic expression plasmid pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km has the most obvious effect on promoting AKP activity. The nucleotide sequence of the genomic expression plasmid pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km is shown in SEQ ID NO.3.

Based on a general technical concept, the present invention also provides a method for preparing alkaline phosphatase with high enzymatic activity, comprising the following steps: cultivating the above-mentioned AKP heterologously expressed engineering bacteria, extracting and separating the obtained fermented liquid, and using acetone Purify by precipitation method to obtain the alkaline phosphatase with high enzymatic activity. Preferably, the acetone concentration adopted by the acetone precipitation method is 37.5%-50% (v/v), and the acetone treatment time is 5-30min; more preferably, the acetone concentration adopted by the acetone precipitation method is 50% (v/v ), the acetone treatment time was 30min.

The present invention utilizes Red/ET homologous recombination technology combined with gene modification and site-directed mutation technology to construct an AKP heterologous expression engineering strain with high expression, high activity and high stability plasmid in B. subtilis, and directly isolates the fermentation broth Purification of AKP enzyme protein by propanol precipitation. In previous experiments, there was no research on the inflammatory response of Escherichia coli-derived AKP in cells. By treating IPEC-J2 cells, the expression levels of genes related to cell inflammation were detected. AKP can promote the intestinal health of mammals, including preventing and reducing intestinal inflammation and bacterial translocation, regulating the growth of intestinal bacteria and local intestinal pH, which can reduce the inflammatory response and protect the intestinal health of livestock and poultry.

Compared with prior art, the beneficial effect of the present invention is:

1. The AKP heterologous expression engineering bacterium obtained by the present invention uses the fermented concentrated enzyme protein as an enzyme preparation, AKP has good thermal stability, high enzyme activity, and has an inhibitory effect on inflammation, which can enhance piglet immunity and disease resistance, and promote The growth and development of animals provides sustainable development momentum for the pig industry, brings good economic benefits, and improves people's quality of life.

2. The construction method of AKP heterologous expression engineering bacteria is easy to operate and the operation cycle is short.

3. A method for preparing alkaline phosphatase with high enzyme activity, and the prepared AKP has high enzyme activity.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are For some embodiments of the present invention, those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is the screening of the tetracycline resistance promoter of AKP in E.coli;

Fig. 2 is to improve the enzymatic activity of AKP by removing signal peptide;

Fig. 3 is to improve the enzymatic activity of AKP by point mutation;

Figure 4 is the plasmid stability of DelSigD ₁₅₃ GD ₃₃₀ N in B. subtilis;

Figure 5 is the expression of AKP in Escherichia coli;

Fig. 6 is the distribution of AKP in fermented liquid;

Figure 7 is the precipitation of AKP with acid, acetone, ethanol and salt;

Fig. 8 is the influence of acetone amount on AKP activity;

Fig. 9 is the influence of incubation time on AKP activity under the situation that acetone concentration (v/v) is 50%;

Figure 10 is the thermal stability of AKP;

Figure 11 is the impact of incubation time on AKP activity at 120°C;

Figure 12 is the impact of AKP on the acid-base environment under the simulated gavage process;

Figure 13 is the effect of AKP on the expression of the nutrient transporter ASCT2 gene;

Figure 14 is the effect of AKP on the expression of tight junction protein CLDN3 gene;

Figure 15 is the effect of AKP on the expression of interleukin-6 gene;

Figure 16 is the effect of AKP on the expression of interleukin-8 gene;

Figure 17 is the effect of AKP on the expression of TNF-α gene.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

Unless otherwise defined, all technical terms used hereinafter have the same meanings as commonly understood by those skilled in the art. The terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the protection scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

Example 1:

An AKP heterologous expression engineering bacterium, using Bacillus subtilis (B.subtilis) as a host, is obtained after the recombinant plasmid carrying the phoA gene of Escherichia coli (E.coli) is transferred into the host cell; Escherichia coli phoA gene The nucleotide sequence is as shown in any one of SEQ ID NO.1-3; The recombinant plasmid is obtained after inserting the phoA gene of Escherichia coli into the pP43NMK vector; the mazE-mazF system has been introduced into the recombinant plasmid, and its nucleotide sequence is as shown in SEQ ID NO.1-3. ID NO.9 is shown.

The construction method of the above-mentioned AKP heterologous expression engineering bacteria comprises the following steps:

1. Screening of AKP gene:

Use the online tool NCBI (https://www.ncbi.nlm.nih.gov/) to search the open reading frame (start codon-stop codon) of the AKP gene from different species such as porcine and microbial sources; porcine Sangon and other eukaryotic AKP genes have introns, which can be obtained by artificial gene synthesis (bioengineering (Shanghai) Co., Ltd., https://mail.sangon.com/) and its open reading frame, microbial source (E.coli , B. subtilis and other microorganisms) AKP gene can obtain its open reading frame by PCR; the open reading frames of three AKP genes such as phoA, phoE and ncdF were collected. Using Red/ET recombination engineering technology, construct expression plasmids containing AKP genes from different species (the vector is pGB), and place the AKP gene behind the inducible promoter (Ptet), and add kana and chloramphenicol resistance to the plasmid Gene and ccdA-ccdB stable system gene. After the AKP gene library was established, the signal peptide cleavage site of the obtained AKP was analyzed by the online analysis software (www.cbs.dtu.dk/services/SignalP/), which laid the foundation for the subsequent construction of high-expression and high-activity AKP engineering strains.

AKP secretion gene DNA from different species was used to construct PGB vector plasmid, and the construct was transferred to heterologous host DH5α. The constructed plasmids are: pGB-Cm-ccdA-Ptet-PhoA (phoA from E.coli, its nucleotide sequence is shown in SEQ ID NO.7), pGB-Cm-ccdA-Ptet-PhoE (phoE from B.subtilis), pGB-Cm-ccdA-Ptet-NudF(nudF from B.subtilis), pGB-Cm-ccdA-Ptet-APN(apn frompig) were transformed into DH5α, inoculated in a 37°C incubator and cultured overnight, The correct construct was screened by kanamycin resistance, transferred to 1mL LB liquid medium, and after culturing in a constant temperature mixer at 900rpm for 12h, each plasmid clone was homogenized at OD 600, and 0.2μg/μL The tetracycline-induced experimental group was used as the control group without adding tetracycline, and the fermentation broth was incubated at 37° C. for 4 hours. AKP activity was compared under tetracycline-induced and non-inducible conditions, and highly active AKP genes were screened to optimize enzyme activity. It can be seen from Fig. 1 that the phoA gene enzyme activity derived from Escherichia coli was significantly increased under tetracycline-induced conditions by the AKP kit, and the activity increased by nearly 2.5 times; while the AKP activity from pigs and Bacillus subtilis did not There was no increase phenomenon, so the phoA gene derived from Escherichia coli was selected for subsequent enzyme activity optimization.

2. Construction of Escherichia coli-Bacillus subtilis shuttle vector (pP43NMK) and recombinant plasmid

The commercially available pP43NMK vector can be directly used. Enzyme cutting technology can also be used to process plasmid pP43NMK-APN-Cm-Km (its nucleotide sequence is shown in SEQ ID NO.8), cut and remove the APN gene, and prepare the shuttle vector (pP43NMK) of Escherichia coli-Bacillus subtilis, Specifically, Escherichia coli DH5α was screened and incubated with Km antibody, and pP43NMK genomic DNA was prepared by alkaline lysis extraction and ethanol precipitation, and the homology was revealed by Pst I digestion. After digestion at 37°C for 3 hours, electrophoresis was run for recovery. The 6845bp fragment was used as a linear vector to obtain the pP43NMK vector.

Use primers P43-phoA-5/P43-phoA-3 (its nucleotide sequence is shown in SEQ ID NO.4, SEQ ID NO.5 respectively), P43-phoE-5/P43-phoE-3, P43- nudF-5/P43-nudF-3 (see Table 1) prepared alkaline Phosphatase phoA, phoE, nudF genes. The nucleotide sequence of the phoA gene is shown in SEQ ID NO.1.

Co-electroporate 2ng PCR product and 2ngpP43NMK vector into L-arabinose-induced DH5α cell overexpression, and directly clone the ETgA operon expressed under the control of the BAD promoter, select transformants with kanamycin, and use alkaline The plasmid was extracted by lysis method, and the correct restriction plasmid was sequenced by Ava I/EcoR I and BamH I/Ava I, and the sequence comparison was performed using SeqMan software to identify the correct recombinant and detect its AKP activity. The recombinant plasmid pP43NMK-PhoA-km was obtained here. The target DNA sequence oligonucleotides used in the present invention are listed in Table 1.

Table 1: Oligonucleotide sequences used in the present invention

3. Construction of plasmid pP43NMK-DelSigphoA-km

In order to further improve the enzymatic activity of the AKP secretory gene phoA and achieve high-level production, the upstream primer P43-DelSig- 5 (its nucleotide sequence is shown in SEQ ID NO.6) and downstream primer P43-phoA-3 (its nucleotide sequence is shown in SEQ ID NO.5) from plasmid pGB-Cm-ccdA-Ptet-PhoA The DelSigphoA gene was prepared in the method, and its nucleotide sequence is shown in SEQ ID NO.2.

2ng of PCR product and 2ng of pP43NMK vector were co-electroporated into L-arabinose-induced overexpression of DH5α cells, and transformants were selected after overnight growth on LB agar plates containing kanamycin at 37°C. The plasmid was extracted by alkaline lysis method, and the transformant was further screened by BsrG I/Apal I, BamH I/Ava I and EcoR I/Pst I to sequence the correct restriction plasmid, and use SeqMan software for sequence comparison to identify the correct recombinant. The DelSigphoA recombinant and its plasmid pP43NMK-DelSigphoA-km were obtained.

In Figure 2, the signal peptide was deleted using Red/ET recombination technology to detect the enzyme activity of DelSigphoA recombinant bacteria. It was found that the enzyme activity increased significantly after deleting the signal peptide, which was 5 times higher than that of PhoA from the data.

4. Construction of plasmid pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km

In the present invention, D ₁₅₃ GD ₃₃₀ N directed mutagenesis can be performed on the active site of Escherichia coli AKP on the basis of removing the signal peptide by utilizing the characteristics of bioinformatics AKP, so as to improve the specific activity of AKP to a greater extent. According to bioinformatics site-directed mutation of the DelSigphoA recombinant gene, the nucleotide sequence of the DelSigD ₁₅₃ GD ₃₃₀ N gene obtained after mutation is shown in SEQ ID NO.3, and the specific operation steps are as follows: (1) pGB-Cm-ccdA-Ptet -PhoA, take 0.5 μL of the plasmid from -20 degrees and add 10 μL of water to dilute for use; (2) use A DelSigphoA-3/A DelSigphoA-5, B DelSigphoA-3/B DelSigphoA-5 and C DelSigphoA-3/C DelSigphoA respectively -5 is a primer (the nucleotide sequences are respectively shown in SEQ ID NO.11～17, see Table 1), and the product obtained in step 1 is used as a template to carry out 50 μL system PCR to obtain the products ADelSigphoA PCR, B DelSigphoA PCR and C For DelSigphoA PCR, perform gel cutting and recovery respectively; (3) Mix 1 μL of each of B DelSigphoA PCR and C DelSigphoA PCR obtained in step (2), mix and dilute 10 times as a PCR template, and simultaneously use B DelSigphoA-5/C DelSigphoA-3 As primers, PCR product B+C was obtained; the product B+C was recovered by gel cutting; (4) the product A DelSigphoA PCR obtained in step (2) and the product B+C obtained in step (3) were each mixed with 1 μL, and mixed Afterwards, dilute 10 times as the PCR template, and simultaneously use A DelSigphoA-5/C DelSigphoA-3 as primers to obtain the PCR product DelSigphoA PCR (A+B+C), and the PCR product is recovered through the column for subsequent use; (5) step (4) 2 μL of the obtained product and pP43NMK vector were mixed and co-transformed into Ara-induced pSC101-BAD-ETgA-tet in DH5G. After recombination, the product plasmid pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km was obtained; The correct clones were sequenced and verified with P43-seq2, phoA-seq and P43-seq4 primers (see Table 1), and verified that the correct clones were transformed into Bacillus subtilis to obtain the DelSigD ₁₅₃ GD ₃₃₀ N mutant and detect its alkaline phosphatase active.

It was found from Figure 3 that the AKP activity of the plasmid constructed with DelSigD ₁₅₃ GD ₃₃₀ N (ie, pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km) was the highest, which was 30 times higher than that of the DelSigPhoA plasmid.

5. Plasmid stability optimization

Adding antibiotics to the fermentation medium can prevent the loss of expression plasmids, but the introduction of antibiotics will disrupt the intestinal balance of animals, which will limit the application of AKP engineering strains. In order not to destroy the intestinal balance of animals and effectively prevent the loss of the plasmid and improve the stability of the plasmid, the _present invention can _also introduce the toxin- Antitoxin system (mazE-mazF plasmid stabilization system), the specific operation is as follows:

Query the mazE-mazF plasmid stabilization system (its nucleotide sequence is shown in SEQ ID NO.9) through the NCBI website, design and synthesize mazEF-5 and mazEF-3 primers (the nucleotide sequences are shown in SEQ ID NO.10, Shown in SEQ ID NO.11) mazEF was obtained by PCR and introduced into pP43NMK-DelSigD by co-electroporating the PCR product and 2ng pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-km into L-arabinose-induced overexpression of DH5α cells In the ₁₅₃ GD ₃₃₀ N-km expression plasmid, pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-Km-Cm-mazEF was obtained. By testing the stability of the plasmid, it was found that the stability of pP43NMK-DelSigD ₁₅₃ GD ₃₃₀ N-Km-Cm-mazEF was significantly improved ( FIG. 4 ).

6. High expression of AKP in Bacillus subtilis

Since Bacillus subtilis (B.subtilis) has the characteristics of super extracellular secretion of protein and metabolites and no production of endotoxin, B.subtilis is used as the host for heterologous expression of AKP. The recombinant plasmid was transformed into B. subtilis by competent transformation method to obtain AKP heterologous expression engineering strain DelSigphoA recombinant and DelSigD ₁₅₃ GD ₃₃₀ N mutant.

Transformation process of Bacillus subtilis: first activate Bacillus subtilis on LB plates in a 37°C constant temperature incubator; transfer the cells to 30°C, and incubate overnight with 1.8mL GMI medium at 800rpm constant temperature mixer; then 1mL Transfer the fermentation broth to 9mL GM I medium, and incubate on a 37°C fast shaker (250rpm/min) for 210min; then transfer 5mL of the culture broth to 45mL GM II, and use a 37°C slow shaker (125rpm/min) After incubation for 90 minutes, collect the competent cells and centrifuge at 5,000 g for 10 minutes; divide 200 μL per tube, add 30 μL of DNA, incubate at 30°C for 1 hour at a speed of 700 rpm/min, and spread 100 μL of bacteria to the corresponding tubes at 9000 rpm. Antibody plate and incubate overnight at 37 °C.

The configuration materials of medium GMI are as follows: K ₂ HPO ₄ 13.4 g/L, KH ₂ PO ₄ 5.7 g/L, (NH ₄ ) ₂ SO ₄ 1.9 g/L, Na ₃ C ₆ H ₅ O ₇ ·2H ₂ O 1g/L, MgSO ₄ ·7H ₂ O 0.2g/L, glucose 0.5 g/L, Casein 0.2g/L, Yeastextract 1 g/L.

The configuration materials of GM II are as follows: K ₂ HPO ₄ 13.4 g/L, KH ₂ PO ₄ 5.7 g/L, (NH ₄ ) ₂ SO ₄ 1.9 g/L, Na ₃ C ₆ H ₅ O ₇ 2H ₂ O 1g /L, MgSO ₄ .7H ₂ O 0.2g/L, glucose 0.5 g/L, Casein 0.04g/L, Yeastextract 0.04g/L, MgCl ₂ 0.51 g/L, CaCl ₂ 0.37 g/L.

It can be observed from Figure 5 that the AKP activity was tested and found that in B. subtilis, the enzyme activity of PhoA derived from Escherichia coli was still the highest, and it was up to 10 times higher than that of PhoA in Escherichia coli.

Example 2:

A kind of preparation method of alkaline phosphatase with high enzymatic activity, concrete operation and experimental steps are as follows:

1. Fermentation broth extraction

In order to clarify whether AKP is secreted intracellularly or extracellularly, the supernatant of the AKP heterologously expressed engineering strains (DelSigphoA recombinant, DelSigD ₁₅₃ GD ₃₃₀ N mutant) obtained in Example 1 and the bacterial cells were subpackaged and ultrasonically disrupted. The bacterial suspension was processed by the instrument, and the AKP activity of the supernatant and the bacterial cell disruption liquid was detected by using the AKP kit.

Operation method: Spread the AKP heterologous expression engineering strain on the solid medium and cultivate overnight in a 37°C incubator, select recombinants by Km resistance, use 1mL Km resistance liquid medium to culture in a 900rpm constant temperature mixer for 12h to expand Increase the bacteria, centrifuge the fermentation broth at 4°C and 9000rpm/min for 10min, separate the supernatant and bacteria, resuspend the bacteria in 1mL LB liquid medium, and then process the suspension with an ultrasonic breaker , to detect the AKP activity of the supernatant and the cell disrupted liquid. It can be seen from Figure 6 that AKP is mainly distributed in the supernatant.

2. Optimization of purification parameters of AKP

The supernatant of the fermentation broth of the AKP heterologous expression engineering strain obtained in step 1 was collected, AKP was precipitated by acid, acetone, ethanol, and salt precipitation, and the AKP activity of the resuspension was detected using an AKP kit.

Operation method: collect 1 mL of the supernatant of the fermentation broth, take (1) acid, and slowly add 2% HCl to the supernatant, so that the final pH of the supernatant is: 4, 4.5, 5, 5.5, 6 and react for 30 minutes; ( 2) Acetone, add an equal amount of 4°C acetone solution to the supernatant and react for 30 minutes; (3) Ethanol, add an equal amount of 4°C ethanol solution to the supernatant and react for 30 minutes; (4) Salt precipitation, add to the supernatant The same amount of saturated NaCl solution was reacted for 30 minutes to precipitate AKP, and the mixture was centrifuged at 4°C and 9000 rpm/min for 30 minutes to collect the precipitate and resuspend the precipitate in 100 μL LB liquid medium. The AKP activity of the resuspension was detected using the AKP kit.

It is found from Figure 7 that compared with the control group, there is no enzyme activity at acidic pHs of 4, 4.5, 5, 5.5, and 6, and under the condition of salt precipitation, AKP is also inactive. Under the treatment of acetone and ethanol, AKP was detected to have a certain activity, but the effect of precipitating enzyme protein was the best under the condition of acetone treatment, and the detection activity was close to that of the stock solution. Therefore, it can be inferred that acetone is the best precipitation reagent for AKP compared with other conditions.

A large amount of fermentation broth is required to collect high-concentration AKP. In order to optimize the amount of acetone used, the ratio of fermentation broth:acetone is 1:0, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6. After 30 min, the mixture was centrifuged at 4°C and 9000 rpm/min for 30 min, the precipitate was collected, and the precipitate was resuspended in 100 μL LB liquid medium, and the AKP activity of the resuspension was detected using the AKP kit. From Fig. 8, it is found that within a certain range, the greater the amount of acetone used, the higher the AKP enzyme activity, so the optimal amount of 50% (v/v) acetone concentration is selected.

In order to optimize the treatment time of acetone, the AKP strain supernatant was treated with the optimal treatment amount of acetone for 5min, 10min, 15min, 20min, 25min and 30min, and the AKP activity of the precipitated protein suspension was detected by the AKP kit.

It is found from Figure 9 that within a certain range, the longer the treatment time is, the higher the activity of AKP is, and the optimal treatment time is 30 min.

3. Thermal stability of AKP

In order to determine whether AKP can be concentrated and purified at high temperature, the thermal stability of AKP was tested. The AKP heterologous expression engineering strain (DelSigphoA recombinant, DelSigD ₁₅₃ GD ₃₃₀ N mutant) obtained in Example 1 was activated on Km-resistant solid medium, and cultured overnight in a 37°C incubator, using 1mL of Km-resistant liquid Culture medium was amplified and screened for bacteria, and the effects of different temperatures on the enzyme activity of engineering strains expressing AKP heterologously were detected. They were treated at 20°C, 37°C, 60°C, 80°C, 100°C, 120°C for 5 minutes, and the treated solution was Centrifuge at 1500rpm/min for 10min. Since there is a technology that can concentrate alkaline phosphatase at high temperature in a short time, the influence of different treatment time on the AKP activity of the AKP heterologous expression engineered strain at 120°C was detected, and they were placed at 120°C for 0, 5s, 20s, 1 min, 5 min, and centrifuge the treatment solution at 1500 rpm/min for 10 min. The results showed that when the temperature was 60°C, the relative activity of the DelSigphoA recombinant was significantly reduced, and only 40% remained. The enzymatic activity of the DelSigD ₁₅₃ GD ₃₃₀ N mutant did not change significantly. When the temperature was raised to 120° C., only 20% of the enzyme activities of the two AKP heterologous expression engineering strains remained ( FIG. 10 ).

Normal spray drying is actually kept at high temperature for 5 seconds, and the activity of AKP heterologous expression engineered strains is detected by shortening the treatment time at 120°C. After 1 min of treatment, the enzyme activity of the DelsigphoA recombinant was reduced by 60%, while that of the DelsigD ₁₅₃ GD ₃₃₀ N mutant was only reduced by 20%. The results showed that the thermostability of the DelSigD ₁₅₃ GD ₃₃₀ N mutant was more stable than that of DelSigphoA, and it still maintained high enzyme activity after being treated at 120° C. for 1 minute ( FIG. 11 ). And it can be seen that the two AKP heterologous expression engineering strains can be dried at high temperature and maintain high enzyme activity.

4. Simulation of dynamic measurement of AKP activity in vitro

In order to detect the activity changes of AKP affected by acid and alkali when passing through the digestive system of piglets by gavage, the pH of the stomach, small intestine, and large intestine of piglets was simulated externally.

The AKP heterologous expression engineering strain (DelSigphoA recombinant, DelSigD ₁₅₃ GD ₃₃₀ N mutant) obtained in Example 1 was cultured in 20 mL of Km-resistant liquid medium for 12 h to amplify and screen, and the fermentation broth was incubated at 4° C., 9000 rpm/ Centrifuge for 10 min under the condition of min, and take the supernatant. Slowly add 2% HCl to the supernatant, mix well, adjust its pH to 2.3-2.5, take out 1mL, then slowly add 2% NaOH to the supernatant, mix well, adjust its pH to 7.0-8.0, take out After 1mL, slowly add 2% HCl again and mix well, then adjust the pH to 6.0-7.0, take out 1mL, treat for 2h under each condition, then take out the reaction solution, and use the AKP kit to detect the pH of different parts respectively. AKP activity.

Through experiments on DelSigphoA and DelSigD ₁₅₃ GD ₃₃₀ N two AKP heterologous expression engineering strains, AKP kits were used to detect AKP activity at different pHs. The results are shown in Figure 12. It was found that the AKP activity was detected under acidic conditions (stomach: pH=2.3-2.5), and the relative enzyme activity of AKP of DelSigphoA and DelSigD ₁₅₃ GD ₃₃₀ N strains tended to zero; After 7-8) conditions, the AKP activity of the DelSigphoA strain was restored to 20%, while the AKP activity of DelSigD ₁₅₃ GD ₃₃₀ N was restored to 30%; adjusted to the large intestine (pH=6-7) condition, the AKP activity of the DelSigPhoA strain The AKP activity of the plasmid DelSigD ₁₅₃ GD ₃₃₀ N was restored to 55% and 65%. The experimental results show that AKP does not exhibit activity under acidic conditions, but it is not completely denatured. After returning from acidic conditions to alkaline conditions, AKP activity is restored to a certain extent. Therefore, it can be seen that AKP is more stable under weakly alkaline conditions. Strong, low or even no enzyme activity under acidic conditions.

5. Real-time quantitative PCR

The following is the reverse transcription reaction, choose TB Green Premix ExTaq ^TM II (RR820A, Takara) for Real Time PCR reaction. Transcript levels of pro-inflammatory factors were analyzed by real-time qPCR in the Applied Biosystems Real Time PCR System (Thermo Fisher Scientific).

After the reaction, confirm the amplification curve and melting curve of Real Time PCR, and make a standard curve when performing PCR quantification to ensure the specificity of the reaction. β-Actin antibody and cyclophilin-A (CycA) were used as internal reference genes. The amount of PCR product was calculated using the ΔΔCT method as previously described. To determine the stability of the reference gene, PCR amplicons were separated by electrophoresis in a 2% agarose gel, the resulting bands were verified by visualization in an agarose gel, and quantified by a Take3 ^TM multivolume plate.

The enzyme protein filtrates of DelSigphoA and DelSigD ₁₅₃ GD ₃₃₀ N AKP heterologous expression engineering strains were added to IPEC-J2 cell culture. In order to detect the effect of the supernatant of the bacterial strain on the expression of the IPEC-J2 cell transporter gene, the relative gene expression of amino acid transporter 2 (ASCT2) was determined, and Figure 13 shows that compared with the control group, DelSigphoA and DelSigD ₁₅₃ GD ₃₃₀ N had no significant effect on ASCT2, with p values of 0.276 and 0.109, respectively.

In order to detect the effect of AKP on the relative gene expression of tight junction proteins in IPEC-J2 cells, the gene expression of CLDN3 in the treatment group was detected. Figure 14 shows that compared with the control, DelSigphoA (P=0.002) and DelSigD ₁₅₃ GD ₃₃₀ N (p< 0) extremely significantly up-regulated the expression of claudin3 (CLDN3). In order to detect the anti-inflammatory properties of the supernatant of the strains on IPEC-J2 cells, the relative gene expression levels of pro-inflammatory cytokines IL-6, IL-8, and TNF-α were determined. It can be seen from Figure 15 that, compared with the control, DelSigphoA had no significant effect on IL-6 gene expression (P=0.863), while DelSigD ₁₅₃ GD ₃₃₀ N significantly down-regulated its expression (p=0.002). It can be seen from Figure 16 that DelSigphoA (p=0.457), DelSigD ₁₅₃ GD ₃₃₀ N (p=0.054) had no effect on the expression of IL-8. It can be seen from Figure 17 that DelSigphoA (p=0.336) had no significant effect, and DelSigD ₁₅₃ GD ₃₃₀ N (p=0.005) significantly down-regulated the expression of TNF-α gene expression. The beneficial effect of DelSigD ₁₅₃ GD ₃₃₀ N on inflammation suppression is thus evident.

In summary, AKP has promoting effects on mammalian intestinal health, including prevention and reduction of intestinal inflammation and bacterial translocation, regulation of intestinal bacterial growth and local intestinal pH. However, there are few studies on exogenous Escherichia coli-derived AKP treatment of cells to reduce the inflammatory response. In the present invention, by using Red/ET homologous recombination technology, combined with gene modification technology and site-directed mutation technology, an AKP heterologous expression engineering strain with high expression, high activity and high stability plasmid is constructed; B. subtilis with protein capacity serves as a host for overproduction of heterologous proteins, and under the double effect, the expression of AKP protein gene is increased to a greater extent. The activity is increased by adding exogenous AKP to achieve the purpose of regulating the growth of gastrointestinal flora and reducing gastrointestinal inflammation. The present invention conducts further experiments, and the effect experiment is carried out in small intestinal epithelial cells of pigs. The results show that E. coli exogenous AKP can effectively reduce the expression of pro-inflammatory cytokine genes in small intestinal epithelial cells of pigs, and effectively enhance the intestinal barrier structure in an inflammatory state. and function.

sequence listing

<110> Hunan Normal University

<120> A kind of AKP heterologous expression engineering bacteria and its construction method and preparation method of high enzyme activity alkaline phosphatase

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gctggctagg accgaaagca acgtaccatg gcaatatcga taagcccgca gtcacctgta 900

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aggattcaca agaacatacc ggcagtcagt tgcgtattgc ggcgtatggc ccgcatgccg 1320

ccaatgttgt tggactgacc gaccagaccg atctcttcta caccatgaaa gccgctctgg 1380

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gtgatcagac tgccgctctg cgtgattctc ttagcgataa acctgcaaaa aatattattt 180

tgctgattgg cgatgggatg ggggactcgg aaattactgc cgcacgtaat tatgccgaag 240

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ctaccgcaga gttgcagggc gccacgcccg ctgcgctggt ggcacatgtg acctcgcgca 540

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aacaggcaca ggcgcgtggt tatcagttgg tgagcgatgc tgcctcactg aattcggtga 780

cggaagcgaa tcagcaaaaa cccctgcttg gcctgtttgc tgacggcaat atgccagtgc 840

gctggctagg accgaaagca acgtaccatg gcaatatcga taagcccgca gtcacctgta 900

cgccaaatcc gcaacgtaat gacagtgtac caaccctggc gcagatgacc gacaaagcca 960

ttgaattgtt gagtaaaaat gagaaaggct ttttcctgca agttgaaggt gcgtcaatcg 1020

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aagccgtaca acgggcgctg gaattcgcta aaaaggaggg taacacgctg gtcatagtca 1140

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gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa 480

agggaaacac ctactactga tagtatgccg ccattattac gacaagctat cgaattattt 540

gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat atgcggatta 600

gaaaaacaac ttaaatgtga aagtgggtct taaaagcagc ataacctttt tccgtgatgg 660

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atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 780

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 840

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 900

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 960

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1020

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 1080

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 1140

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 1200

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 1260

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 1320

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 1380

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caccatcgaa tggccagatg attaattcct aatttttgtt gacactctat cattgataga 1500

gttattttac cactccctat cagtgataga gaaaagtgaa atgaatagtt cgacaaaaat 1560

ctagaggaag atatatgaaa caaagcacta ttgcactggc actcttaccg ttactgttta 1620

cccctgtgac aaaagcccgg acaccagaaa tgcctgttct ggaaaaccgg gctgctcagg 1680

gcgatattac tgcacccggc ggtgctcgcc gtttaacggg tgatcagact gccgctctgc 1740

gtgattctct tagcgataaa cctgcaaaaa atattatttt gctgattggc gatgggatgg 1800

gggactcgga aattactgcc gcacgtaatt atgccgaagg tgcgggcggc ttttttaaag 1860

gtatagatgc cttaccgctt accgggcaat acactcacta tgcgctgaat aaaaaaaccg 1920

gcaaaccgga ctacgtcacc gactcggctg catcagcaac cgcctggtca accggtgtca 1980

aaacctataa cggcgcgctg ggcgtcgata ttcacgaaaa agatcaccca acgattctgg 2040

aaatggcaaa agccgcaggt ctggcgaccg gtaacgtttc taccgcagag ttgcaggatg 2100

ccacgcccgc tgcgctggtg gcacatgtga cctcgcgcaa atgctacggt ccgagcgcga 2160

ccagtgaaaa atgtccgggt aacgctctgg aaaaaggcgg aaaaggatcg attaccgaac 2220

agctgcttaa cgctcgtgcc gacgttacgc ttggcggcgg cgcaaaaacc tttgctgaaa 2280

cggcaaccgc tggtgaatgg cagggaaaaa cgctgcgtga acaggcacag gcgcgtggtt 2340

atcagttggt gagcgatgct gcctcactga attcggtgac ggaagcgaat cagcaaaaac 2400

ccctgcttgg cctgtttgct gacggcaata tgccagtgcg ctggctagga ccgaaagcaa 2460

cgtaccatgg caatatcgat aagcccgcag tcacctgtac gccaaatccg caacgtaatg 2520

acagtgtacc aaccctggcg cagatgaccg acaaagccat tgaattgttg agtaaaaatg 2580

agaaaggctt tttcctgcaa gttgaaggtg cgtcaatcga taaacaggat catgctgcga 2640

atccttgtgg gcaaattggc gagacggtcg atctcgatga agccgtacaa cgggcgctgg 2700

aattcgctaa aaaggagggt aacacgctgg tcatagtcac cgctgatcac gcccacgcca 2760

gccagattgt tgcgccggat accaaagctc cgggcctcac ccaggcgcta aataccaaag 2820

atggcgcagt gatggtgatg agttacggga actccgaaga ggattcacaa gaacataccg 2880

gcagtcagtt gcgtattgcg gcgtatggcc cgcatgccgc caatgttgtt ggactgaccg 2940

accagaccga tctcttctac accatgaaag ccgctctggg gctgaaataa gtcgacgtca 3000

ggtggcactt ttcggggaaa tgtgcgcgga cccctataga cgtcaggtgg cacttttggc 3060

gaaaatgaga cgttgatcgg cacgtaagag gttccaactt tcaccataat gaaataagat 3120

cactaccggg cgtatttttt gagttatcga gattttcagg agctaaggaa gctaaaatgg 3180

agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt 3240

ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta 3300

cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc ttattcaca 3360

ttcttgcccg cctgatgaat gctcatccgg aattccgtat ggcaatgaaa gacggtgagc 3420

tggtgattg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt 3480

tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc 3540

aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata 3600

tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgatta aacgtggcca 3660

atatggaca cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca 3720

aggtgctgat gccgctggcg attcaggttc atcatgccgt cccagcccgc ctaatgagcg 3780

ggcttttttt tgaacaaaag gctgcagctg gacttctaga gctagcctcg agtgtcaaac 3840

atgagaattc tagagtcaac ataacgaaag gtaaaataca taaggcttac taaaagccag 3900

ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta 3960

tacccgaagt atgtcaaaaa gaggtgtgct atgaagcagc gtattacagt gacagttgac 4020

agcgacagct atcagttgct caaggcatat gatgtcaata tctccggtct ggtaagcaca 4080

accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa 4140

gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg 4200

gactggtga 4209

<210> 8

<211> 9221

<212>DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 8

tagagggaat tgatgaatta tatcaacata ttaagccttt gggcattttg caccccaata 60

catcattaaa agatcagtgg tgggatgaac gagactttgc agtaattgat cccgacaaca 120

atttgattag cttttttcaa caaataaaaa gctaaaatct atttattaatc tgttcagcaa 180

tcgggcgcga ttgctgaata aaagatacga gagacctctc ttgtatcttt tttattttga 240

gtggttttgt ccgttacact agaaaaccga aagacaataa aaattttat cttgctgagt 300

ctggctttcg gtaagctaga caaaacggac aaaataaaaa ttggcaaggg tttaaaggtg 360

gagatttttt gagtgatctt ctcaaaaaat actacctgtc ccttgctgat ttttaaacga 420

gcacgagagc aaaacccccc tttgctgagg tggcagaggg caggtttttt tgtttctttt 480

ttctcgtaaa aaaaagaaag gtcttaaagg ttttatggtt ttggtcggca ctgccgcgcc 540

tcgcagagca cacactttat gaatataaag tatagtgtgt tatactttac ttggaagtgg 600

ttgccggaaa gagcgaaaat gcctcacatt tgtgccacct aaaaaggagc gatttacata 660

tgagttatgc agtttgtaga atgcaaaaag tgaaatcagc tggactaaaa ggcatgcaat 720

ttcataatca aagagagcga aaaagtagaa cgaatgatga tattgaccat gagcgaacac 780

gtgaaaatta tgatttgaaa aatgataaaa atattgatta caacgaacgt gtcaaagaaa 840

ttattgaatc acaaaaaaca ggtacaagaa aaacgaggaa agatgctgtt cttgtaaatg 900

agttgctagt aacatctgac cgagattttt ttgagcaact ggatcctgat aggtggtatg 960

ttttcgcttg aacttttaaa tacagccatt gaacatacgg ttgatttaat aactgacaaa 1020

catcaccctc ttgctaaagc ggccaaggac gccgccgccg gggctgtttg cgttcttgcc 1080

gtgatttcgt gtaccattgg tttacttatt tttttgccaa ggctgtaatg gctgaaaatt 1140

cttacattta ttttacattt ttagaaatgg gcgtgaaaaa aagcgcgcga ttatgtaaaa 1200

tataaagtga tagcggtacc attatgggta agagaggaat gtacgcatgc gcaactcgac 1260

caagacatct ctattactgg ccggcttatg cacagcggcc caaatggttt ttgtaacaca 1320

tgcctcagct gcacaaggtg gctgggttct gcttcttctt ggtttaagac tgccgctttc 1380

actgggcttt atcccggttg aagaggagga tccggcattc tggaatagac aagctgcaca 1440

agcgcttgac gtggcgaaaa agctgcagcc gattcaaacg gcggcgaaga atctgatttt 1500

atttcttggc gacggcatgg gcgtgagcac agttacggcg acacgcattt taaaaggaca 1560

gatgaacgga aagcccggtc cggaaacacc gcttgcaatg gatcgcttcc cgtatttagc 1620

gctgagcaag acgtataacg tggatcgcca agtgccggat tcagcgggaa caacaacagc 1680

gtatttatgc ggcgtgaaga cgaatatgaa agtgatcggc gtgagcgcag cagcacgcta 1740

tgatcagtgc aacacaacgc aaggtaacga ggtgatcagc gtgatgaatc gcgcgaagaa 1800

ggcgggaaaa tcagttggcg tggttacgac gacgcgcgtg caacatgcgt caccggctgg 1860

tgcgtacgca cacacagtga accgcaactg gtatagcgat gcggaccttc cggcggaagc 1920

gaaaaaaaac ggatgtcaag atattagcac acagcttgtg tataatatgg atattgatgt 1980

gattttaggc ggcggccgca aatatatgtt cccggaagga acgccggacc cggaatatcc 2040

cgataacccg cgccaaaatg gcgtgcgcaa agataagcgc aatttagtgc aagaatggca 2100

agcgaaacat caaggcgcac gctacgtgtg gaatcgcaca gcactgattc aagctagcca 2160

agatccgtct ttaacgcatt taatgggttt atttgaaccg ggagacatga agtatgaaaa 2220

agagcgcgat ctgtctcgcg atccgtctct ggtggaaatg acggaagttg cactgcgcct 2280

tctgagccgc aatccgcgcg gctttttttt attcgtggaa ggaggccgca tcgatcatgg 2340

acatcatgag ggcattgcgt accgcgcgct gatcgaaacg gtggtgtttg acacggcgat 2400

cgataaagct ggtcagctga caagcgaaga ggatacactg acgcttgtga cagcggatca 2460

cagccatgtg tttacgtacg gaggctaccc gcttagagga agcagcgttt ttggtttagc 2520

ggacggcaag gcgagcgatg gcaaagcgta tacaagcatt ttatatggaa acggcccggg 2580

ctataaactg agcgaaggcg cacgcccgga tgtggatgaa acgaaatcac gcgacccggc 2640

gtacgtgcaa caagcagcgg tgccgcttgg cgcagaaaca catggaggcg aggatgtggc 2700

agtgtttgca cgcggcccga gagcgcatct ggttcatggc gtgcaagaac aaagctttgt 2760

ggcgcatgtg atggcattcg cggcgtgttt agaaccgtat acatcagact gcgatctgcc 2820

gccgccttct ggtccgacag cagctggtca tccgggcccc gctgcgtgta catctttact 2880

tgcacttctg gctggtgcgc tgcttcttct gcttgcaccg gcgctgcatt gatttaaata 2940

agcttggcga agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga 3000

agccctgggc caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact 3060

ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag 3120

gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc 3180

aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc 3240

agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt 3300

tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta 3360

tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt 3420

tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc 3480

agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc 3540

ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca 3600

gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca 3660

aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg 3720

tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt 3780

ggcagggcgg ggcgtaaccc agcccgccta atgagcgggc ttttttttga acaaaaggct 3840

gcagctggac ttgatatcgc tcacaattcc acacaacata cgagccggaa gcataaagtg 3900

taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 3960

cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 4020

gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 4080

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 4140

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4200

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4260

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4320

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4380

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 4440

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 4500

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 4560

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 4620

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 4680

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 4740

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 4800

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 4860

cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 4920

ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 4980

tgacagctcg agttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc 5040

aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc 5100

gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac 5160

atcaatacaa cttattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc 5220

atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg 5280

ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt 5340

cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca 5400

aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc 5460

tgaatcagga tattcttcta atacctggaa tgctgttttc ccagggatcg cagtggtgag 5520

taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc 5580

cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc 5640

atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc 5700

tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga 5760

atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt 5820

tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 5880

tattagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 5940

cgtctaagaa accatttatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 6000

ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 6060

gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 6120

agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact 6180

gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat 6240

caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc 6300

ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac 6360

gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtgaattcc ttaaggaacg 6420

tacagacggc ttaaaagcct ttaaaaacgt ttttaagggg tttgtagaca aggtaaagga 6480

taaaacagca caattccaag aaaaacacga tttagaacct aaaaagaacg aatttgaact 6540

aactcataac cgagaggtaa aaaaagaacg aagtcgagat cagggaatga gtttataaaa 6600

taaaaaaagc acctgaaaag gtgtcttttt ttgatggttt tgaacttgtt ctttcttatc 6660

ttgatacata tagaaataac gtcattttta ttttagttgc tgaaaggtgc gttgaagtgt 6720

tggtatgtat gtgttttaaa gtattgaaaa cccttaaaat tggttgcaca gaaaaaccccc 6780

atctgttaaa gttataagtg actaaacaaa taactaaata gatgggggtt tcttttaata 6840

ttatgtgtcc taatagtagc atttattcag atgaaaaatc aagggtttta gtggacaaga 6900

caaaaagtgg aaaagtgaga ccatggagag aaaagaaaat cgctaatgtt gattactttg 6960

aacttctgca tattcttgaa tttaaaaagg ctgaaagagt aaaagattgt gctgaaatat 7020

tagagtataa acaaaatcgt gaaacaggcg aaagaaagtt gtatcgagtg tggttttgta 7080

aatccaggct ttgtccaatg tgcaactgga ggagagcaat gaaacatggc attcagtcac 7140

aaaaggttgt tgctgaagtt attaaacaaa agccaacagt tcgttggttg tttctcacat 7200

taacagttaa aaatgtttat gatggcgaag aattaaataa gagtttgtca gatatggctc 7260

aaggatttcg ccgaatgatg caatataaaa aaattaataa aaatcttgtt ggttttatgc 7320

gtgcaacgga agtgacaata aataataaag ataattctta taatcagcac atgcatgtat 7380

tggtatgtgt ggaaccaact tattttaaga atacagaaaa ctacgtgaat caaaaacaat 7440

ggattcaatt ttggaaaaag gcaatgaaat tagactatga tccaaatgta aaagttcaaa 7500

tgattcgacc gaaaaataaa tataaatcgg atatacaatc ggcaattgac gaaactgcaa 7560

aatatcctgt aaaggatacg gattttatga ccgatgatga agaaaagaat ttgaaacgtt 7620

tgtctgattt ggaggaaggt ttacaccgta aaaggttaat ctcctatggt ggtttgttaa 7680

aagaaataca taaaaaatta aaccttgatg acacagaaga aggcgatttg attcatacag 7740

atgatgacga aaaagccgat gaagatggat tttctattat tgcaatgtgg aattgggaac 7800

ggaaaaatta ttttattaaa gagtagttca acaaacgggc cagtttgttg aagattagat 7860

gctataattg ttattaaaag gattgaagga tgcttaggaa gacgagttat taatagctga 7920

ataagaacgg tgctctccaa atattcttat ttagaaaagc aaatctaaaa ttatctgaaa 7980

agggaatgag aatagtgaat ggaccaataa taatgactag agaagaaaga atgaagattg 8040

ttcatgaaat taaggaacga atattggata aatatgggga tgatgttaag gctattggtg 8100

tttatggctc tcttggtcgt cagactgatg ggccctattc ggatattgag atgatgtgtg 8160

tcatgtcaac agaggaagca gagttcagcc atgaatggac aaccggtgag tggaaggtgg 8220

aagtgaattt tgatagcgaa gagattctac tagattatgc atctcaggtg gaatcagatt 8280

ggccgcttac acatggtcaa tttttctcta ttttgccgat ttatgattca ggtggatact 8340

tagagaaagt gtatcaaact gctaaatcgg tagaagccca aacgttccac gatgcgattt 8400

gtgcccttat cgtagaagag ctgtttgaat atgcaggcaa atggcgtaat attcgtgtgc 8460

aaggaccgac aacatttcta ccatccttga ctgtacaggt agcaatggca ggtgccatgt 8520

tgattggtct gcatcatcgc atctgttata cgacgagcgc ttcggtctta actgaagcag 8580

ttaagcaatc agatcttcct tcaggttatg accatctgtg ccagttcgta atgtctggtc 8640

aactttccga ctctgagaaa cttctggaat cgctagagaa tttctggaat gggattcagg 8700

agtggacaga acgacacgga tatatagtgg atgtgtcaaa acgcatacca ttttgaacga 8760

tgacctctaa taattgttaa tcatgttggt tacgtattta ttaacttctc ctagtattag 8820

taattatcat ggctgtcatg gcgcattaac ggaataaagg gtgtgcttaa atcgggccat 8880

tttgcgtaat aagaaaaagg attaattatg agcgaattga attaataata aggtaataga 8940

tttacattag aaaatgaaag gggattttat gcgtgagaat gttacagtct atcccggcat 9000

tgccagtcgg ggatattaaa aagagtatag gttttttg ggataaagta ggtttcactt 9060

tggttcacca tgaagatgga ttcgcagttc taatgtgtaa tgaggttcgg attcatctat 9120

gggaggcaag tgatgaaggc tggcgcctcg tagtaatgat tcaccggttt gtacaggtgc 9180

ggagtcgttt attgctggta ctgctagttg ccgcattgaa g 9221

<210> 9

<211> 700

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 9

atgatccaca gtagcgtaaa gcgttgggga aattcaccgg cggtgcggat cccggctacg 60

ttaatgcagg cgctcaatct gaatattgat gatgaagtga agattgacct ggtggatggc 120

aaattaatta ttgagccagt gcgtaaagag cccgtattta cgcttgctga actggtcaac 180

gacatcacgc cggaaaacct ccacgagaat atcgactggg gagagccgaa agataaggaa 240

gtctggtaat ggtaagccga tacgtacccg atatgggcga tctgatttgg gttgattttg 300

acccgacaaa aggtagcgag caagctggac atcgtccagc tgttgtcctg agtcctttca 360

tgtacaacaa caaaacaggt atgtgtctgt gtgttccttg tacaacgcaa tcaaaaggat 420

atccgttcga agttgtttta tccggtcagg aacgtgatgg cgtagcgtta gctgatcagg 480

taaaaagtat cgcctggcgg gcaagaggag caacgaagaa aggaacagtt gccccagagg 540

aattacaact cattaaagcc aaaattaacg tattgattgg gtagtgttac taactcactt 600

ctattctggt cacaggtttc cacctgacag acccgcaatt ttcaggacaa ttcaatgaat 660

caaatcgacc gtttgctcac tattatgcag cgcctgcgcg 700

<210> 10

<211> 58

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 10

cctaatgact ggcttttata aaggaggaag cttcatgatc cacagtagcg taaagcgt 58

<210> 11

<211> 59

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 11

gcgttttgac acatccacta tatatccgtg tcgactaccc aatcagtacg ttaattttg 59

<210> 12

<211> 59

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 12

cggcccaaat ggtttttgta acacatgcct cagctgcacg gacaccagaa atgcctgtt 59

<210> 13

<211> 21

<212>DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 13

ctgcaactct gcggtagaaa c 21

<210> 14

<211> 45

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 14

gtttctaccg cagagttgca gggcgccacg cccgctgcgc tggtg 45

<210> 15

<211> 44

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 15

cccacaagga ttcgcagcat gattctgttt atcgattgac gcac 44

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 16

catgctgcga atccttgtgg g 21

<210> 17

<211> 59

<212> DNA

<213> Artificial Sequence (ArtificialSequence)

<400> 17

gttgtgtgga attgtgagcg atatcaagtc cagctgcagt tatttcagcc ccagagcgg 59

Claims (7)

1. An AKP heterologous expression engineering bacterium, which is characterized in that bacillus subtilis is usedBacteria (A), (B)B.subtilis) As a host, into which a vector carrying Escherichia coli (E.coli) is transferredE.coli）phoARecombinant plasmids of the genes; the Escherichia coliphoAThe nucleotide sequence of the gene is shown in any one of SEQ ID NO. 1-3;

said vector carrying Escherichia coliphoAThe recombinant plasmid of the gene is composed of Escherichia coli (E.coli)E.coli）phoAThe gene is inserted into a shuttle vector, and the shuttle vector is a pP43NMK vector;

the recombinant plasmid is introduced with a mazE-mazF system, and the nucleotide sequence of the mazE-mazF plasmid stabilizing system is shown in SEQ ID NO. 9.

2.A construction method of AKP heterologous expression engineering bacteria is characterized by comprising the following steps:

(1) Escherichia coli (A), (B) and (C)E.coli）phoAGene inserting shuttle vector, preparing into Escherichia coli by alkaline cracking methodphoARecombinant plasmids of the genes; the Escherichia coliphoAThe nucleotide sequence of the gene is shown in any one of SEQ ID NO. 1-3;

(2) Transforming the recombinant plasmid obtained in the step (1) into bacillus subtilis by using a competent transformation method (a)B.subtilis) Obtaining AKP heterologous expression engineering bacteria;

the shuttle vector is a pP43NMK vector or is prepared by the following method: screening and incubating escherichia coli DH5 alpha by using Km antibody, preparing genome DNA of pP43NMK plasmid by an alkaline lysis extraction method and an ethanol precipitation method, digesting by using Pst I to expose homology, and recovering linear vector by electrophoresis gel running to obtain the shuttle vector.

3. The method according to claim 2, wherein the recombinant plasmid is prepared by a method comprising the steps of: amplifying a plasmid pGB-Cm-ccdA-Ptet-PhoA by adopting a primer pair P43-PhoA-5/P43-PhoA-3, co-electroporating an obtained PCR product and a shuttle vector into overexpression of DH5 alpha cells induced by L-arabinose, directly cloning an ETgA operon expressed under the control of a BAD promoter, selecting a transformant by using kanamycin, extracting the plasmid by adopting an alkaline lysis method, and further screening correct restriction plasmids for the transformant by using Ava I/EcoR I and BamH I/Ava I to obtain a recombinant plasmid pP43NMK-PhoA-km;

the nucleotide sequences of the primer pair P43-PhoA-5/P43-PhoA-3 are respectively shown as SEQ ID NO.4 and SEQ ID NO.5, and the nucleotide sequence of the plasmid pGB-Cm-ccdA-Ptet-PhoA is shown as SEQ ID NO. 7.

4. The construction method according to claim 2, wherein the method for preparing the recombinant plasmid specifically comprises the following steps: amplifying the plasmid pGB-Cm-ccdA-Ptet-PhoA by adopting a primer pair P43-DelSig-5/P43-PhoA-3, co-electroporating the obtained PCR product and a shuttle vector into L-arabinose-induced DH5 alpha cell overexpression, culturing a transformant on an LB agar plate containing kanamycin, extracting the plasmid by adopting an alkaline lysis method, and further screening correct restriction plasmids for the transformant by BsrG I/Apal I, bamH I/Ava I and EcoR I/Pst I to obtain a recombinant plasmid pP43NMK-DelSigphoA-km；

The nucleotide sequences of the primer pair P43-DelSig-5/P43-PhoA-3 are respectively shown as SEQ ID No.6 and SEQ ID No.5, and the nucleotide sequence of the plasmid pGB-Cm-ccdA-Ptet-PhoA is shown as SEQ ID No. 7.

5. The method according to claim 2, wherein the recombinant plasmid is prepared by a method comprising the steps of: plasmid pGB-Cm-ccdA-Ptet-PhoA is taken as a template, and a primer pair A DelSig is respectively adoptedphoA-3/ A DelSigphoA-5，B DelSigphoA-3/ B DelSigphoA-5 and C DelSigphoA-3/ C DelSigphoA-5, amplifying, mixing the obtained PCR product with a shuttle vector, co-transforming into Ara-induced pSC101-BAD-ETgA-tet in DH5G, and recombining to obtain a recombinant plasmid pP43NMK-DelSigD ₁₅₃ G-D ₃₃₀ N；

The primer pair A DelSigphoA-3/ A DelSigphoA-5，B DelSigphoA-3/ B DelSigphoA-5 and C DelSigphoA-3/ C DelSigphoAThe nucleotide sequences of-5 are respectively shown in SEQ ID NO. 12-17.

6. The method of constructing according to any one of claims 2 to 5, wherein the method of preparing the recombinant plasmid specifically comprises the steps of: amplifying the mazE-mazF plasmid stabilization system by adopting a primer pair mazEF-5/mazEF-3, and performing co-electroporation on the obtained PCR product and the recombinant plasmid to the overexpression of the DH5 alpha cells induced by the L-arabinose to obtain the recombinant plasmid;

the nucleotide sequence of the mazE-mazF plasmid stabilization system is shown as SEQ ID No.9, and the nucleotide sequences of the primer pair mazEF-5/mazEF-3 are respectively shown as SEQ ID No.10 and SEQ ID No. 11.

7. A method for preparing high-enzyme activity alkaline phosphatase, which is characterized by comprising the following steps: culturing the AKP heterologous expression engineering bacterium obtained by the construction method of any one of claims 1 or 2-6, extracting and separating the obtained fermentation broth, and purifying by acetone precipitation to obtain the high-enzyme-activity alkaline phosphatase.

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