CN107446942B

CN107446942B - Preparation method and kit of active human AGR2

Info

Publication number: CN107446942B
Application number: CN201710797011.0A
Authority: CN
Inventors: 李大伟; 江文丽; 朱奇; 赫迪希; 武正华
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2017-09-06
Filing date: 2017-09-06
Publication date: 2021-07-13
Anticipated expiration: 2037-09-06
Also published as: CN107446942A

Abstract

The invention relates to the technical field of molecular biology, in particular to a preparation method of active human pre-gradient protein AGR2, which comprises the following steps: (1) inserting the human AGR2 gene into a vector to obtain an AGR2 recombinant expression vector; (2) transforming host cells by the AGR2 recombinant expression vector obtained in the step (1) to obtain a transformant; (3) and (3) culturing the transformant in the step (2) and processing the cultured transformant to obtain the active human AGR 2.

Description

Preparation method and kit of active human AGR2

Technical Field

The invention relates to the technical field of molecular biology, in particular to a preparation method and a kit of active humanized AGR 2.

Background

The xenopus anterior gradient proteins XAG-1, XAG-2, and XAG-3 were first found in xenopus embryonic sections. As the embryo ages, XAG-2 expression increased in the anterior region of the ectoderm in the back of the embryo, which is involved in the development of the mucus glands.

Pre-gradient protein 2 (AGR 2) is a protein expressed in humans by the AGR2 gene and is a homolog of XAG-2 in humans. AGR2 is highly expressed in mucus-secreting tissues and endocrine organs including the lung, stomach, colon, prostate and small intestine. The expression of AGR2 is regulated by androgens and estrogens. The AGR2 protein is a protein disulphide isomerase, and plays an important role in protein folding. AGR2 has a sequence of CXXS active domain that can undergo redox reactions, forming disulfides in substrates such as enteromucin. AGR2 interacts with mucin 2 through the thioredoxin-like domain to form a hetero-disulfide bond with the cysteine residue of mucin 2. The C-terminus of AGR2 has a KTEL domain similar to KDEL and KVEL sequences, associated with endoplasmic reticulum retention.

AGR2 of human origin was first found in estrogen receptor positive breast cancer cells. Later studies showed overexpression of AGR2 in all of esophageal, pancreatic and prostate cancer. In a human body, the high expression of AGR2 protein can cause the down regulation of tumor suppressor protein p53, the activity of vascular endothelial growth factor VEGF and fibroblast growth factor FGF2 is enhanced, and angiogenesis and fibroblast are promoted to coordinate the invasion of tumor cells, so that the AGR2 protein is related to the formation and migration of the tumor cells. The obtained active human AGR2 protein has important theoretical and practical significance for further research of biological function and clinical application.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a method and a kit for preparing an active human AGR2, so as to prepare an active human AGR2, thereby further studying the biological function and clinical application of the human AGR 2.

The invention provides a preparation method of active human pre-gradient protein AGR2, which comprises the following steps: (1) inserting the human AGR2 gene into a vector to obtain an AGR2 recombinant expression vector; (2) transforming host cells by the AGR2 recombinant expression vector obtained in the step (1) to obtain a transformant; (3) and (3) culturing the transformant obtained in the step (2) and treating the cultured transformant to obtain the active human AGR 2.

In one embodiment of the invention, the sequence of the human AGR2 gene is shown in SEQ ID NO: 5, respectively.

In one embodiment of the present invention, the human AGR2 gene can be obtained by a method comprising the following steps: extracting total RNA from human breast cancer cells MCF 7; preparing cDNA corresponding to the total RNA; carrying out AGR2 gene specific PCR amplification by taking the cDNA as a template to obtain a humanized AGR2 gene; wherein, the sequence of the upstream primer of the AGR2 gene specific PCR is shown as SEQ ID NO: 1, specifically 5'-GGATCCATGGAGAAAATTCCAGTGTC-3', wherein the upstream primer of the AGR2 gene specific PCR comprises an enzyme cutting site; the downstream primer sequence of the AGR2 gene specific PCR is shown as SEQ ID NO: 2, specifically 5'-TTACAATTCAGTCTTCAGCA-3'.

In one embodiment of the present invention, the vector in step (1) is prokaryotic expression vector pMAL-c 2.

In one embodiment of the present invention, the step (1) comprises: carrying out enzyme digestion and connection on a human AGR2 gene and the prokaryotic expression vector pMAL-c2 to obtain a recombinant expression vector pMAL-AGR 2-MBP; and deleting an MBP gene and inserting a His tag gene in the pMAL-AGR2-MBP by mutation PCR to obtain an AGR2 recombinant expression vector.

In one embodiment of the present invention, the sequence of the upstream primer of the mutation PCR is shown in SEQ ID NO: 3, specifically 5'-CACCACCACAGAGATACCACAGTCAAACCTGGAGCCA-3'; the sequence of the downstream primer of the mutation PCR is shown as SEQ ID NO: and 4, specifically 5'-GTGGTGGTGCATAATCTATGGTCCTTGTTGGTGAAGTGC-3'.

In one embodiment of the present invention, the step (3) includes: purifying the active human AGR2 by a nickel chromatographic column.

The invention also provides a preparation kit of the active human pre-gradient protein AGR2, which comprises: a reagent and a vector for obtaining the human AGR2 gene, a reagent and a transformation reagent for constructing an AGR2 recombinant expression vector.

In one embodiment of the invention, the reagent for obtaining the human AGR2 gene comprises a total RNA extraction reagent, a reverse transcription reagent, an upstream primer of AGR2 gene specific PCR and a downstream primer of AGR2 gene specific PCR; wherein, the sequence of the upstream primer of the AGR2 gene specific PCR is shown as SEQ ID NO: 1, specifically 5'-GGATCCATGGAGAAAATTCCAGTGTC-3', wherein the upstream primer of the AGR2 gene specific PCR comprises an enzyme cutting site, and the sequence of the downstream primer of the AGR2 gene specific PCR is shown in SEQ ID NO: 2, specifically 5'-TTACAATTCAGTCTTCAGCA-3'.

In one embodiment of the invention, the vector is the prokaryotic expression vector pMAL-c 2.

In one embodiment of the invention, the reagent for constructing the recombinant expression vector of AGR2 comprises a reagent for cutting and connecting the human AGR2 gene and the prokaryotic expression vector pMAL-c2, an upstream primer of mutation PCR and a downstream primer of mutation PCR.

Compared with the prior art, the invention has the following beneficial effects: can simply and efficiently obtain the human AGR2 with activity, and is beneficial to the research of biological function and clinical application of the human AGR 2.

Drawings

FIG. 1 is a flow chart of the construction of pMAL-his-AGR2 provided in the examples of the present invention;

FIG. 2 is a specific PCR map of human AGR2 gene. Lane 21 is DS5000 molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), and lane is electrophoresis result of PCR product with specificity of 22.

FIG. 3 shows the identification map of the specific PCR fragment of human AGR2 gene and pMAL-c2 vector by digestion and ligation. Lane 31DS5000 molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), Lane 32 electrophoresis result of T4 ligase ligation product, Lane 33 electrophoresis result of vector BamHI cleavage product, Lane 34 electrophoresis result of specific PCR fragment BamHI cleavage product.

FIG. 4 shows the restriction enzyme identification map of recombinant plasmid pMAL-his-AGR 2. Lane 41 is DS5000 molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), lane 42 is electrophoresis result of plasmid pMAL-his-AGR2 not digested with enzyme, lane 43 is electrophoresis result of pMAL-his-AGR2 digested with BamHI, lane 44 is electrophoresis result of pMAL-his-AGR2 digested with Xbal.

FIG. 5 is a mutant product identification map. Lane 51 is the DS5000 molecular weight standard (5000 bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom), lane 52 is the electrophoresis result of the mutated product, and lane 53 is the electrophoresis result of pMAL-MBP-AGR 2.

FIG. 6 shows the SDS-PAGE identification of the expression of human AGR2 recombinant protein. Lane 61 is a protein molecular weight standard (180 kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, and 10kDa in this order from top to bottom), lane 62 is the electrophoresis result of pMAL-his-AGR2 without IPTG induction, lane 63 is the electrophoresis result of pMAL-his-AGR2/DH 5. alpha. with IPTG induction, lane 64 is the electrophoresis result of bacterial lysate centrifugation supernatant after IPTG induction of pMAL-his-AGR2/DH 5. alpha., and lane 65 is the electrophoresis result of bacterial lysate centrifugation precipitation after IPTG induction of pMAL-his-AGR2/DH 5. alpha. with IPTG induction.

FIG. 7 shows the results of western blot identification and detection of expression patterns of human AGR2 recombinant protein expression. Lane 71 shows the standard protein molecular weight (180 kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, and 10kDa in this order from top to bottom), lane 72 shows the electrophoresis results of pMAL-his-AGR2 without IPTG induction, lane 73 shows the electrophoresis results of pMAL-his-AGR2/DH 5. alpha. with IPTG induction, lane 74 shows the electrophoresis results of the supernatant after IPTG induction, pMAL-his-AGR2/DH 5. alpha. ultrasonication, and lane 75 shows the electrophoresis results of precipitation after IPTG induction, pMAL-his-AGR2/DH 5. alpha. ultrasonication.

FIG. 8A shows SDS-PAGE detection of the eluate. Lane 81 shows the molecular weight standards of the proteins (180 kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa in the order from top to bottom), and lanes 82 to 87 show the results of electrophoresis of eluents 1 to 6, respectively.

FIG. 8B shows SDS-PAGE of purified human AGR2 recombinant protein. The recombinant protein with a purity of 95% or more was obtained by nickel column affinity chromatography (lane 811).

FIGS. 9A and 9B show the results of western blot analysis of purified human AGR2 recombinant protein.

FIG. 9A shows 18A4 (mouse anti-AGR 2 monoclonal antibody) as the primary antibody and fluorescently labeled goat anti-mouse IgG as the secondary antibody. Lane 91 shows the protein molecular weight standards (180 kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10kDa in the order from top to bottom), lane 92 shows the electrophoresis result of the negative control BSA, and lane 93 shows the electrophoresis result of the purified protein.

FIG. 9B shows the use of mouse anti-his monoclonal antibody as the primary antibody and fluorescently labeled goat anti-mouse IgG as the secondary antibody. Lane 94 shows the protein molecular weight standards (180 kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, and 10kDa in this order from top to bottom), lane 95 shows the electrophoresis result of negative control BSA, and lane 96 shows the electrophoresis result of purified protein.

FIG. 10 shows the results of measuring the concentration of the purified human AGR2 recombinant protein. Lane 101 shows the molecular weight standards of the proteins (180 kDa, 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, and 10kDa in the order from top to bottom), lane 102 shows the result of electrophoresis obtained by diluting the purified protein 30 times, lane 103 shows the result of electrophoresis obtained by diluting the purified protein 60 times, lane 104 shows the result of electrophoresis obtained by diluting the purified protein 120 times, lane 105 shows the result of electrophoresis obtained by diluting the purified protein 1mg/ml, lane 106 shows the result of electrophoresis obtained by diluting the purified protein 0.5mg/ml, and lane 107 shows the result of electrophoresis obtained by diluting the purified protein 0.25 mg/ml.

FIG. 11A, FIG. 11B, FIG. 11C are the results of the activity assay of human AGR2 recombinant protein.

FIG. 11A shows the result of agarose dropping a concentration gradient of 0mg/ml His-AGR2 recombinant protein onto a cell slide cultured with 3T3 cells, and the cells invaded into the agarose after 14 hours.

FIG. 11B shows the result of agarose gel electrophoresis of His-AGR2 recombinant protein with a concentration gradient of 1mg/ml, which was dropped onto a cell slide cultured with 3T3 cells, and the cells invaded into the agarose gel after 14 hours.

FIG. 11C shows the result of agarose gel electrophoresis of His-AGR2 recombinant protein with a concentration gradient of 3mg/ml, dropped onto a cell slide cultured with 3T3 cells, and the cells invaded into the agarose gel after 14 hours.

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

The embodiment of the invention provides a preparation method of active human AGR2, which comprises the following steps: (1) inserting the human AGR2 gene into a vector to obtain an AGR2 recombinant expression vector; (2) transforming host cells by the AGR2 recombinant expression vector obtained in the step (1) to obtain a transformant; (3) and (3) culturing the transformant obtained in the step (2) and treating the cultured transformant to obtain the active human AGR 2.

The preparation method of the active human pre-gradient protein AGR2 provided by the embodiment of the invention is simple and convenient to operate, compared with the AGR2 protein on the market, the method has the advantages that a signal peptide fragment is deleted, the active human AGR2 can be efficiently prepared, and the biological function and clinical application research on the human AGR2 can be favorably carried out.

In one example, the sequence of the human AGR2 gene is as set forth in SEQ ID NO: 5, respectively.

In one example, the human AGR2 gene can be obtained by a method comprising the following steps: extracting total RNA from human breast cancer cells MCF 7; preparing cDNA corresponding to the total RNA; carrying out AGR2 gene specific PCR amplification by taking the cDNA as a template to obtain a humanized AGR2 gene; wherein, the sequence of the upstream primer of the AGR2 gene specific PCR is shown as SEQ ID NO: 1, specifically 5'-GGATCCATGGAGAAAATTCCAGTGTC-3', wherein the upstream primer of the AGR2 gene specific PCR comprises an enzyme cutting site; the downstream primer sequence of the AGR2 gene specific PCR is shown as SEQ ID NO: 2, specifically 5'-TTACAATTCAGTCTTCAGCA-3'.

In one example, the vector in step (1) is prokaryotic expression vector pMAL-c 2.

In one example, the step (1) includes: carrying out enzyme digestion and connection on a human AGR2 gene and the prokaryotic expression vector pMAL-c2 to obtain a recombinant expression vector pMAL-AGR 2-MBP; and deleting an MBP gene and inserting a His tag gene in the pMAL-AGR2-MBP by mutation PCR to obtain an AGR2 recombinant expression vector.

In one example, the upstream primer sequence of the mutant PCR is set forth in SEQ ID NO: 3, specifically 5'-CACCACCACAGAGATACCACAGTCAAACCTGGAGCCA-3'; the sequence of the downstream primer of the mutation PCR is shown as SEQ ID NO: and 4, specifically 5'-GTGGTGGTGCATAATCTATGGTCCTTGTTGGTGAAGTGC-3'.

The escherichia coli expression system is a simple and efficient prokaryotic expression system and has the characteristics of low cost, high protein expression quantity, simplicity in operation and the like. The commonly used PET system can add a histidine tag (his-tag) with small molecular weight at the tail of an expression product, has small influence on the structural function of a target protein and has low immunogenicity. The histidine tag is specifically bound with a Ni2+ affinity chromatography column, and the target protein can be separated and purified by using a nickel column affinity chromatography method. However, the PET system plasmid uses the T7 promoter, and the E.coli strain BL21(DE3) fused with T7 phage RNA polymerase. More importantly, the PET system has the defects of low purity of the purified protein product, more impurities and the like. Yet another commonly used protein purification system, pMAL, inserts a cloned gene downstream of the malE gene expressing Maltose Binding Protein (MBP) to form an MBP fusion protein. The pMAL system uses a tac promoter, i.e., an artificial hybrid of trp promoter and lac promoter, has a high transcription level, and can be used for any Escherichia coli strain, so that DH5 alpha can be used for plasmid transformation, and the operation is simpler. But the MBP protein has large molecular weight and has great influence on the structure and the function of the protein; in the example, the pMAL system is optimized, the malE gene region is deleted, and the histidine tag is inserted, so that the advantages of the two systems are combined, the influence of the tag on the structure and the function of the protein is reduced, and the purity of the target protein is improved.

In one example, the step (3) includes: purifying the active human AGR2 by a nickel chromatographic column; wherein the sequence of the active human AGR2 is shown as SEQ ID NO: and 6.

In one example, the preparation method of the active human AGR2 provided by the embodiment of the present invention further includes the following steps: (5) agarose containing the active human AGR2 at a specific concentration is dripped on a cell climbing sheet for culturing 3T3 cells to detect the activity of the active human AGR 2. The research of the inventor shows that AGR2 promotes angiogenesis and fibroblast to coordinate tumor cell invasion in the extracellular direction, and secreted AGR2 promotes angiogenesis and invasion of vascular endothelial cells and fibroblasts by enhancing the activities of VEGF and FGF 2. In this example, the results of this study can be used to test the activity of the human AGR2 prepared. In one example, agarose containing AGR2 protein prepared at a concentration gradient of 0mg/ml, 1mg/ml, 3mg/ml can be added dropwise to a cell slide in which 3T3 cells are cultured, and AGR2 protein can be prepared to promote 3T3 cells to invade the agarose, and the longer the invasion distance, the better the protein activity.

The embodiment of the invention also provides application of the active humanized AGR2 prepared according to the preparation method in preparing a medicine for treating soft tissue injury.

The research of the inventor shows that the human AGR2 can accelerate the healing of skin wounds by regulating the migration of keratinocytes and fibroblasts, and can be used as a potential replacement therapy of the existing medicines for accelerating the healing of soft tissues, particularly skin wounds.

In one example, the soft tissue injury comprises a skin injury.

The embodiment of the invention provides a preparation kit of active human pre-gradient protein AGR2, which comprises: a reagent and a vector for obtaining the human AGR2 gene, a reagent and a transformation reagent for constructing an AGR2 recombinant expression vector.

In one example, the reagents for obtaining the human AGR2 gene include a total RNA extraction reagent, a reverse transcription reagent, an upstream primer of AGR2 gene specific PCR and a downstream primer of AGR2 gene specific PCR; wherein, the sequence of the upstream primer of the AGR2 gene specific PCR is shown as SEQ ID NO: 1, specifically 5'-GGATCCATGGAGAAAATTCCAGTGTC-3', wherein the upstream primer of the AGR2 gene specific PCR comprises an enzyme cutting site, and the sequence of the downstream primer of the AGR2 gene specific PCR is shown in SEQ ID NO: 2, specifically 5'-TTACAATTCAGTCTTCAGCA-3'.

In one example, the vector is the prokaryotic expression vector pMAL-c 2.

In one example, the reagent for constructing the recombinant expression vector of AGR2 comprises a reagent for cutting and connecting the human AGR2 gene and the prokaryotic expression vector pMAL-c2, an upstream primer of mutation PCR and a downstream primer of mutation PCR.

The technical solution of the embodiment of the present invention will be described in more detail with reference to specific example 1.

Example 1

1. Construction and identification of recombinant plasmid pMAL-his-AGR2

The process for constructing and identifying the recombinant plasmid pMAL-his-AGR2 is shown in FIG. 1. The details are as follows.

1-1 and AGR2 derived from human.

Designing human AGR2 gene upstream and downstream primers according to the gene full-length sequence (NC-000007.14) of human AGR2 in GenBank, and introducing corresponding restriction endonuclease cutting sites, wherein the upstream primer sequence (shown in SEQ ID NO: 1) is 5'-GGATCCATGGAGAAAATTCCAGTGTC-3' (the underlined part is a BamHI cutting site); the sequence of the downstream primer (shown in SEQ ID NO: 2) is as follows: 5'-TTACAATTCAGTCTTCAGCA-3' are provided. The MCF7cDNA containing the full-length sequence of the human AGR2 gene is used as a template, and PCR amplification is carried out by using a specific primer of the human AGR2 gene. The size of the amplified fragment is 528bp, wherein the gene sequence of the human AGR2 is shown in SEQ ID NO. 5. The PCR reaction conditions are 94 ℃ for 2min, 94 ℃ for 15sec, 50 ℃ for 30sec, and 68 ℃ for 1min, and 30 cycles are total; further extension was carried out at 68 ℃ for 10 min. The PCR products were analyzed by 1% agarose gel electrophoresis and recovered. The results of PCR amplification and electrophoresis analysis of human AGR2 are shown in FIG. 2.

1-2, construction and identification of a recombinant expression plasmid pMAL-his-AGR 2.

The PCR product of the human AGR2 gene and pMAL-c2 were digested with BamHI, ligated with T4 ligase at 16 ℃ for 1 hour, and a recombinant plasmid containing the human AGR2 gene was constructed. The results of electrophoretic analysis of the cleaved and ligated products are shown in FIG. 3, lane 32 is the ligated product band, lane 33 is the BamHI cleaved band of pMAL-c2, and lane 34 is the BamHI cleaved band of the PCR product of human AGR 2. The ligation product was transformed to extract plasmid, which was digested with BamHI and Xbal respectively, and the map is shown in FIG. 4, after digestion with BamHI there is a specific DNA band at 550bp (lane 43), after digestion with Xbal there is a specific DNA band at 6.6kb (lane 44). Indicating that the plasmid construction is correct. The plasmid was sent to Bio Inc. for sequencing, and the correctly sequenced plasmid was designated pMAL-MBP-AGR 2. Deleting an MBP fragment from the obtained pMAL-AGR2-MBP by mutation PCR, inserting a His fragment, wherein the sequence of an upstream primer of the mutation PCR (shown in SEQ ID NO: 3) is 5'-CACCACCACAGAGATACCACAGTCAAACCTGGAGCCA-3'; the sequence of the downstream primer of the mutation PCR (shown in SEQ ID NO: 4) is: 5'-GTGGTGGTGCATAATCTATGGTCCTTGTTGGTGAAGTGC-3' are provided. The PCR reaction conditions are 94 ℃ for 2min, 94 ℃ for 15sec, 68 ℃ for 30sec, and 68 ℃ for 6min, and 30 cycles are total; further extension was carried out at 68 ℃ for 15 min. The size of the product after mutation is 5973 bp. The results of electrophoretic analysis of the mutated product are shown in FIG. 5, lane 52 is the mutated product, and lane 53 is the pMAL-MBP-AGR2 plasmid. After the mutant product is recovered, the mutant product is connected for 1h at 16 ℃ under the action of T4 ligase, after the connecting product is transformed into escherichia coli DH5 alpha, a plasmid is extracted and sent to a biological company for sequence determination, and the result shows that the gene sequence is consistent with the sequence published by Genbank. The correctly sequenced plasmid was designated pMAL-his-AGR 2.

2. pMAL-his-AGR2/DH5 alpha is used for inducing expression of the target protein and identifying the expression product.

2-1, expression of human AGR 2.

The induction expression of the pMAL-his-AGR2/DH5 alpha engineering bacteria is that a single colony of pMAL-his-AGR2/DH5 alpha is selected from an Amp + LB plate and inoculated in an Amp resistant LB culture medium, and the bacteria are shaken at 37 ℃ and 200rpm overnight; inoculating in new Amp-resistant LB culture medium at 1:1000 the next day, and shaking at 37 deg.C and 200rpm for 2-3 hr until OD550 is 0.6-0.8; adding 1mM IPTG to induce expression for 4h at 37 ℃, and setting pMAL-his-AGR2/DH5 alpha engineering bacteria non-induced control. Sucking bacterial liquid 300ul, centrifuging at 4 ℃ and 10000rpm for 30s, discarding supernatant, and collecting thalli. Adding 100ul PBS for resuspension, adding 25ul 5 xSDS loading buffer, mixing, and boiling at 95 deg.C for 5 min.

3. Purifying and identifying human AGR 2.

3-1, extraction, identification and expression form analysis of human AGR2, which are specifically as follows.

Extraction of human AGR 2: the expression bacteria were resuspended in PBS and sonicated in a sonicator. The ultrasonic condition is 3s of work, 6s of pause and 400W, and the total ultrasonic time is 10 minutes. During ultrasonic treatment, the expression bacteria are placed on ice to cool the bacteria liquid and prevent protein degradation. After disruption, centrifugation was carried out at 13000rpm for 20 minutes at 4 ℃.

Identification and expression form analysis of human AGR 2: the expression bacteria, the uninduced control, the supernatant and the precipitate are respectively taken for SDS-PAGE electrophoresis. The results of the electrophoretic analysis are shown in FIG. 6, and the expressed bacteria have a specific protein band at 15-25kDa (lane 63), which is consistent with the expected molecular weight of the recombinant protein of AGR2 of human origin. Human AGR2 in the expression strain exists in the precipitate mainly in the form of inclusion body, and a small amount of expression is also carried out in the supernatant. After the gel was subjected to membrane transfer, the recombinant protein was identified by western Blot using a mouse anti-AGR 2 monoclonal antibody as a primary antibody and a fluorescently labeled goat anti-mouse IgG as a secondary antibody, and the result is shown in fig. 7, where a specific fluorescent band (lane 73) is present at 15-25kDa, indicating that the recombinant protein can specifically bind to the anti-AGR 2 monoclonal antibody.

3-2, purification, post-purification identification and content identification of human AGR2

Purification of human AGR 2: the supernatant was collected and passed through a pretreated nickel column, human AGR2 was affinity-adsorbed to Ni2+ by a histidine tag, then unbound impurities were washed off with 10 volumes of binding buffer (0.5M NaCl, 20mM Tris-HCl, 5mM imidazole) and 6 volumes of washing buffer (0.5M NaCl, 20mM Tris-HCl, 60mM imidazole), and human AGR2 recombinant protein was eluted with 6 volumes of elute buffer (0.5M NaCl, 20mM Tris-HCl, 1M imidazole), and the eluates were collected sequentially at 0.5ml per tube. 40ul of each of the first 6 eluate samples was added with 10ul of 5 Xloading buffer, mixed well and boiled at 95 ℃ for 5 min. The boiled samples were labeled in turn as eluents 1 to 6 (lanes 82 to 87), and the samples were subjected to SDS-PAGE analysis, and the results are shown in FIG. 8A, in which the sample having the highest protein concentration was eluent 3 (lane 84), followed by eluents 2 (lane 83) and 4 (lane 85). And (3) putting the eluent 2-4 into a dialysis bag for dialysis, and precipitating high-concentration salt solution in the eluent, wherein the protein in the dialysis bag is the high-purity human AGR2 recombinant protein. The amino acid sequence of the human AGR2 recombinant protein is shown as SEQ ID No: 6, containing a human AGR2 sequence and 6 histidine sequences.

Identification of purified AGR2 of human origin: the purified protein was subjected to SDS-PAGE, and the result is shown in FIG. 8B, which indicates that the recombinant protein having a purity of 95% or more was obtained by nickel column affinity chromatography (lane 811). After gel membrane transfer, a mouse anti-AGR 2 monoclonal antibody and a mouse anti-his monoclonal antibody are used as primary antibodies, a fluorescently-labeled goat anti-mouse IgG is used as a secondary antibody, and the recombinant protein is identified by using western Blot, and the result is shown in fig. 9A and 9B, wherein a specific fluorescent band (lane 93) exists at the 15-25kDa position, which indicates that the purified recombinant protein can be specifically combined with the anti-AGR 2 monoclonal antibody.

Identification of the concentration of purified human AGR 2: the purified human AGR2 is respectively diluted by 30 times, 60 times and 120 times to prepare positive control BSA 1mg/ml, 0.5mg/ml and 0.25mg/ml, and the result is shown in figure 10 by SDS-PAGE analysis, which shows that the concentration of the recombinant protein prepared by the method can reach 30-60 mg/ml.

33. Activity identification of human AGR2 recombinant protein

According to the characteristic that human AGR2 can promote fibroblast migration, agarose with concentration gradient of 0mg/ml, 1mg/ml and 3mg/ml His-AGR2 recombinant protein is dripped on a cell slide for culturing 3T3 cells, and the distance of the cells invading the agarose under the influence of His-AGR2 protein is observed after 14 h. The results are shown in FIG. 11A, FIG. 11B and FIG. 11C, which indicate that the purified recombinant protein of human Pregradin 2 has higher pro-migratory activity.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

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<400> 4

gtggtggtgc ataatctatg gtccttgttg gtgaagtgc 39

<210> 5

<211> 996

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatcacttgg ggaaaggaag gttcgtttct gagttagcaa caagtaaatg cagcactagt 60

gggtgggatt gaggtatgcc ctggtgcata aatagagact cagctgtgct ggcacactca 120

gaagcttgga ccgcatccta gccgccgact cacacaaggc aggtgggtga ggaaatccag 180

agttgccatg gagaaaattc cagtgtcagc attcttgctc cttgtggccc tctcctacac 240

tctggccaga gataccacag tcaaacctgg agccaaaaag gacacaaagg actctcgacc 300

caaactgccc cagaccctct ccagaggttg gggtgaccaa ctcatctgga ctcagacata 360

tgaagaagct ctatataaat ccaagacaag caacaaaccc ttgatgatta ttcatcactt 420

ggatgagtgc ccacacagtc aagctttaaa gaaagtgttt gctgaaaata aagaaatcca 480

gaaattggca gagcagtttg tcctcctcaa tctggtttat gaaacaactg acaaacacct 540

ttctcctgat ggccagtatg tccccaggat tatgtttgtt gacccatctc tgacagttag 600

agccgatatc actggaagat attcaaatcg tctctatgct tacgaacctg cagatacagc 660

tctgttgctt gacaacatga agaaagctct caagttgctg aagactgaat tgtaaagaaa 720

aaaaatctcc aagcccttct gtctgtcagg ccttgagact tgaaaccaga agaagtgtga 780

gaagactggc tagtgtggaa gcatagtgaa cacactgatt aggttatggt ttaatgttac 840

aacaactatt ttttaagaaa aacaagtttt agaaatttgg tttcaagtgt acatgtgtga 900

aaacaatatt gtatactacc atagtgagcc atgattttct aaaaaaaaaa ataaatgttt 960

tgggggtgtt ctgttttctc caaaaaaaaa aaaaaa 996

<210> 6

<211> 175

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Glu Lys Ile Pro Val Ser Ala Phe Leu Leu Leu Val Ala Leu Ser

1 5 10 15

Tyr Thr Leu Ala Arg Asp Thr Thr Val Lys Pro Gly Ala Lys Lys Asp

20 25 30

Thr Lys Asp Ser Arg Pro Lys Leu Pro Gln Thr Leu Ser Arg Gly Trp

35 40 45

Gly Asp Gln Leu Ile Trp Thr Gln Thr Tyr Glu Glu Ala Leu Tyr Lys

50 55 60

Ser Lys Thr Ser Asn Lys Pro Leu Met Ile Ile His His Leu Asp Glu

65 70 75 80

Cys Pro His Ser Gln Ala Leu Lys Lys Val Phe Ala Glu Asn Lys Glu

85 90 95

Ile Gln Lys Leu Ala Glu Gln Phe Val Leu Leu Asn Leu Val Tyr Glu

100 105 110

Thr Thr Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Val Pro Arg Ile

115 120 125

Met Phe Val Asp Pro Ser Leu Thr Val Arg Ala Asp Ile Thr Gly Arg

130 135 140

Tyr Ser Asn Arg Leu Tyr Ala Tyr Glu Pro Ala Asp Thr Ala Leu Leu

145 150 155 160

Leu Asp Asn Met Lys Lys Ala Leu Lys Leu Leu Lys Thr Glu Leu

165 170 175

Claims

1. A method for preparing active human pre-gradient protein AGR2, which is characterized by comprising the following steps:

(1) inserting a human AGR2 gene into a vector which is a prokaryotic expression vector pMAL-c2, obtaining a recombinant expression vector pMAL-AGR2-MBP after enzyme digestion and connection, deleting the MBP gene in the recombinant expression vector pMAL-AGR2-MBP through mutation PCR and inserting a His tag gene to obtain an AGR2 recombinant expression vector;

(2) transforming host cells by the AGR2 recombinant expression vector obtained in the step (1) to obtain a transformant;

(3) culturing the transformant obtained in the step (2) and processing the cultured transformant to obtain active human AGR 2;

the amino acid sequence of the human AGR2 is shown as SEQ ID NO: and 6.

2. The method of claim 1, wherein the step (3) comprises:

purifying the active human AGR2 by a nickel chromatographic column.