CN110305824A - Bacillus subtilis exhibiting organophosphate hydrolase and its preparation method - Google Patents

Abstract

The present invention relates to the Ko subtilis and preparation method thereof for showing organophosphor hydrolytic enzyme, belong to field of biotechnology, for the biological prosthetic of organophosphorus pesticide environmental pollution.There is the nucleotide sequence of coding organophosphor hydrolytic enzyme OPH, capsid protein CotG and own promoter after Ko subtilis host of the invention is engineered.After carrying out raw born of the same parents' culture by nutrition failure method, the fusion protein of capsid protein CotG and organophosphor hydrolytic enzyme OPH are expressed in host and show on the surface of Ko subtilis.The Ko subtilis has the ability of hydrolysis organophosphorus pesticide, also has good resistance to complex environment, can be used as environmentally protective pesticide cleanser for the biological prosthetic of contaminated environment.

Description

Bacillus subtilis exhibiting organophosphate hydrolase and its preparation method

technical field

The invention relates to subtilis spores displaying organophosphorus hydrolase OPH and a preparation method thereof, belonging to the field of biotechnology, and used for bioremediation of organophosphorus pesticide environmental pollution.

Background technique

Some organophosphorus compounds represented by organophosphates can cause harm to the body by inhibiting the activity of cholinesterase in the body. Nerve agents in chemical warfare agents and most civilian pesticides are designed and manufactured based on this principle. For each country, the protection and pollution control of organophosphorus compounds are related to national and public safety. Traditional treatment is mainly through physical and chemical degradation. Although good results have been achieved, it has also brought some problems, such as not being able to be directly used on human bodies or precision instruments, and decontamination agents are often toxic or corrosive, causing secondary pollution to the environment. Organophosphorus hydrolase can catalyze the hydrolysis of organophosphorus compounds. It has the advantages of fast action, mild conditions, non-toxicity and non-corrosion. detection and other fields.

The application of organophosphate hydrolase can reduce the use of chemical substances, reduce logistics burden and environmental pollution, but there are still some technical difficulties in actual production and application. For example: the separation and purification of enzymes is cumbersome, and also affects the activity of enzymes; organophosphate hydrolases are sensitive to temperature and pH changes, and have low storage stability, which is not conducive to use in natural environments or even under harsh conditions. Moreover, pure enzymes need to be combined with saline solution, foam base, etc. when used. Therefore, there is a demand for developing a more stable organophosphate hydrolase, so that the use of the organophosphate hydrolase is more convenient and has a wider range of applications.

Subtilis spore display technology refers to the display of foreign proteins on the surface of spores by means of genetic engineering or adsorption, while maintaining their relatively independent spatial conformation and original biological activity. The excellent stress resistance of spores improves the storage stability of exogenous proteins, which is suitable for application in complex conditions; spore display technology avoids the separation and purification of enzymes and reduces production costs. Therefore, the subtilis spore display technology has developed rapidly in recent years, and has shown good application prospects in the fields of vaccine preparation and biocatalysis. However, there is no universally applicable surface display system in the prior art, because whether the foreign protein can be effectively displayed is affected by various factors, among which the size and characteristics of the target protein are the main factors that determine the effectiveness of the spore display of Bacillus subtilis factor (see Wang He et al., Appl Microbiol Biotechnol (2017), 101:933-949). However, there are no reports of spores displaying organophosphate hydrolases.

Contents of the invention

The purpose of the present invention is to solve the technical problems of poor stability and cumbersome separation and purification procedures of organophosphorus hydrolase when applied in the actual environment, and provide subtilis spores displaying organophosphorus hydrolase and a preparation method thereof.

The Bacillus subtilis described in the present invention has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on March 8, 2018. The preservation number is CGMCC No.15427, and the preservation address is Beichen West Road, Chaoyang District, Beijing. No. 1 Courtyard No. 3, Institute of Microbiology, Chinese Academy of Sciences.

The technical solution adopted by the present invention to solve the above problems: display organophosphate hydrolase spore subtilis, the surface of the subtilis spore displays the fusion protein of organophosphate hydrolase OPH and capsid protein CotG, has the biological activity of OPH to degrade organophosphorus compounds, and is effective for complex The environment has excellent stress resistance; among them, the amino acid sequence of organophosphate hydrolase OPH is:

MSAQAMRSIRARPIT

ISEAGFTLTHEDISAARQDSCVLGQSSSVAQSSSGKGCERIARQSGWRANDCRCVDFRYRS RRQFIGRGFAGCRRSYLAATGLWFDPPLSMRLRYVEELTLVLPAVRFNMASKYTGIRAGII KVATTGKATPFQELVLKAAARASLATGVPVTTHTAASQRDGERGRPPFLSPKLEPSRVCIG HSDDTDDLSYLTALLRGYLIGLDHIPHSAIGLEDNASASPLLGIRSWQTRALLIKALIDQGY MKQILVSNDWLFGFSSYVTNIMDVMDRVNPDGMAFIH，衣壳蛋白CotG的氨基酸序列为： MGHYSHSDIEEAVKSAKKEGLKDYLYQEPHGKKRSHKKSHRTHKKSRSHKKSYCSHKKS RSHKKSFCSHKKSRSHKKSYCSHKKSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRS YKKSRSYKKSYCSHKKKSRSYKKSCRTHKKSYRSHKKYYKKPHHHCDDYKRHDDYDSK KEYWKDGNCWVVKKKYK。 For the first time, organophosphate hydrolase was displayed on the surface of subtilis spores, and the excellent stress resistance of spores was used to improve the stability of organophosphate hydrolase under complex conditions, and promote its application in real life.

The steps of the method for preparing subtilis spores displaying organophosphate hydrolase of the present invention are as follows:

(1) Extract the Bacillus subtilis genome as a template, and amplify the nucleotide sequence of the capsid protein CotG and its own promoter by PCR; The shuttle plasmid vector was subjected to double enzyme digestion; the two enzyme digestion products were mixed at a ratio of 1:3 to 1:5, and connected with nucleic acid ligase to construct a recombinant protein containing the capsid protein CotG and its own promoter nucleotide sequence. Plasmid; the above-mentioned recombinant plasmid is introduced into the E. coli cloning strain host by chemical transformation method, and a single clone is selected after antibiotic selection and culture; the above-mentioned single clone is cultured and amplified, and the recombinant plasmid is extracted for enzyme digestion electrophoresis verification and gene sequencing identification; In the step, the capsid protein CotG from Bacillus subtilis itself is selected as the anchor protein fused with the organophosphate hydrolase OPH, which has a high abundance in mature spores, and its modification will not affect the structure of the spores ;In addition, in order to reduce the impact of foreign proteins on the host and bud formation, the promoter of the capsid protein CotG itself is used in this step, so that the expression and regulation of the protein are closer to the natural state;

(2) Optimize the gene coding sequence of the organophosphate hydrolase OPH according to the codon preference of E. coli and Bacillus subtilis, and synthesize the nucleotide sequence of the organophosphate hydrolase OPH by artificial synthesis: atgagcgcgcaggcgatgcg tagcattcgt gcgcgtccga ttaccattag cgaagcgggc tttaccctga cccatgaagatattagcgcg gcgcgtcagg atagctgcgt gctgggccag agcagcagcg tggcgcagag cagcagcggcaaaggctgcg aacgtattgc gcgtcagagc ggctggcgtg cgaacgattg ccgttgcgtg gattttcgttatcgtagccg tcgtcagttt attggccgtg gctttgcggg ctgccgtcgt agctatctgg cggcgaccggcctgtggttt gatccgccgc tgagcatgcg tctgcgttat gtggaagaac tgaccctggt gctgccggcggtgcgtttta acatggcgag caaatatacc ggcattcgtg cgggcattat taaagtggcg accaccggcaaagcgacccc gtttcaggaa ctggtgctga aagcggcggc gcgtgcgagc ctggcgaccg gcgtgccggtgaccacccat accgcggcga gccagcgtga tggcgaacgt ggccgtccgc cgtttctgag cccgaaactggaaccgagcc gtgtgtgcat tggccatagc gatgataccg atgatctgag ctatctgacc gcgctgctgcgtggctatct gattggcctg gatcatattc cgcatagcgc gattggcctg gaagataacg cgagcgcgagcccgctgctg ggcattcgta gctggcagac ccgtgcgctg ctgattaaag cgctgattga tcagggctatatgaaacaga ttctggtgag caacgattgg ctgtttggct ttagcagcta tgatgatgacca gatcg tgtgaacccg gatggcatgg cgtttatca ttaa, and amplified by PCR; use restriction endonucleases to carry out double enzyme digestion to the nucleotide sequence of above-mentioned amplification and the recombinant plasmid constructed in step (1) respectively; Mix at a ratio of 1:3 to 1:5, use nucleic acid ligase to connect the organophosphate hydrolase gene to the C-terminus of the capsid protein CotG gene, and construct the capsid protein CotG, CotG's own promoter, and the organophosphate hydrolase OPH core A recombinant plasmid with a nucleotide sequence; the above-mentioned recombinant plasmid was introduced into the E. coli cloning strain host by chemical transformation method, and a single clone was selected after antibiotic screening and cultivation; the above-mentioned single clone was cultured and amplified, and the recombinant plasmid was extracted for enzyme digestion electrophoresis verification and Gene sequencing identification; in this step, in view of the technical problem that the organophosphate hydrolase OPH is easy to form inclusion bodies during the recombinant expression process, the coding gene was modified with codon preference, and the gene sequence is original;

(3) Introduce the recombinant plasmid constructed in step (2) into the expression host of Bacillus subtilis by electroporation, select a single clone after screening and culturing with antibiotics; culture and amplify the above-mentioned single clone, and extract the recombinant plasmid for enzyme digestion and electrophoresis Verification; this step constructs the subtilis engineering strain used to display organophosphate hydrolase OPH, and its source is the food-grade safe strain Bacillus subtilis approved by the U.S. Food and Drug Administration and the Ministry of Agriculture of China;

(4) The Bacillus subtilis expression host constructed in the step (3) is carried out cell growth culture by nutrient depletion method, culture fluid is collected after cultivating in DSM medium for 1-2 days, DSM medium is 8g nutrient broth medium, 1g potassium chloride, 0.25g magnesium sulfate heptahydrate, 2mg manganese chloride tetrahydrate, adjust pH to 7.0 with sodium hydroxide, distilled water to 1L, autoclave at 121℃ for 20min, add 1ml 1M calcium nitrate sterilized by filter membrane solution and 1ml 1mM ferric sulfate;

( ⁵ ) After diluting the above culture solution by 106 with sterilized water, take 100-200 microliters and smear it on the screening plate. After ⁶ , take 100-200 microliters and smear it on the screening plate, count the number of spores after overnight culture, and the results show that the spore formation rate of the engineered strain reaches 60-70%; this spore production rate is close to that of Bacillus subtilis in the natural state , indicating that the expression and display of the organophosphate hydrolase OPH will not affect the host's spore formation;

(6) Purify the spores in the culture medium in step (4), collect the bacteria by centrifugation at 4000-20000g, discard the supernatant and resuspend with 2mg/ml lysozyme solution, incubate at 37°C for 30min-1h, and collect the spores by centrifugation at 4000-20000g , wash with 1M NaCl and 1M KCl respectively, and then wash with pure water for 3 to 5 times, collect spores by centrifugation at 4000-20000g, and resuspend in 10ml of PBS buffer;

(7) Resuspend the purified spores in step (6) in one-tenth volume of stripping buffer, 0.1M DTT, 0.5% SDS, 0.1M NaCl; incubate at a high temperature above 70°C for more than 30 minutes, and the obtained spore coat The shell protein was purified by histidine affinity column after desalting by dialysis, and the purified protein was analyzed by Western blot and dot blot experiments. Compared with the control group, the extracted protein of the recombinant spores had significant chemiluminescence, and the results showed that each spore The expressed fusion protein molecules are on the order of 10 ⁵ ; experiments in this step show that the organophosphate hydrolase OPH can be successfully expressed into the recombinant spores;

(8) Incubate the purified spores in step (6) with mouse histidine antibody, wash with PBS and incubate with goat anti-mouse-FITC secondary antibody. Observation and analysis under a focusing microscope showed that the recombinant spores had significant fluorescence compared with the control group, indicating that the fusion protein was successfully displayed on the surface of the subtilis spores; experiments in this step showed that the organophosphate hydrolase OPH could be successfully displayed on the surface of the recombinant spores;

(9) Using paraoxon as the reaction substrate, add 20% of the spore suspension prepared in step (6) to the reaction environment of 75mM Tris-HCl, 50μM CoCl ₂ , pH=8.0, react at 37°C for 2min, and use ultraviolet spectroscopy The reaction solution A ₄₁₀ was detected by a photometer, and the results showed that the Saccharomyces subtilis exhibiting organophosphate hydrolase had significant hydrolysis activity, reaching 15.81U/mg;

Subtilis spores were treated with organic reagents, high temperature, freeze-thaw, acidic, alkaline, and protease to detect their activity. The results showed that the hydrolytic activity of subtilis spores exhibiting organophosphate hydrolase was higher than that of organic reagents such as methanol or ether, and high temperature of 40-60 °C. , -15～-20℃ freeze-thaw, pH 3～5, pH 9～11, trypsin or proteinase K environment, the tolerance is significantly enhanced; experiments at this step show that recombinant spores displaying organophosphate hydrolase OPH have the ability to hydrolyze The activity of organophosphorus pesticides, and the tolerance to various adverse environments have been significantly improved, P less than 0.001.

Beneficial effects of the present invention:

1. The engineered strain of Bacillus subtilis improved by genetic recombination can successfully express and display the organophosphate hydrolase OPH on the surface of the spores, and the obtained Bacillus subtilis has catalytic activity for hydrolyzing organophosphorus pesticides, and can be used as a mild and environmentally friendly pesticide scavenger For bioremediation of contaminated environments;

2. Compared with pure enzymes, the stability of spores exhibiting organophosphorus hydrolase OPH in various harsh environments is enhanced, and the preparation of spores is simple, which saves the complicated separation and purification steps of pure enzymes and reduces production The cost is conducive to the promotion and application in real life;

3. As the dormant body of the engineering strain, the spores can germinate and regenerate autonomously under suitable conditions, and can achieve sustainable and recyclable utilization.

Description of drawings

Figure 1 Schematic diagram of recombinant plasmid pHY300PLK-cotG-opdcb

1% agarose electrophoresis diagram of enzyme digestion identification of the recombinant vector in Figure 2

In the figure: Lane 1 is the result of HindIII and BamHI double enzyme digestion electrophoresis of recombinant plasmid pHy300PLK-cotG-opdcb; Lane 2 is the result of BamHI single enzyme digestion electrophoresis of recombinant plasmid pHy300PLK-cotG-opdcb; HindIII and BamHI double-enzyme electrophoresis results; lane 4 is the BamHI single-enzyme electrophoresis result of recombinant plasmid pHY300PLK-cotG; lane 5 is 1kb DNA Marker.

Fig. 3 shows the count plate diagram of the recombinant strain of OPH on the surface

In the figure: the first column is the total bacteria of the strain pHY300PLK-cotG/DB104 cell culture medium; the second column is the spores of the strain pHY300PLK-cotG/DB104 cell culture medium; the third column is the strain pHY300PLK-cotG-opdcb/DB104 cell growth The total bacteria in the culture solution; the fourth column is the spores in the spore-forming culture solution of the strain pHY300PLK-cotG-opdcb/DB104.

Figure 4 Western blot of capsid protein of recombinant spores

In the figure: Lane 1 is the protein Marker; Lane 2 is the capsid protein of the spores of the recombinant strain pHY300PLK-cotG-opdcb/DB104; Lane 3 is the capsid protein of the spores of the recombinant strain pHY300PLK-cotG-opdcb/DB104.

Figure 5 Capsid protein dot blot of recombinant spores

In the figure: group 1 is the capsid protein of recombinant strain pHY300PLK-cotG/DB104 spores; group 2 is the standard protein carrying histidine tag.

Figure 6 Immunofluorescence microscopic examination of recombinant spores

In the figure: I is the bright-field microscope image; II is the fluorescence microscope image; III is the integrated image; a is the recombinant spore sample; b is the wild spore sample.

Figure 7 Flow cytometric detection chart of recombinant spores

In the figure: a is the result of flow cytometry detection of spores of recombinant strain pHY300PLK-cotG/DB104; b is the result of flow detection of spores of recombinant strain pHY300PLK-cotG-opdcb/DB104; the ordinate is the number of spores, and the abscissa is the fluorescence value.

Figure 8 Activity detection chart of recombinant spores

In the figure: group 1 is the wild spore sample; group 2 is the recombinant spore sample; the ordinate is the enzyme activity per mg of spore dry weight.

Figure 9 Tolerance detection chart of recombinant spores

In the figure: group 1 is untreated; group 2 is methanol treatment; group 3 is ether treatment; group 4 is freeze-thaw treatment; group 5 is high temperature treatment; group 6 is alkaline treatment; group 7 is acid treatment; group 8 is Trypsin treatment; Group 9 is proteinase K treatment; Ordinate is the percentage of original enzyme activity; represents the recombinant spore group, Represents the pure enzyme group.

Detailed ways

The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments.

The methods in the following examples, if not specified, are conventional methods; the percentages in the following examples, if not specified, are mass percents.

Main test material

High Fidelity DNA Polymerase (Thermo Fisher Scientific), dNTP Mix (Thermo Fisher Scientific), PCR Master Mix 2× (Thermo Fisher Scientific), Plasmid Extraction Reagent (Thermo Fisher Scientific), DNA Gel Purification Kit (Thermo Fisher Scientific) , Restriction Enzyme (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DTT (Thermo Fisher Scientific), Protein Molecular Weight Standard (Thermo Fisher Scientific), DNA Ladder (Fermentas), Peptone (UK OXOID), Yeast Extract (OXOID), Tris Base (Amresco), Glycine (Amresco), Skimmed Milk Powder (BD), HRP Substrate Chemiluminescent Detector (Tiangen), Agarose (Biowest), Agar (Biowest), Dimethyl Thiazole Di Phenyltetrazolium bromide MTT (Sigma), Coomassie Brilliant Blue R-250 (Beijing Lianxing Biological Company, China), sodium dodecylsulfonate SDS (Amresco), vector plasmid pHY300PLK (TaKaRa), cloned strain Escherichia coli DH5α (TIANGEN), Bacillus subtilis DB104 (The Tenth Research Institute of the Academy of Military Medical Sciences of the Chinese People's Liberation Army), anti-His mouse monoclonal antibody (Tiangen), horseradish peroxidase-labeled goat anti-mouse IgG antibody (Sino Biological ), FITC-labeled anti-mouse goat antibody (ZSGB-BIO), phycoerythrin-labeled anti-His mouse monoclonal antibody (abcam).

Example 1

Acquisition of Display Vectors for Organophosphate Hydrolase

1. Construction of recombinant vector containing capsid protein gene

The genome of Bacillus subtilis DB104 was extracted, and the capsid protein cotG (SEQ ID NO: 4) and its promoter gene were obtained by PCR reaction using the genome as a template, and the primers were cotG-F (5-gccttt gaattc agtgtccctagctccgag-3' (SEQ ID NO :6)) and cotG-R (5-ctattg ggatcc tgaacccccacctcctttgtatttctttttgacta-3' (SEQ ID NO: 7)). By PCR, a flexible peptide chain (Gly-Gly-Gly-Gly-Ser) was introduced into the CotG end of the capsid protein. The PCR amplification conditions were pre-denaturation at 95°C for 5 min, followed by 35 cycles of amplification: 95°C for 30 s, 55°C for 30 s, 72°C for 2 min, and finally 72°C for 10 min. After the PCR product was purified by a purification kit, it was digested and purified using restriction endonucleases EcoRI and BamHI.

The Escherichia coli clone strain DH5α containing the shuttle plasmid pHY300PLK was cultivated, and the plasmid vector was extracted after amplification. The vector was digested and purified with restriction enzymes EcoRI and BamHI. A ligation reaction was performed between the digested DNA fragment containing the capsid protein cotG gene and the digested vector fragment (see Figure 1 for a schematic diagram of the recombinant plasmid). The ligated product was transformed using E. coli cloning strain DH5α competent. The screening culture was carried out on a solid medium containing tetracycline, and the obtained transformants were cultured and amplified for enzyme digestion identification and sequencing analysis. The electrophoresis results of enzyme digestion identification are shown in lanes 3 and 4 of FIG. 2 . The results showed that the structure and sequence of the recombinant vector were correct, containing a 1060bp insert segment, and no gene mutation was identified by sequencing.

2. Construction of recombinant vector containing organophosphate hydrolase gene

The organophosphate hydrolase gene opdcb (SEQ ID NO: 3) was obtained by artificial synthesis, and a histidine tag His×6 was added to the end of the gene to facilitate subsequent purification and identification (Beijing Aoke Dingsheng Biotechnology Co., Ltd.). The target protein gene was amplified by PCR, and the primers were opdcb-F (5-actg ggatcc agcgcgcaggcgatgcgtag-3' (SEQ ID NO: 8)) and opdcb-R (5-agag aagctt tta gtggtggtggtggtggtg-3' (SEQ ID NO: 9) ), the PCR amplification conditions were pre-denaturation at 95°C for 5 minutes, followed by 35 cycles of amplification: 95°C for 30s, 55°C for 30s, 72°C for 2min, and finally 72°C for 10min. After the PCR product was purified by a purification kit, it was digested and purified using restriction endonucleases HindIII and BamHI.

The Escherichia coli clone strain DH5α containing the recombinant plasmid pHY300PLK-cotG was cultivated, and the recombinant plasmid was extracted after amplification. The vector was digested and purified with restriction endonucleases HindIII and BamHI. A ligation reaction is carried out between the digested DNA fragment containing the gene of organophosphorus hydrolase opdcb and the digested carrier fragment.

The second ligation product was transformed using E. coli cloning strain DH5α competent. The solid medium containing tetracycline was used for screening and culturing, and the obtained transformants were cultured and amplified for enzyme digestion identification and sequencing analysis. The electrophoresis results of enzyme digestion identification are shown in lanes 1 and 2 in Figure 2. The results showed that the structure and sequence of the recombinant vector were correct, containing a 915bp insert segment, and no gene mutation was identified by sequencing.

Example 2

Obtaining recombinant strains displaying organophosphate hydrolases

DB104 is extracellular neutral protease (nprE) and serine protease (aprA) deficient Bacillus subtilis, which according to FUJIO KAWAMURA and ROY H.DOI, Construction of a Bacillus subtilis Double Mutant Deficient in Extracellular Alkaline and Neutral Proteases, Journal of Bacteriology, Vol .160, No.1, p.442-444, the method described in October, 1984 was prepared.

The display host Bacillus subtilis DB104 was cultivated to the plateau stage, transferred to the competent growth medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 0.5M sorbitol) and cultivated to an _OD600 of Between 0.85 and 0.95. Bacteria were collected after 10 minutes of ice bathing, and pre-cooled electroshock buffer (0.5M sorbitol, 0.5M mannitol, 10% glycerol) was used to wash the bacterial cells, and repeated 4 times. 1:40 resuspended in electroporation buffer, aliquoted in 60 μl, and stored at -80°C until use.

The Escherichia coli clone strain DH5α containing the recombinant plasmid pHY300PLK-cotG-opdcb was cultivated, and the plasmid that had undergone two gene recombination was extracted after amplification. Take Bacillus subtilis DB104 competent cells to dissolve in an ice bath, add 50ng of recombinant vector to ice bath for 10min, and transfer to a pre-cooled 2mm electroporation cup. The recombinant vector was introduced into Bacillus subtilis DB104 competent cells by electroporation, the electroporation parameter was 2.5kv, and the electric shock was 1 time. Add 1ml of preheated recovery medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 0.5M sorbitol, 0.38M mannitol) immediately after the electric shock, 37℃, 150rpm shaking recovery culture 3h. After the bacteria were collected, the solid medium containing tetracycline was used for screening and culture, and the transformant pHY300PLK-cotG-opdcb/DB104 was identified by PCR. Conditions were pre-denaturation at 95°C for 5 minutes, followed by 25 cycles of amplification: 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, and finally 72°C for 10 min.

Example 3

Culture and purification of recombinant spores

The recombinant strain pHY300PLK-cotG-opdcb/DB104 was inoculated in sporulation medium (DSM: 8% nutrient broth, 0.1% potassium chloride, 0.025% magnesium sulfate heptahydrate, 0.01mM manganese chloride, 1mM nitric acid Calcium, 0.01mM ferric sulfate, pH to 7.0) for 48h. Calculate the total number of strains in the medium by plate counting method, and calculate the number of spores after incubating the culture medium at 80°C for 15 minutes. The counting results (see Fig. 3) showed that the concentration of spores in the cell culture medium was (9.35±0.83)×10 ⁶ cfu/ml, and the cell growth rate was (61.61±16.09)%.

Spores were collected by high-speed centrifugation, resuspended in 2mg/ml lysozyme solution, incubated at 37°C for 30min, washed with 1M NaCl and 1M KCl, and washed 3 times with pure water. Resuspend the spore sample in pure water for use.

Example 4

Expression and Display Identification of Organophosphate Hydrolase in Recombinant Spores

Add capsid protein stripping buffer (0.1M DTT, 0.5% SDS, 0.1M NaCl) to the spore suspension, incubate at 70°C for 30min, and dialyze the supernatant to desalt for 4h, repeat 3 times. The dialysate was purified using a histidine column, and the purified solution was ultrafiltered with a 10K ultrafiltration tube to desalt and then freeze-dried, and the obtained dry powder protein was resuspended in ultrapure water. The above protein samples were subjected to SDS-PAGE electrophoresis, and the obtained electrophoretic gel was subjected to electrotransfer, 110V transfer for 90min, and the protein was transferred to a 0.2 μm PVDF membrane. Then block with 5% bovine serum albumin solution for 1 hour, incubate with mouse histidine antibody for 1 hour, wash with TBS-T for 5 minutes, repeat 4 times, incubate with goat anti-mouse-HRP secondary antibody for 1 hour, wash with TBS-T for 5 minutes, repeat 4 times , ECL chromogenic solution was added dropwise to the membrane to develop color and photographed and recorded. The results of western blot identification are shown in Figure 4. The results showed that the recombinant strain pHY300PLK-cotG-opdcb/DB104 could express organophosphate hydrolase and assemble it into recombinant spores.

Dot blot experiments were performed using the above protein purified samples. Dilute the protein sample with the standard protein GFP-6×His carrying histidine tag, take 2 μl of the protein sample and its dilution, standard protein and its dilution, and add them dropwise to a 0.2 μm PVDF membrane, with 5% BSA The solution was blocked for 1 hour, incubated with mouse histidine antibody for 1 hour, washed with TBS-T for 5 minutes, repeated 4 times, incubated with goat anti-mouse-HRP secondary antibody for 1 hour, washed with TBS-T for 5 minutes, repeated 4 times, and added dropwise to the membrane to display ECL. The color solution was developed and photographed and recorded, and the results of dot blot identification are shown in Figure 5. The results showed that each spore of the recombinant strain pHY300PLK-cotG-opdcb/DB104 could express about 8.74×10 ⁴ OPH molecules on average.

The spore suspension was washed three times with PBS, incubated with 3% bovine serum albumin solution at room temperature for 30min, centrifuged at 10000g for 5min, and the supernatant was discarded. Resuspend with mouse histidine antibody, incubate overnight at 4°C, and wash 3 times with PBS. Incubate for 2 h with goat anti-mouse-FITC secondary antibody, and wash 3 times with PBS. About 20 μl of the processed sample was dropped onto a glass slide, placed under a laser confocal microscope for observation, and photographed and recorded. The results of immunofluorescence microscopy are shown in Figure 6. The results showed that the organophosphate hydrolase expressed into the spores could be localized on the surface of the recombinant spores. The spore suspension was washed three times with PBS, incubated with 3% bovine serum albumin solution at room temperature for 30min, centrifuged at 10000g for 5min, and the supernatant was discarded. Resuspend with mouse histidine monoclonal antibody, incubate for 2 hours, and wash 3 times with PBS. The processed samples were taken for flow cytometric detection and recorded, and the flow cytometric detection results are shown in Figure 7. The results showed that the average fluorescence intensity of the recombinant spores displaying OPH was (55.93±3.54), and the positive rate was 5.65%, which were significantly higher than those of the control group (P<0.001).

Example 5

Identification of Organophosphate Hydrolase Activity in Recombinant Spores

With paraoxon as the reaction substrate, 200 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension, 700 μl of assay buffer (75 mM Tris-HCl, 50 μM CoCl ₂ , pH=8.0) and 100 μl of 20 mM paraoxon were sequentially added Methanol solution, mix well, and react at 37°C for 2min. After centrifugation, take the supernatant to measure A ₄₁₀ after diluting 3 times with the living solution, and repeat three times. The recombinant spore pHY300PLK-cotG/DB104 suspension was used as a negative control and the influence of natural hydrolysis was deducted, and the recombinant spore enzyme activity of organophosphate hydrolase was calculated according to the p-nitrophenol standard curve. Another 1ml of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension was lyophilized and weighed. The enzyme activity unit (U) of recombinant spores is defined as: the amount of recombinant spores required to release 1 nmol of p-nitrophenol per minute at 37°C. The enzyme specific activity of the recombinant spore is defined as: the unit of enzyme activity per milligram of the recombinant spore (U/mg). The results of the activity test are shown in Figure 8. The results show that the activity of the recombinant spores to hydrolyze paraoxon is 15.81U/mg dry weight of spores, which is significantly improved compared with the control group (p<0.001). That is, the recombinant spores produced by the recombinant strain pHY300PLK-cotG-opdcb/DB104 have the ability to degrade organophosphate substrates, and the organophosphate hydrolase can be displayed on the surface of the recombinant spores in a biologically active form.

Take 100 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, add an equal amount of methanol or ether, and incubate at 37°C for 1 hour, detect its activity by the above method, repeat three groups and record. Take 100 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, incubate at 40°C for 1 h, detect its activity by the above method, repeat three groups and record. Take 100 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, incubate at -20°C for 1 hour, detect its activity after dissolution, and repeat three groups and record. Take 100 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, add 0.25% trypsin respectively, incubate at 37°C for 1 hour, detect its activity by the above method, repeat three groups and record. Take 100μl recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, add 50U proteinase K respectively, incubate at 37°C for 1h, detect its activity by the above method, repeat three groups and record. Take 100 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, detect its activity in Gly-NaOH buffer solution with pH 10 by the above method, repeat three groups and record. Take 100 μl of recombinant spore pHY300PLK-cotG-opdcb/DB104 suspension and OPH pure enzyme sample, detect its activity in pH 4 Na ₂ HPO ₄ -CitriAcid buffer solution by the above method, repeat three groups and record. After the activity was detected by spectrophotometry, the percentage of the residual activity to the original activity was calculated, and the tolerance test results are shown in FIG. 9 . The experimental results showed that the enzyme activity of recombinant spores pHY300PLK-cotG-opdcb/DB104 displaying OPH was significantly more resistant to methanol, ether, freeze-thaw, high temperature, pH 4, trypsin, and proteinase K (P<0.001).

Claims (2)

1. A subtilis spore displaying organophosphorus hydrolase, characterized in that the surface of the subtilis spore displays the fusion protein of organophosphate hydrolase OPH and capsid protein CotG, has the biological activity of OPH degrading organophosphorus compounds, and is excellent in complex environments的抗逆性；其中，有机磷水解酶OPH的氨基酸序列为：MSAQAMRSIRARPITISEAGFTLTHEDISAARQDSCVLGQSSSVAQSSSGKGCERIARQSGWRANDCRCVDFRYRSRRQFIGRGFAGCRRSYLAATGLWFDPPLSMRLRYVEELTLVLPAVRFNMASKYTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTAASQRDGERGRPPFLSPKLEPSRVCIGHSDDTDDLSYLTALLRGYLIGLDHIPHSAIGLEDNASASPLLGIRSWQTRALLIKALIDQGYMKQILVSNDWLFGFSSYVTNIMDVMDRVNPDGMAFIH，衣壳蛋白CotG的氨基酸序列为：MGHYSHSDIEEAVKSAKKEGLKDYLYQEPHGKKRSHKKSHRTHKKSRSHKKSYCSHKKSRSHKKSFCSHKKSRSHKKSYCSHKKSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRSYKKSRSYKKSYCSHKKKSRSYKKSCRTHKKSYRSHKKYYKKPHHHCDDYKRHDDYDSKKEYWKDGNCWVVKKKYK。 2. A method for preparing subtilis spores exhibiting organophosphate hydrolase, characterized in that the steps of the method for preparing subtilis spores are as follows: (1) Extract the Bacillus subtilis genome as a template, and amplify the nucleotide sequence of the capsid protein CotG and its own promoter by PCR; The shuttle plasmid vector was subjected to double enzyme digestion; the two enzyme digestion products were mixed at a ratio of 1:3 to 1:5, and connected with nucleic acid ligase to construct a recombinant protein containing the capsid protein CotG and its own promoter nucleotide sequence. Plasmid: the above-mentioned recombinant plasmid is introduced into the E. coli cloning strain host by chemical transformation method, and a single clone is selected after being screened and cultivated by antibiotics; the above-mentioned single clone is cultured and amplified, and the recombinant plasmid is extracted for enzyme digestion electrophoresis verification and gene sequencing identification; (2) Optimize the gene coding sequence of the organophosphate hydrolase OPH according to the codon preference of E. coli and Bacillus subtilis, and synthesize the nucleotide sequence of the organophosphate hydrolase OPH by artificial synthesis: atgagcgcgcaggcgatgcg tagcattcgt gcgcgtccga ttaccattag cgaagcgggc tttaccctga cccatgaagatattagcgcg gcgcgtcagg atagctgcgt gctgggccag agcagcagcg tggcgcagag cagcagcggcaaaggctgcg aacgtattgc gcgtcagagc ggctggcgtg cgaacgattg ccgttgcgtg gattttcgttatcgtagccg tcgtcagttt attggccgtg gctttgcggg ctgccgtcgt agctatctgg cggcgaccggcctgtggttt gatccgccgc tgagcatgcg tctgcgttat gtggaagaac tgaccctggt gctgccggcggtgcgtttta acatggcgag caaatatacc ggcattcgtg cgggcattat taaagtggcg accaccggcaaagcgacccc gtttcaggaa ctggtgctga aagcggcggc gcgtgcgagc ctggcgaccg gcgtgccggtgaccacccat accgcggcga gccagcgtga tggcgaacgt ggccgtccgc cgtttctgag cccgaaactggaaccgagcc gtgtgtgcat tggccatagc gatgataccg atgatctgag ctatctgacc gcgctgctgcgtggctatct gattggcctg gatcatattc cgcatagcgc gattggcctg gaagataacg cgagcgcgagcccgctgctg ggcattcgta gctggcagac ccgtgcgctg ctgattaaag cgctgattga tcagggctatatgaaacaga ttctggtgag caacgattgg ctgtggttggct ttagcagcta tgatggact atcg tgtgaacccg gatggcatgg cgtttatca ttaa, and amplified by PCR; use restriction endonucleases to carry out double enzyme digestion on the nucleotide sequence of the above-mentioned amplification and the recombinant plasmid constructed in step (1) respectively; Mix at a ratio of 1:3 to 1:5, use nucleic acid ligase to connect the organophosphate hydrolase gene to the C-terminus of the capsid protein CotG gene, and construct the capsid protein CotG, CotG's own promoter, and the organophosphate hydrolase OPH core A recombinant plasmid with nucleotide sequence; the above recombinant plasmid was introduced into the E. coli cloning strain host by chemical transformation method, and a single clone was selected after antibiotic screening and cultivation; the above single clone was cultured and amplified, and the recombinant plasmid was extracted for enzyme digestion electrophoresis verification and Gene sequencing identification; (3) Introduce the recombinant plasmid constructed in step (2) into the Bacillus subtilis expression host by electroporation, select a single clone after antibiotic screening and culture; culture and amplify the above-mentioned single clone, and extract the recombinant plasmid for enzyme digestion electrophoresis verify; (4) The Bacillus subtilis expression host constructed in the step (3) is carried out cell growth culture by nutrient depletion method, after culturing in DSM medium for 1 to 2 days, the culture fluid is collected, DSM medium is 8g nutrient broth medium, 1g potassium chloride, 0.25g magnesium sulfate heptahydrate, 2mg manganese chloride tetrahydrate, adjust pH to 7.0 with sodium hydroxide, distilled water to 1L, autoclave at 121℃ for 20min, add 1ml 1M calcium nitrate sterilized by filter membrane solution and 1ml 1mM ferric sulfate; ( ⁵ ) After diluting the above culture solution by 106 with sterilized water, take 100-200 microliters and smear it on the screening plate. After ⁶ , take 100-200 microliters and smear it on the screening plate, count the number of spores after overnight culture, and the results show that the spore generation rate of the engineering strain reaches 60-70%; (6) Purify the spores in the culture medium in step (4), collect the bacteria by centrifugation at 4000-20000g, discard the supernatant and resuspend with 2mg/ml lysozyme solution, incubate at 37°C for 30min-1h, and collect the spores by centrifugation at 4000-20000g , wash with 1M NaCl and 1M KCl respectively, and then wash with pure water for 3 to 5 times, collect spores by centrifugation at 4000-20000g, and resuspend in 10ml PBS buffer; (7) Resuspend the purified spores in step (6) in one tenth volume of stripping buffer, 0.1M DTT, 0.5% SDS, 0.1M NaCl; incubate at a high temperature above 70°C for more than 30min, and obtain the spore coat The shell protein was purified by histidine affinity column after desalting by dialysis, and the purified protein was analyzed by Western blot and dot blot experiments. Compared with the control group, the extracted protein of the recombinant spores had significant chemiluminescence, and the results showed that each spore The expressed fusion protein molecules reach the order of 10 ⁵ ; (8) Incubate the purified spores in step (6) with mouse histidine antibody, wash with PBS and incubate with goat anti-mouse-FITC secondary antibody. Observation and analysis under a focusing microscope, compared with the control group, the recombinant spores had significant fluorescence, indicating that the fusion protein was successfully displayed on the surface of the subtilis spores; (9) Using paraoxon as the reaction substrate, add 20% of the spore suspension prepared in step (6) to the reaction environment of 75mM Tris-HCl, 50μM CoCl ₂ , pH=8.0, react at 37°C for 2min, and use ultraviolet spectroscopy The reaction solution A ₄₁₀ was detected by a photometer, and the results showed that the Saccharomyces subtilis exhibiting organophosphate hydrolase had significant hydrolysis activity, reaching 15.81U/mg; Subtilis spores were treated with organic reagents, high temperature, freeze-thaw, acidic, alkaline, and protease to detect their activity. -15～-20℃ freezing and thawing, pH3～5, pH9～11, trypsin or proteinase K environment, the tolerance was significantly enhanced, P<0.001.