阅读说明：本技术 重组人结合珠蛋白β亚基蛋白的表达和纯化方法 (Expression and purification method of recombinant human haptoglobin beta subunit protein ) 是由 段瑞峰 邓炳楠 张源 蒲玲玲 刘伟丽 王新兴 王天辉 陈照立 于 2019-10-21 设计创作，主要内容包括：本发明公开了一种重组人结合珠蛋白β亚基蛋白的表达和纯化方法。本发明人工合成人结合珠蛋白全长基因,利用PCR技术扩增人结合珠蛋白β亚基基因,然后利用原核表达系统将该基因在大肠杆菌Shuffle T7-B中进行表达,随后利用His标签可溶性蛋白纯化试剂盒进行蛋白纯化,获得重组人结合珠蛋白β亚基的重组蛋白。根据本发明所述方法制备的重组人结合珠蛋白β亚基蛋白具有可溶性蛋白产率高及操作简便的特点,可用于进一步研究该蛋白体外结合血红蛋白的功能及应用于清除血浆中游离血红蛋白。(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.)

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.

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