CN115851658B - Mutant of mantis shrimp sensitization protein AK and application thereof - Google Patents
Mutant of mantis shrimp sensitization protein AK and application thereof Download PDFInfo
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Abstract
The invention provides a mutant of a mantis shrimp sensitization protein AK and application thereof, wherein the mutant is obtained by deleting alanine at 197 th position of the amino acid sequence of the mantis shrimp sensitization protein AK, and the amino acid sequence of the mantis shrimp sensitization protein AK mutant is shown as SEQ ID NO: 1. According to the mantis shrimp sensitization protein AK mutant, the gene sequence of the mantis shrimp AK mutant is obtained by overlapping extension polymerase chain reaction technology; connecting the mutant gene with a recombinant expression vector, and transferring the recombinant expression vector into a host cell to induce expression to obtain a mantis shrimp AK mutant D-A197; compared with AK, the D-A197 has obviously reduced IgE binding activity, is expected to be used for immunotherapy or immunoprophylaxis, and improves the safety index of the preventive treatment.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a mantis kou sensitization protein AK mutant and application thereof.
Background
Allergic diseases are a global problem that severely affects human health, and in recent years, the incidence of allergies caused by food has increased year by year worldwide, and food allergy has become a global concern for food safety and public health, called "second wave" allergy epidemic following asthma.
The middle-mouth mantis shrimp, which is also called as skin shrimp, has important nutritive value and economic value, is deeply favored by coastal residents, but the allergic reaction caused by the middle-mouth mantis shrimp increases year by year. Currently, allergen-specific immunotherapy is a clinically accepted immunotherapy measure, and the tolerance of the organism is continuously stimulated to induce the tolerance of the organism through low-dose and long-time exposure to the allergen, but side effects are also generated. The allergen preparation with low sensitization is utilized, so that side effects in immunotherapy can be greatly reduced, and the safety coefficient of the therapy is improved.
The transformation of the mantis shrimp sensitization protein is not reported, and arginine kinase (ARGININE KINASE, AK) is a main allergen of shrimp and can cause wide cross reaction as a ubiquitous allergen in shellfish. Therefore, the engineering of the shrimp sensitization protein AK is worthy of intensive research.
Disclosure of Invention
The present invention aims to solve at least to some extent one of the technical problems in the above-described technology. To this end, a first object of the present invention is to provide a mutant of mantis shrimp sensitization protein AK.
The second object of the present invention is to propose a gene encoding a mutant of mantis shrimp sensitization protein AK.
A third object of the present invention is to propose a recombinant vector.
A fourth object of the present invention is to propose a method for preparing mutants of mantis shrimp sensitization protein AK.
The fifth object of the invention is to propose the use of a mutant of mantis shrimp sensitization protein AK.
In a first aspect of the present invention, there is provided a mutant of a mantis shrimp sensitization protein AK, wherein the mutant is obtained by deleting alanine at position 197 of the amino acid sequence of the mantis shrimp sensitization protein AK, and the amino acid sequence of the mantis shrimp sensitization protein AK mutant is shown as SEQ ID NO: 1.
According to the embodiment of the invention, the amino acid alanine at 197 of the amino acid sequence of the mantis shrimp sensitization protein AK is deleted, thus a hyposensitization mutant can be obtained, the binding activity of the mutant to IgE is reduced, and the mutant can be used for immunotherapy or immunoprophylaxis.
In a second aspect of the present invention, there is provided a gene encoding a mutant of mantis shrimp sensitization protein AK, the nucleotide sequence of the gene being as set forth in SEQ ID NO: 2.
In a third aspect of the present invention, there is provided a recombinant vector comprising a gene encoding a mutant of the above mantis shrimp sensitization protein AK.
In a fourth aspect of the present invention, there is provided a method for preparing a mutant of mantis shrimp sensitization protein AK, comprising the steps of:
(1) Taking a plasmid obtained by the pET-22b-AK strain as a template, and carrying out SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO:10 is primer to carry out overlapping extension PCR first round amplification to obtain a first product;
(2) With the first product template, SEQ ID NO: 7. SEQ ID NO:8, performing overlapping extension PCR second round amplification on the primer to obtain a second product, and performing amplification recovery on the second product to obtain a mutant gene;
(3) Cloning the mutant gene into a prokaryotic expression vector to construct a recombinant vector;
(4) Transferring the recombinant vector into host bacteria, inducing the expression and purifying protein to obtain the mutant.
Optionally, the preparation of the pET-22b-AK strain comprises:
(1) Taking mantis shrimp cDNA as a template, and SEQ ID NO: 5. SEQ ID NO:6, carrying out PCR amplification on the primer to obtain a third product;
(2) Connecting the third product with pEASY-T1 plasmid to obtain a mantis shrimp pEASY-T1-AK plasmid;
(3) Using pEASY-T1 plasmid as template, SEQ ID NO: 7. SEQ ID NO:8, carrying out PCR amplification by using the primer to obtain a fourth product;
(4) Connecting the fourth product with pEASY-T1 plasmid to obtain a connecting product, converting the connecting product into Trans-T1 competent cells, and selecting single bacterial colony for colony PCR verification;
(5) Activating the strain with correct sequence and the expression vector pET-22b strain at 37 ℃ overnight, collecting thalli, extracting plasmids for double enzyme digestion, connecting enzyme digestion products, constructing a recombinant vector and transferring the recombinant vector into host bacteria to obtain the pET-22b-AK strain.
In a fifth aspect of the present invention, according to an embodiment of the present invention, the present invention also provides an application of the mutant of the mantis shrimp sensitization protein AK in preparing a medicament for preventing or treating shrimp allergic diseases.
According to the embodiment of the invention, the mantis koturi sensitization protein AK mutant can greatly reduce side effects in immunotherapy and improve the safety index of the therapy in the application of the prepared medicine and antigen-specific therapy.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows clone expression of mantis shrimp AK gene, wherein M is nucleic acid Marker, FIG. 1 (A) shows AK gene clone, FIG. 1 (B) shows colony PCR verification, FIG. 1 (C) shows pEASY-T1-AK plasmid double enzyme digestion electrophoresis pattern, and FIG. 1 (D) shows pET-22B plasmid double enzyme digestion electrophoresis pattern;
FIG. 2 shows SDS-PAGE (A) and Western blot (B) analysis of mantis shrimp AK;
FIG. 3 shows SDS-PAGE (A) and Western blot (B) analysis of mantis shrimp AK mutant D-A197;
FIG. 4 is an analysis of serum IgE binding activity of mantis shrimp AK and mutant D-A197 against mantis shrimp allergy sufferers.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. In order to simplify the present disclosure, specific embodiments or examples are described below. They are, of course, merely examples and are not intended to limit the invention. In addition, one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials, as examples of the various specific processes and materials provided by the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques in the fields of chemistry, molecular biology, etc., which are within the ability of a person skilled in the art. In addition, unless otherwise indicated, herein, nucleic acids are written in a 5 'to 3' direction from left to right, and amino acid sequences are written in an amino-to carboxy-terminal direction from left to right.
The invention is described below by way of illustrative specific examples, which are not intended to limit the scope of the invention in any way. Specifically described are: the reagents used in the present invention are commercially available unless otherwise specified.
EXAMPLE 1 cloning of mutant D-A197 of mantis shrimp sensitization protein AK
(1) Design and Synthesis of AK primer
The Primer 5.0 software was used to design the upstream and downstream primers (F, R) of mantis shrimp AK, and the NdeI and SalI restriction enzyme cleavage sites (F ', R') were added to the upstream and downstream primers, respectively. Primers used in the experiments were synthesized by Xiaomen Rui platinum Biotechnology Co.
F: 5’ -ATGGCTGACGCTGCTGTTATTGA-3’ ; （SEQ ID NO: 5）
R: 5’ -CATCTCCTTCTCCATCTTGATGAGC-3’ ; （SEQ ID NO: 6）
F’: 5’ -CATATGATGGCTGACGCTGCTGTTATTGA-3’ ; （SEQ ID NO: 7）
R’: 5’ -GTCGACCATCTCCTTCTCCATCTTGATGAGC-3’ ; （SEQ ID NO: 8）
(2) Cloning of mantis shrimp AK
Extraction of total RNA of Mantis shrimp according to the method of the instruction of the kit, and the extracted RNA is stored at-80 ℃ for standby after the concentration and purity of the RNA are detected. cDNA was synthesized according to PRIMESCRIPT TM II 1st Strand cDNA synthesis kit instructions. PCR amplification was performed using mantis shrimp cDNA as a template, and the amplification system is shown in Table 1.
TABLE 1 PCR amplification System
The PCR product is analyzed by 1% agarose gel electrophoresis, and after gel cutting, the PCR product is purified by a DNA purification kit, recovered and stored at the temperature of minus 20 ℃ for standby. The recovered fragment was ligated with pEASY-T1 vector at 25℃for 20 min, and the ligation system is shown in Table 2. The connection product is transformed into Trans-T1 competent cells, single colony is selected for colony PCR verification, positive cloned seeds are selected to be sequenced by Xiaomen Rui platinum biotechnology Co., ltd, the obtained sequence is an open reading frame of mantis shrimp sensitization protein AK, as shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.
TABLE 2pEASY-T1 ligation System
(3) Construction of pET-22b-AK recombinant plasmid
PCR amplification was performed using mantis shrimp pEASY-T1-AK plasmid as a template and F 'and R' as primers, respectively, with the amplification system being shown in Table 1. The amplified product was analyzed by 1% agarose gel electrophoresis, the target gene was recovered using a DNA gel recovery kit, and the recovered product was ligated with pEASY-T1 vector at 25℃for 20 min, the ligation system being shown in Table 2. The ligation products were transformed into Trans-T1 competent cells, single colonies were picked for colony PCR validation, and positive clones were selected to the Xiaomen Rui platinum Biotechnology Co.Ltd for sequencing.
The strain with correct sequence and the expression vector pET-22b strain are activated at 37 ℃ overnight, and the thalli are collected to extract plasmids. The plasmid was double digested with NdeI and SalI at 37℃for a period of 1 h and the cleavage system is shown in Table 3. And (3) after a large number of enzyme digestion, recovering the vector and AK genes by using a DNA gel recovery kit.
Table 3 double cleavage System
The cleavage products were mixed and then ligated at 16℃over 16 h to construct pET-22b-AK recombinant plasmid, the ligation system being as shown in Table 4. The ligation products were transformed into E.coli BL21 (DE 3) competent cells, and positive clones were picked up and sent to Xiamen Rui-platinum Biotechnology Co.Ltd for sequencing after colony PCR verification.
Table 4 expression vector ligation System
(4) Cloning of mantis shrimp AK mutant D-A197 and construction of expression plasmid
The plasmid obtained from the pET-22b-AK strain was used as a template, a primer (F mutants,Rmutants) was designed based on the deleted position of alanine at position 197, and the first round of overlap extension PCR amplification was performed using F '/R mutants and F mutants/R' as primers, respectively, with the amplification system shown in Table 5.
Fmutants: 5’ -GCTTCCTGCAGGCTAACGCTT-3’ ; （SEQ ID NO: 9）
Rmutants: 5’ -CAAGCGTTAGCCTGCAGGAAG-3’ ; （SEQ ID NO: 10）
TABLE 5 first round overlap extension PCR amplification System
After the amplified product is analyzed by 1% agarose gel electrophoresis, the target fragment is recovered by a DNA gel recovery kit and stored at-20 ℃ for standby. The second round of overlap extension PCR amplification was performed using the recovered DNA product as template and F '/R' as primer, and the amplification system was as shown in Table 6.
TABLE 6 second round overlap extension PCR System
The amplified product is separated by 1% agarose gel electrophoresis, and then a large amount of amplification and recovery of mutant full-length genes are carried out. The recovered mutant gene was ligated with pEASY-T1 vector at 25℃for 20 min, see Table 2 for ligation system. The connection product is transformed into Trans-T1 competent cells, positive clones are picked after colony PCR verification and sent to Xiamen Rui Chen platinum biotechnology Co-Ltd for sequencing, and the obtained sequence is a mantis shrimp sensitization protein AK mutant D-A197 gene sequence as shown in SEQ ID NO. 2, and the amino acid sequence is shown in SEQ ID NO. 1.
The correctly sequenced strain was activated overnight at 37℃and the cells were collected to extract the plasmid. The plasmids were digested with NdeI and SalI at 37℃for 1h times, respectively, and the digestion system is shown in Table 3. And (3) recovering the mutant full-length genes by using a DNA gel recovery kit after a large number of enzyme cuts. The cleavage products were mixed and then ligated at 16℃over 16 h, the ligation system being shown in Table 4. The ligation products were transformed into E. coliBL21 (DE 3) competent cells, and positive clones were picked up and transferred to Xiaomen Rui platinum Biotechnology Co.Ltd for sequencing after colony PCR verification.
EXAMPLE 2AK preparation of mutant D-A197
(1) Induction expression of AK and mutant D-A197
Strains containing AK target gene and mutants D-A197 gene, which were sequenced correctly, were inoculated into 20mL LB medium containing 1mM Amp and cultured overnight. The following day, the culture solution is transferred into a LB culture medium containing 1mM Amp according to the ratio of 1:1000, and when the culture solution is cultured to OD 600 =0.6-0.8 at 37 ℃, IPTG with the final concentration of 0.5mM is added to induce expression 4 h. After centrifugation (12000 g, 10 min), the cells were collected, resuspended in sonication buffer (200 mM NaCl, 20 mM Tris-HCl, pH 8.0) and sonicated and centrifuged (12000 g, 20 min) to show that AK and D-A197 are both expressed in pellet. The pellet was collected, resuspended in 4M urea-containing sonication buffer, dialyzed against 2M and 0M urea-containing sonication buffer, and the supernatant was collected after centrifugation (12000 g, 20 min) for SDS-PAGE and Western blot analysis, and the results are shown in FIGS. 2 and 3. FIG. 2 shows SDS-PAGE (A) and Western blot (B) analysis of AK, wherein M is a protein Marker, lane 1 is pET-22B empty, lane 2 is total bacterial liquid after ultrasonic treatment of pET-22B-AK strain, lane 3 is supernatant after ultrasonic treatment of strain, lane 4 is precipitation after ultrasonic treatment of strain, lane 5 is supernatant after precipitation denaturation, and lane 6 is supernatant after AK renaturation; FIG. 3 shows SDS-PAGE (A) and Western blot (B) analysis of D-A197, wherein M is a protein Marker, lane 1 is the whole post-sonicated supernatant of the D-A197 strain, lane 2 is the post-sonicated supernatant of the strain, lane 3 is the post-sonicated pellet of the strain, and lane 4 is the post-renatured supernatant of the D-A197 strain.
(2) Purification of AK and mutant D-A197
The supernatant after protein renaturation in (1) is loaded on a Ni 2+ -NTA affinity chromatography column which is balanced by ultrasonic buffer solution in advance at the speed of 1 mL/min, and unbound protein is continuously washed by the ultrasonic buffer solution. Eluting the target protein with 100 mM imidazole-containing ultrasonic buffer until the A 280 of the eluent is less than 0.05, collecting the purified part after SDS-PAGE analysis, concentrating, replacing PBS buffer, adjusting the concentration to 1.0 mg/mL, and storing at-80 ℃ for later use.
Example 3 AK and mutant D-A197 and Mantis shrimp allergy patient serum Dot blot analysis
Mu.L of AK protein, D-A197 protein and BSA were each prepared at a protein concentration of 1.0 mg/mL, spotted on nitrocellulose membrane (0.7X10.7: 0.7 cm per cell), and left to stand for air-drying. The dried nitrocellulose membrane is soaked in 5% of skimmed milk and placed on a shaker for incubation at room temperature of 1.5 h. After blocking, the nitrocellulose membrane was washed 3 times with TBST (17 g NaCl,20 mL 1M Tris-HCl, pH 8.0,1 mL Tween-20 in 2L distilled water), 5min each time. Nitrocellulose membranes were incubated overnight in serum of allergic patients with mantis shrimps diluted 1:2 with TBST, respectively. The next day, nitrocellulose membranes were removed, washed 5 times in TBST, 5min each, and incubated with horseradish peroxidase-labeled goat anti-human IgE antibody in TBST at 1:20000 on a shaker for 1: 1 h. Taking out nitrocellulose membrane, washing 7 times in TBST, each time 7 min, inversely fastening the membrane in a color development liquid, incubating 2 min, photographing by using a chemiluminescence fluorescent gel imager, and respectively obtaining different serum of the patient suffering from the mantis shrimp allergy with the numbers 1-6 as shown in figure 4 (A).
As can be seen from FIG. 4 (A), AK has strong specific binding with the serum of the patient suffering from the oromantis allergy, while D-A197 has a significant decrease in specific binding with the serum of the patient suffering from the oromantis allergy. Gray scale analysis and significance analysis were performed using Image J software and Prism 8 software, respectively, as shown in fig. 4 (B), and the results showed that the fluorescence intensity of the IgE reaction of D-a197 with the serum of the patient allergic to mantis shrimps was significantly lower than AK. From this, it was found that D-A197 has significantly reduced IgE binding ability to serum of patients suffering from an allergic mantis, as compared with AK.
In summary, the application utilizes a molecular cloning technology to obtain an open reading frame of the mantis shrimp sensitization protein AK, removes the 197 th alanine of the mantis shrimp AK gene by a genetic engineering technology, and transfers the mutant gene into host bacteria after being connected with an expression vector pET-22b carrier, thereby successfully obtaining the mantis shrimp AK mutant D-A197. Compared with AK, the combination ability of D-A197 and IgE in serum of a patient with hypersensitive mantis shrimp is obviously reduced, and the medicine for preventing or treating hypersensitive diseases of shrimp aquatic products can be developed and prepared, so that the safety index of treatment is improved.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (3)
1. A mutant of a mantis shrimp sensitization protein AK, which is characterized in that alanine at 197 th position of the amino acid sequence of the mantis shrimp sensitization protein AK is deleted, and the amino acid sequence of the mantis shrimp sensitization protein AK mutant is shown as SEQ ID NO: 1.
2. A gene encoding a mutant of the mantis shrimp sensitization protein AK according to claim 1, wherein the nucleotide sequence of the gene is set forth in SEQ ID NO: 2.
3. A recombinant vector comprising the gene of claim 2.
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