CN101967194B - Recombinant human cell restin MHD fusion protein and preparation method thereof - Google Patents

Abstract

The invention discloses a recombinant human cell restin MHD fusion protein and a preparation method thereof. The fusion protein comprises a human cell restin MHD structure domain, and an N end of the human cell restin MHD structure domain is connected with a purification label sequence, the enzyme digesting sites of thrombase and enterokinase and a Nus sequence. In the invention, the transformant of the restin-MHD fusion protein is obtained by constructing a pronucleus expression vector pET44a(+)-Restin-MHD-MHD and transferring the pronucleus expression vector Escherichia coli, the expression of the transformant is induced in the Escherichia coli, and the restin-MHD fusion protein is obtained by separating and purifying. Bioactivity verification proves that the restin fusion protein has a cell cycle arrest effect on tumuor cells and can be used for preparing antitumor medicaments.

Description

A kind of recombinant human cell resting factor MHD fusion protein and its preparation method

technical field

The invention belongs to the field of biotechnology and relates to the expression of a recombinant fusion protein, in particular to a recombinant human cell resting factor MHD fusion protein and a preparation method thereof.

Background technique

Restin is a new member of the melanoma associated antigen (MAGE) family cloned by Zhu et al. in 1999. The full length of the Restin gene is 1475bp, encoding 219 amino acids. The gene product has different localizations in different cell lines and is expressed in both the cytoplasm and the nucleus.

The 8-25 amino acids at the N-terminal of the Restin protein is a typical binary nuclear localization signal sequence, and the 42-52 amino acids are the casein kinase II phosphorylation site, and the phosphorylation of this site also affects the nuclear localization of the Restin protein;88 The -176th amino acid is the homology domain MHD of the MAGE family, and the theoretical pI value of this domain is 9.99, so the Restin protein is positioned as a basic MAGE protein.

Studies have shown that Restin is related to cell cycle differentiation, overexpressing Restin in cells to observe the cell growth cycle, and found that G ₁ phase arrest, suggesting that it may be involved in the process of cell differentiation. The literature shows that Necdin, a highly homologous protein of Restin, also has the biological function of cell growth cycle arrest, and the 191-222 amino acids in its MHD play a crucial role in cell growth inhibition. Protein sequence comparison found that the MHD of another homologous protein of Restin, MAGE-D1, also contains this sequence, indicating that this sequence is highly conserved in evolution; and the study of MAGE-D1 found that it is also involved in the process of cell differentiation. The 92-122 amino acids in Restin MHD have a homology of 48% with this conserved sequence, suggesting that it plays an important role in the process of Restin producing cell cycle arrest.

In view of the important biological functions of Restin, obtaining high-purity and active Restin protein can be used as a gene drug to inhibit the tumor cell cycle, and has the prospect of being applied to tumor therapy. However, due to the complex secondary structure of the mRNA of the restin gene, it is not conducive to the exogenous expression of the prokaryotic system; and the molecular weight of the full-length Restin protein is relatively large, which is 24.4kD, which will also affect the efficiency of the gene drug in the later stage; in addition, the Restin-MHD polypeptide can For Π-like MAGE molecules, it has a wider target effect than Restin.

Contents of the invention

The problem to be solved by the present invention is to provide a recombinant human cell resting factor MHD fusion protein and its preparation method, clone and express the melanoma-associated antigen homology domain of Restin, and verify the effect of the fusion protein on the tumor cell cycle blocking effect.

The present invention is achieved through the following technical solutions:

A recombinant human cell quiescence factor MHD fusion protein, including the human cell quiescence factor MHD domain, the N-terminus of the human cell quiescence factor MHD domain is also sequentially connected with a purification marker sequence, thrombin and enterokinase cleavage site point, and the Nus sequence.

The amino acid sequence of the MHD structural domain of human cell resting factor is shown in SEQ.ID.NO.1.

The purification tag sequence is a 6×His tag sequence.

The amino acid sequence of the recombinant human resting factor MHD fusion protein is shown in SEQ.ID.NO.2.

A prokaryotic expression vector expressing recombinant human cell resting factor MHD fusion protein, the prokaryotic expression vector is pET44a ₍₊₎ -Restin- _MHD , which is expressed as SEQ.ID.NO. The human cell quiescence factor MHD gene fragment shown in 3 was cloned into pET44a ₍₊₎ expression vector.

A preparation method of recombinant human cell resting factor MHD fusion protein, comprising the following steps:

1) Using the cDNA comprising the complete coding frame of the human Restin gene as a template, using the primer pair P as primers, PCR amplifies the human Restin- _MHD gene fragment, and the primer pair P is:

Upstream primer P1: cg ggatcc aa gaaaagtctc ct;

Downstream primer P2: ccc aagctt g actttcagtt tgc;

2) The human Restin- _MHD gene fragment amplified by PCR was digested with Bam HI/Hind III to obtain a fragment with sticky ends; and the fragment was ligated with the pET44a ₍₊₎ vector digested with Bam HI/Hind III to obtain Expression vector pET44a ₍₊₎ -Restin- _MHD ;

3) Transfect the expression vector pET44a ₍₊₎ -Restin- _MHD into competent Escherichia coli, and screen for positive transformants expressed by the target gene;

4) After culturing the positive transformants in a 37°C incubator for 12 hours, pick colonies with neat edges and good growth status, inoculate them in LB culture medium containing 100 mg/L ampicillin, and culture them overnight at 37°C on a shaking table; Inoculate in fresh LB culture solution containing 100 mg/L ampicillin at a volume ratio of :100, continue to culture at 37°C until the bacterial density reaches OD ₆₀₀ =0.4-0.6, add 0.1-2.5 mM IPTG to induce culture for 4 hours, so that the expression The vector pET44a ₍₊₎ -Restin- _MHD is expressed, and then the bacteria are collected by centrifugation;

5) Add the lysate at a ratio of 10mL/g of bacterial precipitate, resuspend the bacteria, lyse the bacteria in an ice bath, then centrifuge, and collect the supernatant;

Described lysate is the Tris·Cl of pH 8.5, concentration 50mmol/L, the mixed solution of the β-mercaptoethanol of 5mmol/L and the PMSF of 1mmol/L;

6) Load the collected supernatant on the metal chelate affinity chromatography column, wash the column with 10 times column volume of buffer A and elute at a flow rate of 0.5mL/min, then use 2 times column volume of buffer B Wash the column, and finally elute the bound protein with buffer C, and collect the elution peak;

Described buffer solution A is the Tris·Cl of pH 8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixed solution of the β-mercaptoethanol of 5mmol/L and the imidazole of 20mmol/L; Described buffer solution B is pH 8.5, a mixed solution of Tris Cl with a concentration of 20mmol/L, KCl of 1mol/L and β-mercaptoethanol of 5mmol/L; the buffer C is Tris Cl with a pH of 8.5 and a concentration of 20mmol/L, 100mmol The KCl of /L, the mixed solution of the β-mercaptoethanol of 5mmol/L and the imidazole of 100mmol/L;

Applying the collected elution peaks to ion-exchange chromatography, eluting the bound protein with eluent D, collecting the elution peaks to obtain purified recombinant human resting factor MHD fusion protein;

The eluent D is a mixed solution of pH 7.4, Tris Cl and 1 mmol/L NaCl at a concentration of 50 mmol/L.

Compared with the prior art, the present invention has the following beneficial technical effects:

1. Since Restin is a basic protein, its theoretical pI value is 9.05, and the theoretical PI value of the Restin domain is 9.99, which determines that the protein is difficult to express. And the present invention obtains the fusion protein of the recombinant human cell resting factor MHD domain with Restin fusion protein by constructing the prokaryotic expression vector pET44a ₍₊₎ -Restin- _MHD , and transforming Escherichia coli, carrying out induction and expression It has the biological activity of inhibiting the tumor cell cycle, has the arresting effect on the cell cycle of tumor cells, and can be used to prepare antitumor drugs.

The prokaryotic expression vector pET44a ₍₊₎ -Restin- _MHD constructed in the present invention is based on the cloning of the human Restin-MHD gene domain, cloned into the expression vector pET44a ₍₊₎ through the Bam HI/Hind III double restriction site, and ligated A sequence that can be recognized by thrombin and enterokinase, and the constructed Restin-MHD fusion protein also contains a 6×His purification tag sequence, which facilitates the purification of the fusion protein; and when necessary, passes the restriction of thrombin and enterokinase The enzyme digestion reaction can remove the marker sequence, so that the Restin- _MHD polypeptide can be obtained. Restin- _MHD polypeptide can target Π-type MAGE molecules, and has a wider range of target effects than Restin.

The fusion protein is expressed by the prokaryotic Escherichia coli system, which has the advantages of being economical and easy to expand and cultivate. The fusion protein can greatly increase the expression amount, the purification tag is easy to purify, high yield, and high purity are the advantages of the fusion protein.

Description of drawings

Fig. 1 is the electrophoresis detection figure of the Restin- _MHD gene fragment obtained by PCR amplification;

Fig. 2 is the plasmid map of pET44a ₍₊₎ carrier;

Fig. 3 is the BamH I and Hind III double-digestion identification electrophoresis figure of pET44a ₍₊₎ -Restin- _MHD expression vector;

Fig. 4 is the result figure of SDS-PAGE detection Restin- _MHD fusion protein expression;

Fig. 5 is the result graph of the soluble expression of Restin- _MHD fusion protein detected by Western Blot;

Fig. 6 is the result figure after SDS-PAGE detection Restin _-MHD fusion protein is purified;

Fig. 7 is the result figure that flow cytometry detects Restin- _MHD fusion protein to Hela tumor cell cycle inhibition;

Figure 8 is a graph showing the growth curves of Hela tumor cells stimulated by Restin- _MHD fusion protein and normal cultured tumor cells.

Detailed ways

The present invention realizes the preparation of recombinant human Restin melanoma-associated antigen homology domain through biotechnology, constructs the prokaryotic expression vector pET44a(+)-Restin- _MHD , and transforms it into E. Induced expression, separation and purification to obtain the Restin melanoma-associated antigen homology domain fusion protein. SDS-PAGE and Western Blot analysis of the Restin domain fusion protein showed the same molecular weight results; and biological activity verification showed that the Restin domain fusion protein had a cell cycle arrest effect on tumor cells. The present invention will be described in detail below, which is an explanation of the present invention rather than a limitation.

1. Cloning of human Restin- _MHD gene

The human Restin gene (NM 014061) was searched in GenBank, and its melanoma-associated antigen domain was found to be 88-176 aa by using bioinformatics homology comparison. According to its mRNA nucleotide sequence, design and artificially synthesize specific primer P, and introduce BamHI and Hind III into both ends of the Restin- _MHD coding sequence; primer P is specifically:

Upstream primer P1: cg ggatcc aa gaaaagtctc ct 22;

Downstream primer P2: ccc aagctt g actttcagtt tgc 23;

1 μM ATRA (all-trans retinoic acid) was used to induce the differentiation of HL-60 cells, and the cells were collected after 4 days, and the total RNA was extracted by RT-PCR technology and then reverse-transcribed to obtain cDNA;

Using the cDNA obtained by reverse transcription as a template and primer P as a primer, the Restin- _MHD cDNA sequence containing the complete ORF (coding frame) was amplified according to the following procedure: 95°C for 30s, 56°C for 30s, 72°C for 60s, 30s cycle, 10min at 72°C. The amplified PCR product was recovered and amplified by gel, and identified by agarose gel electrophoresis. The results are shown in Figure 1. Lane M is DNA Marker, and lane 1 is the Restin- _MHD gene with cloning sites at both ends of 280 bp. The PCR product was subjected to agarose gel electrophoresis, and after gel cutting and recovery, it was connected to the pMD 18T vector to construct the pMD 18T-R _MHD recombinant plasmid, transformed into E.coli DH5α competent cells, and transferred to a plate containing Amp (100 μg/mL). Incubate overnight at 37°C. The next day, the single clone was picked for expansion and culture, and the plasmid was extracted for enzyme digestion identification. After the identification was correct, it was sent to Beijing Aoke Biological Company for sequencing. The sequencing result is shown in SEQ.ID.NO.3, and the comparison with GenBank showed that human Restin- The _MHD gene sequence was cloned correctly.

2. Design and construction of the expression vector pET44a(+)-Restin- _MHD :

In order to facilitate the smooth expression of the human Restin- _MHD gene and the purification and separation of the Restin- _MHD fusion protein, the present invention selects the pET44a ₍₊₎ prokaryotic expression vector, and clones the human Restin- _MHD gene into the downstream of the T7 promoter. An E. coli protein Nus.Tag fusion partner and tag with extremely high solubility when overexpressed and obtained through a large number of systematic screening can further improve the effective expression of the recombinant fusion protein; the downstream of Nus.Tag has the encoding thrombin and Enterokinase recognizes the sequence of the amino acid site, so that the fusion protein Restin-MHD can be cleaved with a later tag; and the purified His tag contained allows the fusion protein to be purified by chromatographic separation after being expressed.

Specifically, it is constructed through the following steps:

a. Enzyme digestion of vector plasmid and human Restin gene

The correctly sequenced pMD 18T-R _MHD plasmid was digested with BamH I/Hind III, and the human Restin- _MHD gene fragment with cohesive ends was recovered with a kit; the prokaryotic cell expression plasmid pET44a ₍₊₎ was purchased from Novagen, The plasmid map is shown in Figure 2, where T7 is the T7 promoter/primer site, 2426-2442bp; Nus.Tag is: Nus tagged protein, 834-2318bp; His.Tag is: Histidine tagged protein, 2325 -2342bp, 801-818bp and 512-529bp; polyclonal restriction site: 530-724bp; lacI: lac Z allele, 2833-3915bp; Ap: ampicillin resistance gene, 5870-6730bp; f1 ori is: f1 start site, 6852-7299bp. The prokaryotic expression vector pET44a ₍₊₎ was double digested with BamH I/Hind III, and the large fragment of the linearized plasmid was recovered by the kit.

b. Ligate the digested human Restin- _MHD sequence and the recovered pET44 a ₍₊₎ linear fragment with T4 DNA ligase, transform the ligated product into competent DH5α cells, culture in a 37°C incubator for 12 hours, and pick a single clone colony to inoculate In the LB culture solution containing ampicillin (Amp) 100mg/L, cultured on a shaker at 37°C for 12 hours, the bacteria were collected and the plasmid was extracted for enzyme digestion identification. The electrophoresis results of BamH I/Hind III double enzyme digestion identification are shown in Figure 3. The recombinant plasmids shown in lanes 1 and 2 were digested to obtain the expected insertion fragment of about 270bp in size, indicating that the human Restin- _MHD sequence was correctly inserted into the pET44a(+) vector, and the expression vector pET44a(+)-Restin- _MHD was successfully constructed .

3. Transformation and induced expression of expression vector pET44a(+)-Restin- _MHD

The recombinant expression vector pET44a(+)-Restin- _MHD was transformed into the competent expression host Escherichia coli BL21(DE3), cultured in a 37°C incubator for 12 hours, and the colonies with neat edges and good growth were picked and inoculated in the cells containing Ampicillin 100mg/L LB culture medium, shaking culture overnight at 37°C on a shaker. The next day, inoculate in fresh LB culture solution containing 100 mg/L ampicillin at a volume ratio of 1:100, continue shaking culture at 37°C until the bacterial density reaches OD ₆₀₀ =0.4-0.6, and add IPTG to a final concentration of 0.1 , 0.5, 1.0, 1.5, 2.0mmol/L, induced culture for 4h, so that the expression vector pET44a(+)-Restin- _MHD was expressed, and then the bacteria were collected by centrifugation.

4. Identification of Restin- _MHD fusion protein

a.10% SDS-PAGE (polyacrylamide gel electrophoresis) detection of the expression of Restin _-MHD fusion protein

The uninduced pET44a(+)-Restin- _MHD /BL21 recombinant cells were used as a control, and collected by centrifugation after 4 hours of IPTG induction, the bacteria were dissolved with lysate, and 50 μl of 5× loading buffer was added and boiled for 10 minutes. The total protein was subjected to 10% SDS-PAGE electrophoresis. After Coomassie Brilliant Blue staining, obviously induced newborn bands can be seen, with a molecular weight of about 76kDa; the specific results are shown in Figure 4: in the figure, lane M is the protein marker, which are 97.2, 66.4, 44.3, 29.0, 20.1, 14.3kDa respectively; Lane 1 is uninduced pET44a(+)-Restin- _MHD /BL21 recombinant cells, and lanes 2 to 6 are 0.1, 0.5, 1.0, 1.5, 2.0 mM IPTG induced expression for 4 hours; compared with other lanes, lanes 2 to 6 After IPTG induction, a Restin- _MHD fusion protein with a molecular weight of approximately 76 kDa was produced.

b. Western detection of Restin- _MHD fusion protein

Collect the pET44a(+)-Restin- _MHD /BL21(DE3) recombinant bacteria induced by IPTG, extract the bacterial cytoplasmic supernatant and inclusion body (collect the supernatant and precipitate after bacterial lysis), and analyze the fusion Restin- _MHD protein by Western Blot expression. After the SDS-PAGE electrophoresis for fusion protein expression detection is over, remove the gel, follow the Bio-Rad product instructions, put the gel near the cathode side, and the nitrocellulose (NC) membrane near the anode side, in the pre-cooled transfer buffer 100V constant voltage electrophoresis in solution (25mM Tris, 192mMGlycine, 20% methanol) for 60 minutes, and the protein was transferred to the NC membrane; , 0.05% Tween-20) after washing, immersed in blocking solution (TBST containing 2% BSA) at room temperature to block for 1 h, washed 3 times in TBST at room temperature, 5 min each time; then added mouse His tag antibody, incubated at room temperature for 1 h, washed in TBST at room temperature 3 times, 5 min each time; then add luciferase-labeled secondary antibody, incubate at room temperature for 1 h, wash 3 times with TBST at room temperature, 5 min each time, scan the membrane with an infrared protein scanner, and it can be seen in the cytoplasmic supernatant and inclusion bodies. The molecular weight of the Restin-MHD fusion protein band after labeled induction was consistent with that detected by SDS-PAGE electrophoresis, indicating that the Restin-MHD fusion protein was soluble. The specific results are shown in Figure 5, wherein, lane M is the protein Marker, respectively 97.2, 66.4, 44.3, 29.0, 20.1, 14.3kDa; lane 1 is the uninduced pET44a(+)-Restin- _MHD /BL21 recombinant cells; Lane 2 is the inclusion body of pET44a(+)-Restin-MHD/BL21 induced with 1.0mM IPTG; Lane 3 is the cytoplasmic supernatant of pET44a(+)-Restin-MHD/BL21 induced with 1.0mM IPTG; comparison Swimming lanes 1, 2, and 3 can be seen: the Restin-MHD fusion protein is expressed in both the cytoplasmic supernatant and the inclusion body precipitate.

According to the expression of the pET44a(+) vector, the Restin- _MHD fusion protein starts from the amino terminal and contains Nus sequence, thrombin and enterokinase cleavage sites, His tag, and MHD domain in sequence, containing 685 amino acids. The sequence is shown in SEQ.ID.NO.2, wherein the amino acid sequence of the MHD domain is shown in SEQ.ID.NO.1; the theoretical molecular weight of the Restin- _MHD fusion protein is 75.78kDa, which is detected by SDS-PAGE and Western Blot unanimous.

c. Purification of Restin- _MHD fusion protein

Expand the pET44a(+)-Restin-MHD/BL21 recombinant bacteria, collect the bacteria by centrifugation after IPTG induction, and add the lysate (pH 8.5, 50mmol/L Tris Cl, 5mmol/L β-mercaptoethanol, 1mmol/L PMSF), resuspend the bacteria, lyse the bacteria in an ice bath (1min each time, 5-10 times), centrifuge at 12,000r/min for 10min, separate the supernatant and precipitate, transfer the supernatant to a new bottle, -20 ℃ frozen.

Metal chelate affinity chromatography: regenerated NI-NTA column, buffer A (pH 8.5, 20mmol/LTris Cl, 100mmol/L KCl, 5mmol/L β-mercaptoethanol, 20mmol/L imidazole) to equilibrate the NI-NTA layer Analyze the column, put the supernatant on the column, keep the flow rate of 0.5mL/min, wash the column with 10 times column volume of buffer A to wash away impurity proteins; 2 times column volume of buffer B (pH 8.5, 20mmol/LTris. Cl, 1mol/L KCl, 5mmol/L β-mercaptoethanol) to wash the column to wash away the nucleic acid; L imidazole) to elute the binding protein, collect the elution peak, and polyacrylamide gel electrophoresis shows that the eluted protein contains the target protein.

Further purification by ion-exchange chromatography: Ion-exchange chromatography buffer (pH 7.4, 50mmol/LTris ci) equilibrated the Q Sapharose HP chromatography column, and added the Restin- _MHD fusion protein initially purified by the Ni-NTA chromatography column to the Q On the Sapharose HP gel medium, wash the chromatography column with buffer, elute the bound protein with elution buffer (pH 7.4, 50mmol/L Tris Cl, 1mol/L NaCl), collect the elution peak, polyacrylamide The eluted proteins were identified by gel electrophoresis, and the results are shown in Figure 6: Among them, lane M is the protein marker, which is 97.2, 66.4, 44.3, 29.0, 20.1, 14.3kDa; lane 1 is the uninduced pET44a(+)- Restin- _MHD /BL21 recombinant cells; Lane 2 is 1.0mMIPTG induced expression for 4h; Lane 3 is the purified Restin-MHD fusion protein. The purified recombinant human cell quiescent factor domain was subpackaged and freeze-dried for use.

d. The cycle arrest of tumor cells by Restin _-MHD fusion protein

Hela cells cultured to the logarithmic growth phase by conventional methods were made into cell suspension, the cell concentration was 1×10 ⁵ /ml, divided into 3 groups, 3 bottles in each group, the first group was blank control, and the second group was added buffer PBS control, the third group was added to the Restin- _MHD fusion protein group (PBS diluted Restin- _MHD fusion protein to a final concentration of 1 μg/ml), and the cells were collected after 48 hours of action, fixed with 70% ethanol, stained with propidium iodide (PI), Then the cell cycle of Hela cells was detected by flow cytometry. The detection results of the three groups of samples are shown in Figure 7, in which the cell cycle ratios of the first and second groups were normal, and the G1 phase of the Restin- _MHD fusion protein stimulation group in the third group was significantly increased, indicating that Hela cells were stimulated by Restin- Cell cycle arrest occurred after _MHD fusion protein stimulation.

The Hela cells were routinely cultured to the logarithmic phase, and the cells were divided into 12 groups, with 3 bottles in each group, of which 7 groups were stimulated with Restin- _MHD fusion protein at a final concentration of 1 μg/ml, and the other 6 groups were not added with Restin- _MHD fusion protein. as a comparison. One vial was taken from each of the cells in the stimulation group and the control group every day, and each vial was counted 4 times, the average value was taken, and the average value of the three vials was accumulated. The counting was continued for 7 days, and the culture solution containing the fusion protein at a final concentration of 1 μg/ml was changed after three days without counting. The cell growth curve was drawn according to the counting results, and the results are shown in Figure 8. It can be clearly seen that the growth of the fusion protein stimulation group is slower than that of the normal culture group as a control.

Claims (3)

1. recombinant human cell tranquillization factor M HD fusion rotein; It is characterized in that; Comprise people's cell tranquillization factor M HD structural domain, also be connected with purifying flag sequence, zymoplasm and enteropeptidase restriction enzyme site in turn at the N of people's cell tranquillization factor M HD structural domain end, and the Nus sequence;

The aminoacid sequence of described recombinant human cell tranquillization factor M HD fusion rotein is shown in SEQ.ID.NO.2.

2. the prokaryotic expression carrier of an express recombinant people cell tranquillization factor M HD fusion rotein is characterized in that this prokaryotic expression carrier is pET44a ₍₊₎-Restin- _MHD, be through Bam HI/Hind III restriction enzyme site will be shown in SEQ.ID.NO.3 people's cell tranquillization factor M HD gene fragment, be cloned into pET44a ₍₊₎Expression vector.

3. the preparation method of a recombinant human cell tranquillization factor M HD fusion rotein is characterized in that, may further comprise the steps:

1) be template with the cDNA that comprises whole person Restin genes encoding frame, with primer to P as primer, pcr amplification people Restin- _MHDGene fragment, described primer to P is:

Upstream primer P1:cgggatccaa gaaaagtctc ct;

Downstream primer P2:cccaagcttg actttcagtt tgc;

2) with the people Restin-of pcr amplification _MHDGene fragment obtains the fragment with sticky end with Bam HI/Hind III double digestion; And with the pET44a of this fragment and Bam HI/Hind III double digestion ₍₊₎The carrier connection obtains expression vector pET44a ₍₊₎-Restin- _MHD

3) with expression vector pET44a ₍₊₎-Restin- _MHDTransfection is to competent intestinal bacteria, and screening obtains the positive transformant of destination gene expression;

4) with positive transformant picking neat in edge, bacterium colony that growth conditions is good after 37 ℃ of thermostat containers are cultivated 12h, be inoculated in the LB nutrient solution that contains penbritin 100mg/L 37 ℃ of overnight cultures of shaking table; Next day, the volume ratio with 1: 100 was inoculated in the fresh LB nutrient solution that contains penbritin 100mg/L, and 37 ℃ are continued to be cultured to bacterial density and reach OD ₆₀₀=0.4～0.6, add the IPTG inducing culture 4h of 0.1～2.5mM concentration, make expression vector pET44a ₍₊₎-Restin- _MHDObtain expressing centrifugal then collection bacterium;

5) add lysate in the ratio of 10mL/g bacterial precipitation, resuspended bacterium ultrasonicly under ice bath is split bacterium, spinning then, and collect supernatant;

Described lysate is the TrisCl of pH 8.5, concentration 50mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the PMSF of 1mmol/L;

6) supernatant of collecting is splined on the metal chelate affinity chromatography post, washes the flow velocity wash-out of post, wash post with the buffer B of 2 times of column volumes then, use damping fluid C elution of bound albumen at last, collect elution peak with 0.5mL/min with the buffer A of 10 times of column volumes;

Described buffer A is the TrisCl of pH 8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 20mmol/L; Described buffer B is the TrisCl of pH 8.5, concentration 20mmol/L, the mixing solutions of the KCl of 1mol/L and the beta-mercaptoethanol of 5mmol/L; Described damping fluid C is the TrisCl of pH 8.5, concentration 20mmol/L, the KCl of 100mmol/L, the mixing solutions of the beta-mercaptoethanol of 5mmol/L and the imidazoles of 100mmol/L;

The elution peak of collecting is splined on ion exchange chromatography,, collects elution peak, obtain purified recombinant people cell tranquillization factor M HD fusion rotein with the albumen of elutriant D elution of bound;

Described elutriant D is the TrisCl of pH 7.4, concentration 50mmol/L and the mixing solutions of 1mmol/LNaCl.

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