CN110724185A - Expression and purification method of recombinant human haptoglobin beta subunit protein - Google Patents
Expression and purification method of recombinant human haptoglobin beta subunit protein Download PDFInfo
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Abstract
The invention discloses an expression and purification method of recombinant human haptoglobin beta subunit protein. The inventor synthesizes the human haptoglobin full-length gene in a synthetic way, utilizes the PCR technology to amplify the human haptoglobin beta subunit gene, then utilizes a prokaryotic expression system to express the gene in escherichia coli Shuffle T7-B, and then utilizes a His label soluble protein purification kit to carry out protein purification, so as to obtain the recombinant protein of the recombinant human haptoglobin beta subunit. The recombinant human haptoglobin beta subunit protein prepared by the method has the characteristics of high yield of soluble protein and simplicity and convenience in operation, and can be used for further researching the function of the protein for binding hemoglobin in vitro and removing free hemoglobin in blood plasma.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to an expression and purification method of recombinant human haptoglobin beta subunit protein.
Background
Haptoglobin (Hp) is widely present in serum and other body fluids of human beings and many mammals, and has the main function of transporting hemoglobin to the liver for metabolism by combining with free hemoglobin (Hb) to form a Haptoglobin-hemoglobin complex, wherein the hemoglobin contains iron ions, and the Haptoglobin-bound plasma free hemoglobin not only can enable the hemoglobin to be recycled after being degraded in vivo, but also can prevent the loss of the hemoglobin and iron from the kidney and the damage to the kidney. Haptoglobin is an important endogenous protective factor of the body against damage by free hemoglobin, and is synthesized mainly by hepatocytes and secreted into plasma. Haptoglobin can circulate in plasma for 3.5 days, but once bound to free hemoglobin can bind to the CD163 receptor of monocytes and be transported to the liver within 10-20 minutes to be degraded in lysosomes. Long-term hypoxia, burns, dialysis treatment, etc. can cause the level of free hemoglobin in blood plasma to rise, and in extreme cases, can cause the depletion of bound globin, thereby endangering the health of the body. Studies have shown that the longer the blood used for transfusion is kept in vitro, the higher its free hemoglobin concentration will be and the less the contained haptoglobin will be, thus being detrimental to the transfused patient. The binding sites for haptoglobin binding to hemoglobin are its beta subunit, so it is of great interest to conveniently obtain the beta subunit of human haptoglobin to bind and clear free hemoglobin.
Disclosure of Invention
The invention aims to provide a method for expressing and purifying a recombinant human haptoglobin beta subunit pronucleus with high yield of soluble protein and simple and convenient operation, which is favorable for further researching the function of the protein-conjugated hemoglobin and application of the protein-conjugated hemoglobin in removing free hemoglobin in blood plasma.
The invention realizes the aim through the following technical scheme: provides a recombinant human haptoglobin gene, the nucleotide sequence of which is shown in an accessory SEQ NO. 1.
The amino acid sequence of the recombinant human haptoglobin beta subunit is shown in an accessory SEQ NO. 2.
The expression method of the recombinant human haptoglobin beta subunit comprises the following steps:
constructing the synthesized human haptoglobin beta subunit gene on a prokaryotic expression vector pET32a (+), constructing a recombinant expression vector pET32a (+) -HP beta, converting the successfully constructed recombinant expression vector into a Shuffle T7-B competent cell, preparing a solid agarose gel plate, and screening to obtain a recombinant engineering strain; and selecting the recombinant engineering strain clone, culturing the recombinant engineering strain clone in an LB culture medium, and adding a protein expression inducer IPTG to perform induced expression on the human recombinant haptoglobin beta subunit protein.
The expression vector pET32a (+) contains a T7 promoter, a thioredoxin fusion expression structural domain and a protein purification His tag sequence. The fusion expression with thioredoxin can increase the solubility of the human haptoglobin beta subunit recombinant protein, and the fusion expression with His label makes the purification operation of the soluble protein simple and convenient.
The recombinant human haptoglobin beta subunit gene is obtained by PCR amplification, PCR amplification is carried out by utilizing a PCR technology and taking a synthesized human haptoglobin full-length gene as a template, and the sequence of an upstream primer is as follows: CAAGGCCATGGCTCGGATCCTGGGTGGACACC (the underlined part is the Nco I cleavage site); the downstream primer sequence is as follows: GTGCTCGAGTTAGTTCTCAGCTATGGTCTTC (underlined part is Xho I cleavage site).
The method for constructing the recombinant human haptoglobin beta subunit prokaryotic expression vector comprises the following steps: carrying out double enzyme digestion on the full-length human haptoglobin beta subunit gene obtained by PCR amplification and an expression vector pET32a (+) by using Nco I and Xho I endonucleases respectively, purifying enzyme digestion products by agarose gel electrophoresis and a DNA purification kit, then connecting the two purified products by using T4DNA ligase to construct a recombinant expression vector pET32a (+) -HP beta, and then transforming a Shuffle T7-B competent cell by using the vector to obtain an expression strain.
The gene recombination engineering strain is Shuffle T7-B-HP beta.
The conditions for inducing expression of the recombinant human haptoglobin are as follows: culturing at 37 deg.C and 150rpm for about 3h until the OD600 of the bacterial liquid reaches 0.5-0.7, and preferably the OD600 is close to 0.6, adding IPTG with final concentration of 1mM to induce expression for 4h, wherein the recombinant human haptoglobin induced to express is a mixture of soluble protein and inclusion body.
The purification method of the recombinant human haptoglobin for induced expression comprises the following steps: ultrasonically crushing thalli, diluting a supernatant containing Soluble protein by a Soluble Binding Buffer after centrifugation, loading the supernatant on a Ni agarose gel chromatographic column, washing the column by the Soluble Binding Buffer, eluting by the Soluble attachment Buffer containing 500mM imidazole concentration, and collecting an eluent; the eluate was concentrated using a 10kDa ultrafiltration tube and exchanged for PBS solution.
The purification method of the recombinant human haptoglobin for induced expression comprises the following components: 20mM Tris-HCl (pH7.9), 500mM NaCL, 10mM imidazole; the SolubleElution Buffer comprises the following components: 20mM Tris-HCl (pH7.9), 500mM NaCL, 500mM imidazole.
The recombinant human haptoglobin beta subunit protein obtained by induced expression and purification can be used for functional research of free hemoglobin bound by the recombinant human haptoglobin beta subunit protein and application of the recombinant haptoglobin beta subunit protein in binding and removing free hemoglobin in plasma.
The invention has the outstanding advantages that:
an engineering strain of Escherichia coli capable of stably inducing and expressing recombinant protein of beta subunit of human haptoglobin is constructed by using a genetic engineering method. The fusion expression of the beta subunit of human haptoglobin and thioredoxin can increase the solubility of the recombinant protein of the beta subunit of human haptoglobin. Disulfide bond isomerase DsbC expressed in a cell model of the Shuffle T7-B strain is favorable for the recombinant human haptoglobin beta subunit to form a correct disulfide bond and help the protein to be folded correctly. The expression product can be conveniently purified by a Ni agarose gel column, the operation is simple, and the cost is low. The obtained soluble recombinant human conjugated globin beta subunit can be used for researching the function of in vitro conjugated hemoglobin and removing free hemoglobin in blood plasma.
Drawings
FIG. 1 shows the identification of the synthetic human haptoglobin gene fragment inserted into the UC57 plasmid after double digestion with EcoRI and SphI endonucleases (the fragment size corresponds to the expected size);
lane M: representing a DNA Marker; lane 1: pUC57-HP after double digestion of EcoRI and SphI endonucleases; the size of pUC57 plasmid is 2710bp, and the size of synthetic gene is 1062 bp;
FIG. 2 is a (partial) sequencing of the pET32a (+) -HP β plasmid (sequencing results show that this plasmid has correctly inserted the HP β subunit gene fragment);
FIG. 3 is an electrophoresis picture of the induced expression of recombinant human haptoglobin beta subunit protein using the Shuffle T7-B-HP beta strain;
wherein lane M shows protein Marker, KDa represents protein molecular mass; lane 1: protein electrophoretograms of pET32a (+) -HP beta strain without induction; lane 2: protein electropherograms after induction of pET32a (+) -HP beta strain 1mM IPTG (protein specificity increased significantly at about 45kDa after induction);
FIG. 4 is a non-denaturing purified electrophoretogram of the beta subunit of recombinant human haptoglobin protein;
wherein lane M shows protein Marker, KDa represents protein molecular mass; lane 1: the purified recombinant human haptoglobin β subunit (molecular weight approximately 45KDa, consistent with the expected molecular weight).
Detailed Description
Example 1
Construction of recombinant expression vector pET32a (+) -HP beta
1.1 obtaining human haptoglobin beta subunit gene by using chemically synthesized DNA template and PCR method
Synthesized in general biosystems (Anhui) LtdThe human haptoglobin DNA template is inserted into a pUC57 cloning vector, the sequence length is 1062bp, and is shown in an accessory SEQ NO. 1. The full-length gene segment of the human haptoglobin beta subunit is obtained by amplification by taking the protein as a template. The sequence of the upstream primer is as follows: CAAGGCCATGGCTCGGATCCTGGGTGGACACC (the underlined part is the Nco I cleavage site); the downstream primer sequence is as follows:
GTGCTCGAGTTAGTTCTCAGCTATGGTCTTC (underlined part is Xho I cleavage site). And (3) PCR reaction system: 1. mu.L of each of the upstream and downstream primers, 0.2. mu.L of plasmid template, 12.5. mu.L of 2xPfu PCR MasterMix (Tiangen Biochemical technology Co., Ltd., KP201), ddH2O10.3 mu L; pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, 30 cycles, and extension at 72 ℃ for 5 min. 6. mu.L of the PCR product was subjected to 1.2% agarose gel electrophoresis and identified as having a clear band at about 0.7Kb, which is consistent with the expected size of the human haptoglobin β subunit gene fragment.
1.2 construction of expression vector pET32a (+) -HP β
(1) Purifying the PCR product by using a DNA purification kit, simultaneously carrying out Nco I and Xho I double enzyme digestion on pET32a (+) plasmid and the purified PCR fragment of the human haptoglobin beta subunit gene, carrying out enzyme digestion at 37 ℃ for 2h, then purifying by using the DNA purification kit of Tiangen Biochemical technology Co., Ltd and recovering the DNA fragment subjected to enzyme digestion; the plasmid fragment after enzyme digestion and purification was ligated to the human haptoglobin gene fragment using the rapid enzyme ligation reagent from Byuntian Biotechnology Inc. for 40 min at room temperature. The connector system is 1.5 mu L of pET32a (+) plasmid solution, 3 mu L of human haptoglobin gene beta subunit gene fragment solution, 5 mu L of quick connection buffer solution and 0.5 mu L of quick T4DNA ligase.
(2) The ligation product was transformed into Shuffle T7-B competent cells to obtain transformed colonies. The transformation conditions were: mu.L of the ligation product was added to 100. mu.L of Shuffle T7-B competent cell solution, gently mixed, allowed to stand in an ice bath for 30 minutes, followed by heat shock at 42 ℃ for 90s, and then quickly transferred to an ice bath to allow the cells to cool for 3min without shaking the centrifuge tube. 900. mu.L of sterile LB medium (without antibiotics) was added, mixed well and placed on a shaker at 37 ℃ for 45min (150rpm), then 100. mu.L of transformed competent cells were aspirated and plated on LB plates containing ampicillin resistance, and cultured overnight in an inverted state at 37 ℃.
(3) And selecting a single colony for culture, performing PCR verification, screening a positive clone colony for sequencing, and obtaining a genetic engineering bacterium for expressing pET32a (+) -HP beta, namely, Shuffle T7-B-pET32a (+) -HP beta, wherein the sequencing result shows that the nucleotide sequence of the inserted human haptoglobin gene is correct.
Example 2
Expression and identification of recombinant human haptoglobin
2.1 Single colony of recombinant genetically engineered bacterium Shuffle T7-B-pET32a (+) -HP beta was inoculated into 10mL LB medium (containing 100. mu.g/mL ampicillin), and cultured overnight at 37 ℃ with shaking at 150 rpm.
2.2 inoculating the overnight cultured broth into 10mL of fresh LB medium (containing 100. mu.g/mL of ampicillin) at a ratio of 1:9,
culturing at 37 deg.C and 150rpm for about 3h until the OD600 of the bacterial liquid reaches 0.5-0.7, and preferably the OD600 is close to 0.6, adding IPTG with final concentration of 1mM to induce expression for 4h, wherein the recombinant human haptoglobin induced to express is a mixture of soluble protein and inclusion body. Meanwhile, an engineering bacterium ShuffleT7-B-pET32a (+) -HP beta is set to carry out no IPTG induction group for comparison.
2.3 after the induction culture, the culture broth was centrifuged at 12,000g for 1min at 4 ℃ to discard the supernatant and collect the lower layer of the bacterial pellet. Resuspend the cells with 1mL PBS buffer, and crush them with ultrasound until the cells become clear from turbidity. Centrifuging at 4 deg.C for 5min at 12,000g, and sucking the supernatant into a new centrifuge tube to obtain supernatant; the pellet was resuspended in 100. mu.L PBS to obtain a pellet fraction. The result of 12% SDS-PAGE electrophoresis and Coomassie blue staining of the resuspended cells with PBS is shown in FIG. 2, where there is a protein band with increased specific expression at about 45kDa, which is consistent with the expected molecular weight of the beta subunit of recombinant human haptoglobin, and the beta subunit of recombinant human haptoglobin induced to express is a mixture of soluble protein and inclusion body protein.
Example 3
Purification of recombinant human haptoglobin beta subunit protein
3.1 according to the method of example 2, 200mL of recombinant gene engineering strain Shufflet7-B-pET32a (+) -HP beta is induced and expressed, a proper amount of precooled PBS buffer solution is used for resuspending the thalli, ice bath ultrasonic crushing is carried out until the bacterial liquid is clear from turbidity, then centrifugation is carried out for 5min at 12,000g, and the supernatant is collected. The supernatant solution was added to an equal volume of a precooled solution Binding Buffer (20mM Tris-HCl (pH7.9), 500mM NaCl, 10mM imidazole) and purified using the Kangji's His-tagged protein purification kit (Soluble protein).
3.2 loading the supernatant containing Soluble protein and diluted by equal times with the Soluble Binding Buffer on a Ni agarose gel chromatographic column, firstly washing the column with 15 times of column volume of the Soluble Binding Buffer, then eluting with a Soluble interaction Buffer (20mM Tris-HCL (pH7.9), 500mM NaCl, 500mM imidazole.) containing 500mM imidazole concentration, and collecting the eluent; and (3) concentrating the eluent by 10x each time by using a PBS (phosphate buffer solution) ultrafiltration tube, and replacing the eluent by the PBS solution for 3 times, namely the purified protein solution. The results of 12% SDS-PAGE and Coomassie blue gel staining are shown in FIG. 3, and a specific protein band at about 45kDa corresponds to the expected size of the recombinant human haptoglobin β subunit protein. Through further mass spectrometric identification, the recombinant protein is verified to be a human haptoglobin beta subunit protein. Finally, the purified protein is stored in a refrigerator at-70 ℃.
The recombinant human haptoglobin beta subunit protein which is induced to express and purified is applied to researching the hemoglobin binding function of the recombinant haptoglobin beta subunit protein and clearing free hemoglobin in blood plasma.
Appendix SEQ NO.1
Synthetic recombinant human haptoglobin gene sequences
CTAGCTAGCATGAGTGCCCTGGGAGCTGTCATTGCCCTCCTGCTCTGGGGACAGCTTTTTGCAGTGGACTCAGGCAATGATGTCACGGATATCGCAGATGACGGCTGCCCGAAGCCCCCCGAGATTGCACATGGCTATGTGGAGCACTCGGTTCGCTACCAGTGTAAGAACTACTACAAACTGCGCACAGAAGGAGATGGAGTGTACACCTTAAACAATGAGAAGCAGTGGATAAATAAGGCTGTTGGAGATAAACTTCCTGAATGTGAAGCAGTATGTGGGAAGCCCAAGAATCCGGCAAACCCAGTGCAGCGGATCCTGGGTGGACACCTGGATGCCAAAGGCAGCTTTCCCTGGCAGGCTAAGATGGTTTCCCACCATAATCTCACCACAGGTGCCACGCTGATCAATGAACAATGGCTGCTGACCACGGCTAAAAATCTCTTCCTGAACCATTCAGAAAATGCAACAGCGAAAGACATTGCCCCTACTTTAACACTCTATGTGGGGAAAAAGCAGCTTGTAGAGATTGAGAAGGTTGTTCTACACCCTAACTACTCCCAGGTAGATATTGGGCTCATCAAACTCAAACAGAAGGTGTCTGTTAATGAGAGAGTGATGCCCATCTGCCTACCTTCAAAGGATTATGCAGAAGTAGGGCGTGTGGGTTATGTTTCTGGCTGGGGGCGAAATGCCAATTTTAAATTTACTGACCATCTGAAGTATGTCATGCTGCCTGTGGCTGACCAAGACCAATGCATAAGGCATTATGAAGGCAGCACAGTCCCCGAAAAGAAGACACCGAAGAGCCCTGTAGGGGTGCAGCCCATACTGAATGAACACACCTTCTGTGCTGGCATGTCTAAGTACCAAGAAGACACCTGCTATGGCGATGCGGGCAGTGCCTTTGCCGTTCACGACCTGGAGGAGGACACCTGGTATGCGACTGGGATCTTAAGCTTTGATAAGAGCTGTGCTGTGGCTGAGTATGGTGTGTATGTGAAGGTGACTTCCATCCAGGACTGGGTTCAGAAGACCATAGCTGAGAACTAACTCGAGCGG
Appendix SEQ NO.2
Recombinant human haptoglobin beta subunit amino acid sequence
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMARILGGHLDAKGSFPWQAKMVSHHNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPNYSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYVMLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGSAFAVHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN
The 162-407 portion of the sequence is the beta subunit amino acid sequence of human haptoglobin, the 1-109 portion of the sequence is the thioredoxin portion, and the 117-122 portion of the sequence is the His protein tag portion. The molecular weight of the recombinant protein calculated by the DS Gene 1.1 software is 44.75 KDa.
Sequence listing
<110> institute of environmental and operational medicine of military medical research institute of military science institute
<120> expression and purification method of recombinant human haptoglobin beta subunit protein
<130>2019.10.18
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Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
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Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
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Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
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Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
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Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
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Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
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Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
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Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
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Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
130 135 140
His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
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Ala Arg Ile Leu Gly Gly His Leu Asp Ala Lys Gly Ser Phe Pro Trp
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Gln Ala Lys Met Val Ser His His Asn Leu Thr Thr Gly Ala Thr Leu
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Ile Asn Glu Gln Trp Leu Leu Thr Thr Ala Lys Asn Leu Phe Leu Asn
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His Ser Glu Asn Ala Thr Ala Lys Asp Ile Ala Pro Thr Leu Thr Leu
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Tyr Val Gly Lys Lys Gln Leu Val Glu Ile Glu Lys Val Val Leu His
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Pro Asn Tyr Ser Gln Val Asp Ile Gly Leu Ile Lys Leu Lys Gln Lys
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Val Ser Val Asn Glu Arg Val Met Pro Ile Cys Leu Pro Ser Lys Asp
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Tyr Ala Glu Val Gly Arg Val Gly Tyr Val Ser Gly Trp Gly Arg Asn
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Ala Asn Phe Lys Phe Thr Asp His Leu Lys Tyr Val Met Leu Pro Val
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Ala Asp Gln Asp Gln Cys Ile Arg His Tyr Glu Gly Ser Thr Val Pro
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Glu Lys Lys Thr Pro Lys Ser Pro Val Gly Val Gln Pro Ile Leu Asn
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Glu His Thr Phe Cys Ala Gly Met Ser Lys Tyr Gln Glu Asp Thr Cys
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Tyr Gly Asp Ala Gly Ser Ala Phe Ala Val His Asp Leu Glu Glu Asp
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Thr Trp Tyr Ala Thr Gly Ile Leu Ser Phe Asp Lys Ser Cys Ala Val
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ctagctagca tgagtgccct gggagctgtc attgccctcc tgctctgggg acagcttttt 60
gcagtggact caggcaatga tgtcacggat atcgcagatg acggctgccc gaagcccccc 120
gagattgcac atggctatgt ggagcactcg gttcgctacc agtgtaagaa ctactacaaa 180
ctgcgcacag aaggagatgg agtgtacacc ttaaacaatg agaagcagtg gataaataag 240
gctgttggag ataaacttcc tgaatgtgaa gcagtatgtg ggaagcccaa gaatccggca 300
aacccagtgc agcggatcct gggtggacac ctggatgcca aaggcagctt tccctggcag 360
gctaagatgg tttcccacca taatctcacc acaggtgcca cgctgatcaa tgaacaatgg 420
ctgctgacca cggctaaaaa tctcttcctg aaccattcag aaaatgcaac agcgaaagac 480
attgccccta ctttaacact ctatgtgggg aaaaagcagc ttgtagagat tgagaaggtt 540
gttctacacc ctaactactc ccaggtagat attgggctca tcaaactcaa acagaaggtg 600
tctgttaatg agagagtgat gcccatctgc ctaccttcaa aggattatgc agaagtaggg 660
cgtgtgggtt atgtttctgg ctgggggcga aatgccaatt ttaaatttac tgaccatctg 720
aagtatgtca tgctgcctgt ggctgaccaa gaccaatgca taaggcatta tgaaggcagc 780
acagtccccg aaaagaagac accgaagagc cctgtagggg tgcagcccat actgaatgaa 840
cacaccttct gtgctggcat gtctaagtac caagaagaca cctgctatgg cgatgcgggc 900
agtgcctttg ccgttcacga cctggaggag gacacctggt atgcgactgg gatcttaagc 960
tttgataaga gctgtgctgt ggctgagtat ggtgtgtatg tgaaggtgac ttccatccag 1020
gactgggttc agaagaccat agctgagaac taactcgagc gg 1062
Claims (9)
1. A gene of recombinant human haptoglobin beta subunit is characterized in that the nucleotide sequence of the gene is shown as accessory SEQ NO. 1.
2. A recombinant human haptoglobin beta subunit protein is characterized in that the amino acid sequence of the protein is shown as an accessory SEQ NO. 2.
3. A method of expressing recombinant human haptoglobin β subunit protein of claim 2 in a manner which comprises the steps of: firstly, inserting a human haptoglobin beta subunit gene into a prokaryotic expression vector pET32a (+), constructing a recombinant expression plasmid pET32a (+) -HP beta, converting the recombinant expression plasmid into escherichia coli Shuffle T7-B to obtain a recombinant human haptoglobin beta subunit engineering strain, culturing the engineering strain, and then adding IPTG (isopropyl thiogalactoside) to perform inducible expression on the recombinant human haptoglobin beta subunit; expression vector pET32a (+) contains the T7 promoter, a thioredoxin fusion expression domain and a protein purification His tag sequence.
4. The method of claim 3, wherein the full-length sequence of the human haptoglobin gene is chemically synthesized and inserted into the human haptoglobin subunitIn the cloned vector pUC 57; the gene sequence of the recombinant human haptoglobin beta subunit is a product obtained by performing PCR amplification by using a PCR technology and a chemically synthesized human haptoglobin full-length gene as a template, and the upstream primer sequence of the recombinant human haptoglobin beta subunit is as follows: CAAGGCCATGGCTCGGATCCTGGGTGGACACC, the underlined part is the Nco I cleavage site; the downstream primer sequence is as follows: GTGCTCGAGTTAGTTCTCAGCTATGGTCTTC the crosshatch is an Xho I cleavage site.
5. The method of claim 3, wherein the prokaryotic expression vector is constructed by the method comprising: the method comprises the following steps of carrying out double digestion on a chemically synthesized full-length human haptoglobin beta subunit gene and an expression vector pET32a (+) by using Nco I endonuclease and Xho I endonuclease respectively, purifying digestion products by agarose gel electrophoresis and a DNA purification kit, connecting the two purification products by using T4DNA ligase to construct a recombinant expression vector pET32a (+) -HP beta, and transforming Shuffle T7-B competent cells by using the vector to obtain an expression strain.
6. The method of claim 3, wherein the recombinant genetically engineered strain is Shuffle T7-B-pET32a (+) -HP β.
7. The method of claim 3, wherein the conditions for inducing the recombinant human haptoglobin β subunit are: culturing at 37 deg.C and 150rpm for about 3h until OD600 of bacterial liquid reaches 0.5-0.7, adding IPTG with final concentration of 1mM to induce expression for 4h, wherein the recombinant human haptoglobin beta subunit induced expression is mixture of soluble protein and inclusion body.
8. The method of claim 3, wherein the recombinant human haptoglobin β subunit is purified by: ultrasonically crushing thalli, diluting a supernatant containing Soluble protein by using a solvent Binding Buffer after centrifugation, loading the supernatant onto a Ni agarose gel chromatographic column, washing the column by using the solvent Binding Buffer, eluting by using the solvent Elution Buffer containing 500mM imidazole concentration, and collecting an eluent; the eluate was concentrated using a 10kDa ultrafiltration tube and exchanged for PBS solution.
9. The method of claim 8, wherein the Binding Buffer comprises: 20mM Tris-HCl: pH7.9, 500mM NaCL, 10mM imidazole; the solvent Elution Buffer comprises the following components: 20mM Tris-HCl pH7.9, 500mM NaCL, 500mM imidazole.
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