CN113637659B

CN113637659B - Acidocella acetylhexosidase Amuc_2109 protein prepared by genetic engineering and preparation method and application thereof

Info

Publication number: CN113637659B
Application number: CN202110880439.8A
Authority: CN
Inventors: 张敏; 钱锴钺; 王永中; 盛康亮; 王明珠
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2021-08-02
Filing date: 2021-08-02
Publication date: 2023-10-03
Anticipated expiration: 2041-08-02
Also published as: CN113637659A

Abstract

The application relates to the technical field of protein engineering, and particularly discloses a genetically engineered Acetohexosidase Amuc_2109 protein, a preparation method and application thereof, wherein the Amuc_2109 protein has any one of the following sequences: (1) an amino acid sequence shown as SEQ ID NO. 1; (2) An amino acid sequence obtained by substituting, deleting and/or adding one or more amino acid sequences to the amino acid sequence of (1), and a homologous protein sequence having the same function as the amino acid sequence of (1). The application proves the medicinal value of Amuc_2109 protein through purifying Amuc_2109 wild type and mutant protein and playing a role in improving DSS induced colonitis.

Description

Acidocella acetylhexosidase Amuc_2109 protein prepared by genetic engineering and preparation method and application thereof

Technical Field

The application belongs to the technical field of protein engineering, and particularly relates to an Acetohexosidase Amuc_2109 protein prepared by genetic engineering, and a preparation method and application thereof.

Background

Akkermansia muciniphila A. Luciphilia is a gram-negative anaerobic bacterium belonging to the phylum Verrucomica and stably residing in the outer layer of the human gastrointestinal tract (GI), A. Luciphilia improves colitis, and in addition, SCFAs produced by A. Luciphilia fermentation are inversely related to inflammation. The outer membrane protein Amuc_1100 can maintain structure at high temperature, interact with TLR2, and improve intestinal barrier function. Muciniphila can increase the number of goblet cells and the production of antibacterial peptides enhances intestinal barrier function. Thus, in recent years, a. Muciniphila has received a great deal of attention in human intestinal health. In the human gut, a. Muciniphila is involved in the degradation of mucins by secretion of glycoside hydrolases, proteases, sulfatases and sialidases. The mechanism by which muciniphila degrades and is beneficial to mucus is not clear. The muciniciphilia genome encodes 11N-acetyl-beta-hexasamidases, two alpha-1-2-fucosidases, four sialidases and two beta-gallcosidases.

Muciniciphilia metabolizes mucins by expressing a large number of sulfatases and Glycoside Hydrolases (GH), such as α -focussidases, α -sialidases, β -galactosidases, β -acetylhexosidases and α -acetylglucosidases. Among them, β -acetylhexosidase is responsible for releasing β -1,3 and β -1, 4-n-acetylglucosamine (GlcNAc) bound to the glycan structure. However, structural data for representative enzymes of a. Muciniphila for degradation of mucins remain scarce. The molecular mechanism by which muciniciphilia can both degrade and benefit mucus is not known.

Based on the above, a genetically engineered Acetylhexosidase Amuc_2109 protein of Acidovorax and a preparation method and application thereof are provided.

Disclosure of Invention

The application aims to overcome the defects of the prior art and provides an Acetohexosidase Amuc_2109 protein prepared by genetic engineering, and a preparation method and application thereof.

The application realizes the above purpose through the following technical scheme:

the application provides a genetically engineered Acetohexosidase Amuc_2109 protein of Acetobacter, which is characterized by having any one of the following sequences:

(1) An amino acid sequence as shown in SEQ ID NO. 1;

(2) An amino acid sequence obtained by substituting, deleting and/or adding one or more amino acid sequences to the amino acid sequence of (1), and a homologous protein sequence having the same function as the amino acid sequence of (1).

As a further improvement of the present application, one or more mutations in D246A, D62A, H163A, K A, R132A, D247A exist in the amino acid sequence shown in SEQ ID NO.1, which is obtained by substituting, deleting and/or adding one or more amino acid sequences to the amino acid sequence having the amino acid sequence of (1).

As a further improvement of the present application, the gene encoding the protein has a nucleotide sequence as shown IN SEIQ IN NO. 2.

The application also provides application of the Acetohexosidase Amuc_2109 protein prepared by the genetic engineering in preparation of a medicament for treating ulcerative colitis.

The application also provides an application of the modified Acremonium Amuc_2109 in a DSS-induced colitis molecular mechanism.

The application also provides a preparation method of the Acetohexosidase Amuc_2109 protein prepared by the genetic engineering, which is characterized by comprising the following steps:

s1, using cDNA of A.muciniphila as a template, obtaining a gene of Amuc_2109 protein through a PCR amplification technology, and constructing the gene on a pET-22b vector to obtain the Amuc_2109 recombinant plasmid.

S2, expressing the Amuc2109 recombinant plasmid by using an escherichia coli prokaryotic expression system, and purifying by using a Ni-NTA column and affinity chromatography to obtain the Amuc_2109 protein of the Achroman.

The application has the beneficial effects that: the Amuc_2109 protein is purified and obtained, the crystal structure of the compound is obtained, the catalytic mechanism of the compound is elucidated from the molecular level, and structural evidence is provided for degrading mucin. Furthermore, it was demonstrated in animal experiments that wild type amyc_2109 has an effect of improving DSS-induced colitis, whereas its functional mutant does not have this effect, which may be linked to the loss of catalytic activity of the mutant.

In summary, in our application, we demonstrate that the Amuc_2109 protein has pharmaceutical value by purification of Amuc_2109 wild type and mutant proteins, their structural characterization, and their role in improving DSS-induced colitis.

Drawings

FIG. 1 is a graph showing the results of Ni-NTA column purification of recombinant Amuc_2109 protein;

FIG. 2 is a graph showing the results of the size exclusion of recombinant Amuc_2109 protein;

FIG. 3 is a diagram of animal experiment design;

FIG. 4 is a photograph of colon length and a histogram of colon length statistics;

FIG. 5 is a graph of H & E staining results of the colon;

FIG. 6 shows qPCR results;

FIG. 7 is a Venn chart showing the sequencing results of three fecal microorganisms;

fig. 8 is a graph of the composition of the intestinal microbiota altered by amuc_2109 at the Phylum (phy um) level.

Detailed Description

The present application will be described in further detail with reference to the accompanying drawings, wherein it is to be understood that the following detailed description is for the purpose of further illustrating the application only and is not to be construed as limiting the scope of the application, as various insubstantial modifications and adaptations of the application to those skilled in the art can be made in light of the foregoing disclosure.

1. Material

The methods used in this example are conventional methods known to those skilled in the art unless otherwise indicated, and the materials such as reagents used are commercially available products unless otherwise indicated.

2. Method of

2.1 Amuc_2109 protein construction, expression and purification

2.1.1 construction of recombinant plasmids

(1) Extracting total RNA of the A.muciniphila, reversely transcribing the total RNA into cDNA, using the cDNA as a template, amplifying a DNA fragment of the Amuc_2109 gene shown as SEQ ID NO.1 by a PCR method, and respectively adding NdeI and XhoI enzyme cutting sites at two ends to obtain a target gene PCR product.

The PCR amplification primers are as follows:

5’-GGAATTCCATATGCTCCCTGCCCTGATCG-3(Sense Primer)

5’-CTCGAGATATTTCTGGACTGGGGA-3’(Anti Sense Primer)

(2) The PCR product of the target gene and the pET-22b vector recovered by digestion with NdeI and XhoI are digested with the following systems:

the double enzyme digestion reaction is completed in a constant temperature water bath kettle at 37 ℃, and the specific operation is that reactants with corresponding volumes are added into an EP pipe with the volume of 1.5ml according to the following enzyme digestion system, the EP pipe orifice is sealed by a sealing film, and then the mixture is placed in the constant temperature water bath kettle at 37 ℃ for enzyme digestion for 3 hours. The cleavage system is shown in Table 1-1.

TABLE 1-1 double cleavage System for PCR products and vectors

And respectively carrying out gel recovery on the PCR product and the carrier after double enzyme digestion, wherein the gel recovery process is the same as that of the PCR product, and detecting whether the recovery is successful or not by using 0.8% gel electrophoresis after the gel recovery is finished.

(3) Ligation of target Gene and vector

The ligation of the target fragment and the vector was accomplished mainly using T4 DNA ligase, which was T4 DNA ligase (Thermo) used in this experiment, and was performed at 25℃for 3 hours. The connection system is shown in tables 1-2.

TABLE 1-2 connection System

(4) Transformation of DH5 alpha competent cells by recombinant plasmids

a. DH 5. Alpha. Competent strain was removed from the-80℃refrigerator and rapidly thawed on ice for 8min.

b. The thawed 200. Mu.l of competent cells were transferred to the above-described ligation system using a pipette, gently mixed, and placed on ice for 30min.

c, heat shock is carried out for 80s in a water bath kettle at the temperature of 42 ℃ and the water bath kettle is placed on ice for 8min.

d. 500. Mu.l of LB culture solution is taken in a super clean bench and added into the heat-shocked cells, and the cells are incubated for 40min on a shaking table at 37 ℃ and 140 rpm.

e.5000rpm, centrifuging for 2min, discarding 400 μl supernatant, blowing and sucking to precipitate, uniformly coating the mixed bacterial liquid on LB solid medium with glass rod, placing the solid medium into a constant temperature incubator at 37deg.C for normal culture, inverting the plate after 30min, and continuing overnight culture.

f. The monoclonal strain on the overnight culture plate was picked up and placed in a small tube of 4ml LB liquid medium, at 37℃and 220rpm, and cultured overnight.

(5) Recombinant testing

And (3) the clones positive through bacterial liquid PCR and double enzyme digestion identification are sent to a company for sequencing, and the monoclonal transformed BL21 strain which is successfully sequenced is subjected to test expression.

2.2 Amuc_2109 recombinant plasmid test expression and solubility detection

2.2.1 test expression

Transfer of BL 21-transformed monoclonal bacteria to a small tube of 4ml LB medium, addition of antibiotics of the corresponding resistance, and 20. Mu.l in the small tubeCulturing glycerol strain in shaking table at 37deg.C and 220rpm until the strain liquid OD ₆₀₀ Reaching between 0.6 and 0.8. Mu.l of the bacterial liquid was extracted from the culture medium, centrifuged, the supernatant was removed, and 100. Mu.l of 2 Xloading buffer was added to the sediment to resuspend the bacterial cells for fixation as a pre-mutagenesis sample. IPTG with a final concentration of 0.8mM was added to the remaining bacterial liquid, and the resulting mixture was subjected to induction culture at 220rpm in a shaking table at a constant temperature of 37℃for 5hr.

2.2.2 solubility detection

(1) After the test expression result shows that Amuc_2109 protein is expressed, transferring the recombinant plasmid strain containing Amuc_2109 into 200ml LB culture solution to culture, and when the bacterial solution OD600 reaches 0.6-0.8, adding 0.4mM IPTG,16 ℃ and inducing for 20 hours

(2) Collecting fungus blocks, performing ultrasonic crushing, wherein the crushing buffer solution is 50mM Tris-HCl, pH7.0,500mM NaCl, and centrifuging the crushed solution at 10000rpm for 1 hour;

(3) And collecting cell supernatant, purifying Ni-NTA, eluting protein with buffer solution containing imidazole solutions with different gradients, and finally obtaining the protein with higher purity.

(4) The target protein obtained by Ni-NTA affinity chromatography was collected and concentrated to a volume of 2ml using an ultrafiltration concentration tube. The sample was concentrated by centrifugation at 12000g for 30min at 4℃and was applied with 50mM Tris-HCl,500mM NaCl,pH7.0 as buffer at a flow rate of 1.0ml/min. And (3) carrying out ultraviolet on-line monitoring at 280nm to collect target proteins. The molecular sieve column used in this experiment had Hiload 16/60Superdex 75prep grade (120 ml).

2.3Amuc_2109 mutant protein construction, expression and purification

The construction of the mutant adopts a site-directed mutagenesis technology, namely a method for introducing mutation sites into wild genes by utilizing PCR reaction and mismatch primers and specifically cutting off unmutated methylated templates by utilizing methylase (DpnI), so as to obtain mutated genes, wherein the specific experimental steps are as follows:

(1) The mutant primers were as follows:

TABLE 3-1 Point mutation PCR primers

(2) The enzyme for point mutation PCR is PrimeStar enzyme, and the corresponding components are added according to the following reaction system.

TABLE 3-2 Point mutation PCR reaction System

TABLE 3 Point mutation PCR reaction parameters

Note that: a process of 98 ℃ to 72 ℃ and is circulated for 16 times;

the PCR products were gel recovered according to the gel recovery kit instructions.

(3) And (3) carrying out enzyme digestion on the recovered product for 3 hours at 37 ℃ by using DpnI enzyme to remove templates, wherein the enzyme digestion system is as follows:

TABLE 3-4 DpnI cleavage System

(4) And (3) converting the DpnI enzyme-digested product into DH5 alpha strain, selecting a monoclonal for sequencing, and finally expressing the monoclonal converted BL21 strain which is sequenced successfully.

(5) Amuc_2109 mutant protein was expressed according to the same procedure as in 2.2.1, purified using Ni-NTA column, and finally purified using gel filtration chromatography.

Kinetic parameters kcat and Km were determined at 37℃and 50mM Tris-HCl (pH 7.0), 500mM NaCl. Amuc_2109 was added as described above and the pNP-GlcNAc concentration was determined to be 0.5-2.5 mM. Kinetic parameters kcat, km, vmax and kcat/Km were calculated using a Linewaver-Burk, as shown in the following Table:

tables 3-5 kinetic parameters

Enzymes	K _m (mM)	K _cat (S ^-1 )	K _cat /K _m
				wild type-Amuc_2109	0.325±0.055	4.68×10 ^-3 ±0.00033	14.7×10 ^-3 ±0.002
D62A	NA	NA	NA
				R132A	NA	NA	NA
K162A	NA	NA	NA
				H163A	NA	NA	NA
D246A	NA	NA	NA
				D247A	NA	NA	NA

2.4 colitis model Induction and Experimental design

2.4.1 Male C57BL/6 mice (age 6-7 weeks; 20.+ -.2 g) were placed under controlled laboratory conditions (12 hours/photoperiod) and water and food were available. After 2 weeks of adaptive feeding, the mice were randomly divided into four groups, control group (control group), DSS-induced colitis model (DSS), and the group of amuc_2109 (amuc_2109) of DSS-induced mice. There were 6 mice in each group. The mouse colitis model was induced from day 14-21 by using aqueous DSS (3% w/v;36-50 kDa) (see FIG. 3 for experimental design). Mice were perfused with 20 μg protein and an equal amount of PBS.

2.4.2 colon Length Change

Fig. 4A shows a typical appearance of colon tissue, normal color and thickness of colon tissue, no congestion and edema, no adhesion to surrounding tissues, DSS model with congestion and edema of colon tissue, relatively short colon, and relief of colitis symptoms in mice supplemented with the amuc_2109 protein. Colonic shortening is a measure of the severity of colitis. The relative colon length of each group is shown in fig. 4A. As in fig. 4B: the control group showed the longest average colon length (7.57.+ -. 0.4 cm) while the DSS model group had the shortest average colon length (4.98.+ -. 0.7 cm). Amuc_2109 treated group maintained the average colon length (6.0.+ -. 0.7 cm) closest to control group. The average colon length of group D246 was (5.2.+ -. 0.6 cm).

2.4.3 H & E staining

Colon samples were fixed in 4% (v/v) paraformaldehyde tissue fixative for 24H, paraffin embedded sections were dewaxed with xylene, rehydrated by ethanol concentration gradient sequences, stained with hematoxylin and eosin (H & E) following standard laboratory protocols, and observed under light microscopy (magnification x 4/100/200, respectively).

The colon histological images of H & E showed significant tissue damage including epithelial cell damage and leukocyte infiltration in DSS colitis mice. However, tissue damage to the colon was significantly improved by supplementation with amuc_2109 (fig. 5). Colon histological index of mice receiving DSS treatment was significantly increased, and supplementation with amuc_2109 significantly decreased the index. The results indicate that Amuc_2109 protects the integrity of colonic tissue and reduces DSS-induced tissue damage.

2.4.4 qPCR

See FIG. 6 for the expression levels of TNF- α (A), IL-1β (B), IL-6 (C), NLRP3 (D), caspase-1 (E), ASC (F), ZO-1 (G), occludin (H), and claudin-1 (I) mRNA in colon tissue using qRT-PCR.

The expression level of colon TNF-alpha, IL-1 beta and IL-6mRNA in the DSS model group is obviously higher than that in the control group. In contrast, expression levels of colonic TNF-. Alpha.and IL-1β mRNA were significantly lower in Amuc2109 than in the DSS model group (FIGS. 6A-C). Compared with the control group, the expression level of NLRP3, ASC and caspase-1mRNA of the DSS model group is obviously increased. However, amuc2109 treatment significantly down-regulated NLRP3, ASC and caspase-1mRNA expression levels (FIG. 6D-F). The expression level of ZO-1, occludin, claudin-1mRNA was significantly reduced in the DSS group compared to the control group. However, amuc2109 significantly up-regulated the expression level of ZO-1, occludin, claudin-1mRNA after treatment (FIG. 6G-I).

2.4.5 fecal microbiota analysis

The primers sequenced were bacterial V3-V4 region primers:

338F：5’-ACTCCTACGGGAGGCAGCAG-3’

806R:5’-GGACTACHVGGGTWTCTAAT-3’。

the amplified product was detected by agarose gel electrophoresis at 2% mass. The PCR amplified products were quantified using a QuantiFluorTM-ST fluorometer and samples were adjusted as necessary for sequencing, which was performed by Shanghai Meiji Bio Inc.

Sequencing data indicated that the Venn plot showed three sets of overlapping OTU data, indicating that the OTUs of the DSS treated group were different from those of the other groups. Thus, different populations all have their own unique OTUs. There are 187, 27 and 22 different OTUs in the Control group, DSS group and amuc_2109 group, while other OTUs are shared in these groups (see fig. 7).

The histogram of intestinal microbiota community structure shows the relative abundance of this microbial species and its portal level (fig. 8). Firmics (P < 0.01) decreased in abundance by 27.2% and Bactoides (P < 0.01) increased in abundance by 18% compared to the control group. In contrast, after using Amuc_2109, the Bactoidetes (P < 0.1) decreased (6.1%). Firmics and Bactoides are the two most abundant bacterial gates in the gut, and the decrease in Firmics to Bactoides ratio in the intestinal microbiota of the DSS group, amuc_2109 treatment, also reversed this trend compared to the control group (FIG. 7).

2.6 conclusion

Amuc_2109 is a newly discovered glycoside hydrolase. It shows anti-inflammatory activity in DSS-induced colitis mice. Amuc_2109 arrests the progression of colitis by inhibiting excessive production of pro-inflammatory cytokines, inhibiting activation of colonic inflammatory signaling pathways, enhancing the mRNA expression of tight junctions proteins, and modulating intestinal microbiota. These results will help us to understand the mechanism by which amuc_2109 improves colitis, thereby guiding further development of amuc_2109 as an effective pharmaceutical product for preventing IBD.

The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application.

Sequence listing

<110> university of Anhui

<120> an acetylhexosidase Amuc_2109 protein purified from Acidocella, and preparation method and application thereof

<141> 2021-08-02

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 353

<212> PRT

<213> Ackermans (Akkermansia muciniphila)

<400> 1

Met Leu Pro Ala Leu Ile Gly Ile Ser Gly His Glu Val Gly Ala Glu

1 5 10 15

Glu Glu Ala Ala Ile Arg Arg Leu Gln Pro Ala Gly Phe Ile Leu Phe

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Ser Arg Asn Ile Asp Ser Val Glu Gln Val Arg Gly Leu Thr Glu Ser

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Leu Arg Lys Leu Cys Leu His His Pro Val Ile Ala Val Asp Gln Glu

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Gly Gly Arg Val Val Arg Thr Ala Ser Leu Gly Leu Asn Leu Pro Ser

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Pro Ala Ser Leu Ala Arg Leu Gly Ser Val Gly Gly Ile Val Glu Leu

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Gly Ala Val Thr Ala Leu Ala Leu Arg Tyr Leu Gly Val Asn Leu Asn

100 105 110

Phe Ala Pro Val Leu Asp Ile Cys His Asp Pro Ser Ala Ala Asn Ala

115 120 125

Leu Pro Gly Arg Cys Trp Gly Asp Asn Ala Gln Asp Val Ile Ser Arg

130 135 140

Gly Gly Val Tyr Ala Ser Asn Leu Arg Arg Gly Gly Val Gln Ser Cys

145 150 155 160

Gly Lys His Phe Pro Gly Met Gly Arg Ala Leu Ala Asp Pro His Phe

165 170 175

Ser Leu Pro Val Ile Gly Leu Asp Glu Arg Glu Leu Phe Lys Thr Asp

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Leu Leu Pro Phe Leu Ala Leu Cys Pro Ala Leu Ser Ser Ile Met Ser

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Ala His Ile Met Leu Pro Gln Ile Asp Pro Asp Tyr Pro Ala Thr Leu

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Ser Glu Arg Val Ile Arg Gly Leu Leu Arg Asp Arg Leu Gly Phe Arg

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Gly Val Val Phe Thr Asp Asp Leu Cys Met Gly Ala Ile Thr Thr Gln

245 250 255

Tyr Ser Pro Asp Asp Ala Ala Phe Leu Ser Leu Lys Ala Gly Cys Asp

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Leu Pro Leu Ile Cys His Asp Pro Leu Pro Trp Leu Asp Gly Leu Ala

275 280 285

Ser Arg Gln Glu Ser Leu Asn Ala Tyr Asp Arg Trp Asp Ser Phe Lys

290 295 300

Arg Val Glu Lys Leu Ser Asp Ser Leu Cys Phe Pro Phe Pro Glu Lys

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Ala Ser Leu Trp Asp Ser Cys Leu Arg Arg Ala Glu Ala Leu Cys Arg

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Leu Glu Glu Asp Gly Arg Glu Lys Leu Pro Ser Ser Pro Val Gln Lys

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Tyr

<210> 2

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<212> DNA

<213> Ackermans (Akkermansia muciniphila)

<400> 2

tcggttggcg gcatcgtgga actgggcgcg gtgacggctt tagctctccg ctacctgggg 60

gtgaacctga attttgcccc ggtgctggat atttgccatg atccgtccgc agccaacgcg 120

ctgcccggcc gctgctgggg agacaatgcg caggacgtta tttcacgcgg cggcgtttat 180

gcctccaacc tgcgccgggg aggcgtgcag agctgcggca aacattttcc cggcatgggg 240

cgtgccctgg cggatcccca tttcagcctt cccgtgatcg gcctggatga acgggagctg 300

ttcaagacgg atctgctgcc ttttctggct ctgtgtccgg cattgtcctc catcatgtcc 360

gcccatatca tgctgcctca gattgatccc gattatcccg ccaccttgtc tgaacgggtg 420

atcagggggc ttctgcgtga ccgcctcggt ttccggggcg tggtgtttac ggatgatttg 480

tgcatggggg cgattacgac gcagtattca ccggatgacg ccgccttcct gtccctgaag 540

gccggatgcg atcttcccct gatttgccat gaccctcttc cctggctgga tgggttggct 600

tcccgccagg aaagtttgaa cgcctatgac cggtgggatt ctttcaaacg ggtggaaaag 660

ctgagcgact ccctgtgttt cccgtttccg gaaaaggctt ccctgtggga ttcctgcctc 720

cgccgtgcgg aggctctatg ccgcctggaa gaggacggaa gagaaaaact gccttcatcc 780

ccagtccaga aatattga 798

Claims

1. The application of the Acetohexosaminidase Amuc_2109 protein prepared by genetic engineering in preparing medicaments for treating ulcerative colitis is characterized in that the amino acid sequence of the Amuc_2109 protein is shown as SEQ ID NO. 1.