CN112898407A - Preparation method of recombinant camel-derived serum albumin - Google Patents

Abstract

The invention relates to a preparation method of recombinant camel-derived serum albumin, which comprises the steps of plasmid construction, transformation and expression, inclusion body renaturation and purification. The invention adopts a prokaryotic expression system based on escherichia coli, realizes the purpose of successfully expressing recombinant camel-source serum albumin by adopting the prokaryotic expression system through the main steps of plasmid construction, transformation and expression, inclusion body renaturation and purification, and the obtained serum albumin has good water solubility, high purity, high safety, high cost performance and the like, is expected to become a substitute of the human serum albumin, so as to meet the requirement of a patient on the serum albumin.

Description

Preparation method of recombinant camel-derived serum albumin

Technical Field

The invention relates to a preparation method of recombinant camel source serum albumin, which adopts a prokaryotic expression system to successfully express the recombinant camel source serum albumin and relates to the technical fields of plasmid construction, protein expression, inclusion body renaturation and protein purification.

Background

Human Serum Albumin (HSA) is known to the applicant as the major protein in plasma, synthesized in the liver, with a molecular weight of 66.5kDa, and a single-chain peptide consisting of 585 amino acids forming a heart-shaped three-dimensional structure. The main function of human serum albumin is to maintain plasma oncotic pressure and play an important role in substance transport, transporting oxygen and carbon dioxide, metabolites, nutrients, drugs, metal ions, and the like. Due to its important physiological functions, serum albumin is often used to treat acute burn injury, ischemic shock, hypoproteinemia, erythrocythemia, cirrhosis, and the like.

In recent years, the application range of serum albumin has become wider. On one hand, because serum albumin has good water solubility and biocompatibility, long half-life (about 200 days) and low toxicity; on the other hand, serum albumin can be enriched at the tumor site by a targeting mechanism. Tumor tissue is passively targeted by the epr (enhanced specificity and retention) effect and serum albumin is enriched at this site due to the lack of lymphatic vessels in tumor tissue. Through the active targeting effect, the serum albumin can be combined with albumin receptor (gp 60) on the surface of vascular endothelial cells and enriched by intercellular transport to the interstitial site of the tumor. Therefore, serum albumin is increasingly used as a drug carrier in tumor therapy and the like.

According to statistics, the annual demand of serum albumin is about 500 tons, the main source of the serum is human serum at present, but the source has low yield, time and labor consumption, high risk and pathogenicity, and a plurality of strong pathogenic pathogens such as hepatitis virus and Human Immunodeficiency Virus (HIV) can be transmitted through blood, so that a serum albumin production technology with high cost performance and convenience needs to be developed to meet the requirements of patients.

The bactrian camel often inhabits in extreme environments such as grassland, desert, gobi and the like, and has high serum albumin content to maintain basic physiological metabolism and blood osmotic pressure; in addition, the bactrian camel serum albumin has higher homology with human serum albumin and similar structure. Therefore, the recombinant serum albumin (rCSA) based on the bactrian camel has the advantages of high cost performance, mass production, high safety and the like, and is expected to become a substitute for human serum albumin.

However, serum albumin is difficult to produce in prokaryotic expression systems due to its rich disulfide bonds, which inevitably leads to increased production costs. The invention patent applications with application numbers CN201310078906.0 and CN103194482A disclose a plasmid and a recombinant bacterium for expressing human serum albumin. However, the host bacterium adopted by the method is pichia pastoris, and belongs to a eukaryotic expression system.

The development of a method for preparing serum albumin by using a prokaryotic expression system is urgently needed.

Disclosure of Invention

The invention aims to: aiming at the problems in the prior art, the preparation method of the recombinant camel source serum albumin is provided, and a prokaryotic expression system is adopted to successfully express the recombinant camel source serum albumin. Meanwhile, the corresponding recombinant camel source serum albumin, the coding gene thereof, the plasmid thereof and the engineering bacteria thereof are also provided.

The technical scheme for solving the technical problems of the invention is as follows:

a preparation method of recombinant camel serum albumin is characterized by comprising the following steps:

firstly, carrying out PCR amplification on a gene segment for coding recombinant camel source serum albumin, and then recombining the gene segment with a plasmid vector to construct a recombinant plasmid; the amino acid sequence of the recombinant camel source serum albumin is shown as SEQ ID No. 2;

secondly, adopting chemically competent cells prepared by using escherichia coli; transforming the recombinant plasmid into chemically competent cells, and culturing to obtain single colonies; carrying out liquid culture by taking a single colony as a seed, and then adding an inducer to induce and express a target protein to obtain the target protein mainly existing in an inclusion body form;

thirdly, centrifugally collecting precipitate after ultrasonically crushing thalli; then washing the precipitate with a washing buffer solution, and dissolving the precipitate with a dissolving buffer solution to obtain a dissolving solution containing the target protein; purifying the dissolved solution to obtain target protein; adopting a gradual gradient dialysis mode to refold the target protein into a physiological state, namely renaturation;

and fourthly, purifying the renatured target protein to obtain the recombinant camel source serum albumin.

The method adopts a prokaryotic expression system based on escherichia coli, and realizes the purpose of successfully expressing recombinant camel-source serum albumin by adopting the prokaryotic expression system through the main steps of plasmid construction, transformation and expression, inclusion body renaturation and purification.

The technical scheme of the invention is further perfected as follows:

preferably, in the first step, the plasmid vector is pSmart-I; the enzyme cutting sites selected during the recombination construction are as follows: BamHI endonuclease site, and XhoI endonuclease site;

in the second step, the Escherichia coli is BL21(DE3) Escherichia coli strain; the inducer is IPTG;

in the third step, the specific process of washing with the washing buffer is as follows:

resuspending the precipitate with a first wash buffer, then centrifuging and collecting the precipitate, repeating for a predetermined number of times;

then resuspending the precipitate with a second washing buffer solution, then centrifugally collecting the precipitate, and repeating the steps for a preset number of times;

the first wash buffer is: a buffer containing 5 + -0.05 mM DTT, 50 + -0.5 mM Tris-HCl, pH 8.0 + -0.1 mM NaCl, 500 + -5 mM NaCl, 1 + -0.01% Triton X-100, 1 + -0.01 mM EDTA;

the second washing buffer solution is: a buffer containing 5 + -0.05 mM DTT, 50 + -0.5 mM Tris-HCl, pH 8.0 + -0.1 mM NaCl, 500 + -5 mM NaCl, 1 + -0.01 mM EDTA;

the specific process of dissolution with the dissolution buffer is as follows:

resuspending the precipitate with a lysis buffer solution, stirring for a predetermined time, and centrifuging to collect supernatant to obtain a lysis solution containing the target protein;

the lysis buffer is: a buffer solution containing 50 + -0.5 mM of Tris, pH 6.0-6.3, 8 + -0.08M of urea, 1 + -0.01 mM of EDTA and 5 + -0.05 mM of DTT;

in the fourth step, the renatured target protein is purified by using a gel size exclusion column.

Note: IPTG in the second step is i-propyl-beta-D-thiogalactoside.

Preferably, in the third step, the specific process of purifying the dissolution solution to obtain the target protein comprises:

the dissolving solution is firstly subjected to crude extraction and purification of target protein by a nickel column affinity chromatography method, and then a band containing the target protein is identified, separated and collected by an SDS-PAGE method;

the specific process of gradual gradient dialysis is as follows:

respectively dialyzing for a preset time in a buffer solution R1, a buffer solution R2, a buffer solution R3, a buffer solution R4 and a buffer solution R5 in sequence, and then performing ultrafiltration concentration;

the buffer solution R1 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1 mM, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1% of glycerol, 1 plus or minus 0.01mM of DTT and 4 plus or minus 0.04mM of urea;

the buffer solution R2 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT and 2 plus or minus 0.02mM of urea;

the buffer solution R3 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT, 1 plus or minus 0.01mM of urea and 600 plus or minus 6mM of L-arginine;

the buffer solution R4 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT, 0.5 plus or minus 0.005mM of urea and 600 plus or minus 6mM of L-arginine;

the buffer solution R5 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1% of glycerol, 1 plus or minus 0.01mM of DTT and 600 plus or minus 6mM of L-arginine.

By adopting the preferred scheme, the key detail characteristics of each step can be further optimized, so that the recombinant camel-derived serum albumin can be expressed more successfully.

More preferably, in the first step, the nucleotide sequence of the gene fragment is shown as SEQ ID No. 1;

in the second step, the specific process of transforming the recombinant plasmid into chemically competent cells is as follows: respectively placing the prefreezed chemical competent cells and the prefreezed recombinant plasmids on ice for thawing; then adding the recombinant plasmid into the chemically competent cells and uniformly mixing; firstly placing the mixture in an ice bath, then carrying out heat shock, and then placing the mixture in the ice bath for standing; adding LB broth culture medium without antibiotic to culture, and recovering thallus;

the specific process of obtaining single colony through culture is as follows: culturing the recovered thallus on an LB (lysogeny broth) flat plate containing kanamycin to obtain a white single colony;

the specific process of taking a single colony as a seed to carry out liquid culture and then adding an inducer comprises the following steps: picking single bacterial colony and inoculating the single bacterial colony in LB liquid culture medium containing kanamycin for recovery culture; inoculating the recovered bacterial liquid into a fresh LB liquid culture medium containing kanamycin, performing formal culture, and culturing to OD₆₀₀When the preset value is reached, adding an inducer and carrying out induction culture.

More preferably, in the second step, in a specific process of transforming the recombinant plasmid into chemically competent cells: placing in ice bath for 30min, then performing heat shock at 42 deg.C for 45s, and then placing in ice bath for standing for 2 min; culturing at 37 deg.C and 220rpm for 60 min;

in the specific process of obtaining single colonies by culturing: the culture condition is that the inverted culture is carried out for 16h at 37 ℃;

in the specific process of taking a single colony as a seed to carry out liquid culture and then adding an inducer: resuscitating culture conditions at 37 deg.C and 220rpm, and shaking culture overnight; the formal culture conditions were 37 ℃ and 220 rpm; the final concentration of the inducer added was 1mM, and the induction culture was carried out at 15 ℃ and 220rpm for 16 hours.

More preferably, in the third step, the centrifugation conditions after the ultrasonic disruption of the thalli are 12500rpm, 4 ℃ and 30 min;

during the specific course of washing with the wash buffer: centrifuging at 12500rpm and 4 deg.C for 30 min; the predetermined number of times is 3;

during the specific process of lysis with lysis buffer: the preset time is 1 h; centrifuging at 12500rpm and 4 deg.C for 15 min;

in the specific process of purifying the dissolving solution to obtain the target protein: the specific model of the nickel column is GE 5mL Histrap; the nickel column affinity chromatography adopts a binding buffer solution, a washing buffer solution and an elution buffer solution, wherein the binding buffer solution is a buffer solution which is pH7.4 and contains 20mM sodium phosphate, 500mM sodium chloride and 10mM imidazole, the washing buffer solution is a buffer solution which is pH7.4 and contains 20mM sodium phosphate, 500mM sodium chloride and 40mM imidazole, and the elution buffer solution is a buffer solution which is pH7.4 and contains 20mM sodium phosphate, 500mM sodium chloride and 250mM imidazole;

during the specific course of stepwise gradient dialysis: the dialysis temperature is 4 ℃ and the preset time is 8 h; centrifuging for 25min under the ultrafiltration concentration condition of 5000 g;

in the fourth step, the specific model of the gel size exclusion column is GE Superdex 7510/300 incrase; the elution buffer used was 50. + -. 0.5mM sodium phosphate, 500. + -. 5mM sodium chloride at pH 7.0.

With the above preferred scheme, the detailed characteristics of each step can be further optimized.

The invention also proposes:

the amino acid sequence of the recombinant camel source serum albumin is shown as SEQ ID No. 2.

A gene segment for encoding the recombinant camel-derived serum albumin.

Recombinant plasmids having the gene fragments described above.

An engineered bacterium comprising the recombinant plasmid described above.

Compared with the prior art, the method adopts a prokaryotic expression system based on escherichia coli, achieves the purpose of successfully expressing recombinant camel-source serum albumin by adopting the prokaryotic expression system through the main steps of plasmid construction, transformation and expression, inclusion body renaturation and purification, and the obtained serum albumin has good water solubility, high purity, high safety, high cost performance and the like, is expected to become a substitute of the human serum albumin, so as to meet the requirements of patients on the serum albumin.

Drawings

FIG. 1 is a graph showing the results of example 1 of the present invention.

FIG. 2 is a graph showing the results of example 2 of the present invention.

FIG. 3 is a diagram showing the results of SDS-PAGE in example 3 of the present invention.

FIG. 4 is a graph showing the results of the peptide coverage assay of example 3 of the present invention.

FIG. 5 is a diagram showing the result of N-terminal sequencing in example 3 of the present invention.

FIG. 6 is a graph showing the result of Western Blot identification in example 3 of the present invention.

FIG. 7 is a graph showing the results of SDS-PAGE identification of protein expression and inclusion body solubilization in example 4 of the present invention.

FIG. 8 is a graph showing the results of each identification in the nickel column affinity chromatography of example 4 of the present invention.

FIG. 9 is a graph of the results of the fine purification and identification of gel size exclusion column of example 5 of the present invention.

Detailed Description

The invention is described in further detail below with reference to embodiments and with reference to the drawings. The invention is not limited to the examples given.

Example 1

This example is the construction of recombinant plasmids.

The basic process of the embodiment includes: carrying out PCR amplification on a gene segment for coding recombinant camel source serum albumin, and then recombining the gene segment with a plasmid vector to construct a recombinant plasmid; the amino acid sequence of the recombinant camel source serum albumin is shown in SEQ ID No. 2.

Wherein the plasmid vector is pSmart-I; the enzyme cutting sites selected during the recombination construction are as follows: BamHI endonuclease site, and XhoI endonuclease site; the nucleotide sequence of the gene fragment is shown as SEQ ID No. 1.

The following is a specific procedure by way of example:

the Polymerase Chain Reaction (PCR) is used to amplify the gene fragment of interest. BamHI (5392bp) and XhoI (5432bp) endonuclease sites were selected to linearize the plasmid vector. Homologous recombination arm primers are designed at two ends of a target sequence, and sequences of the vector between 5392bp and 5432bp are recombined and replaced. Finally, a recombinant plasmid pSmart-I-CSA is constructed.

The correlation results are shown in fig. 1, where:

the A picture is the result of PCR amplification of a target sequence, and the result shows that the theoretical size of a target fragment is 1818bp, and the actual size in the picture conforms to the theoretical size, which indicates that the target fragment is successfully synthesized;

b picture is the result of taking the bacterium liquid recovered in example 2 to carry out PCR identification, the result shows that 1818bp obtained after PCR amplification is consistent with the size of the target fragment, which indicates that the recombinant plasmid is successfully transformed into the thallus;

the C picture is the empty vector PCR result, and the result shows that the theoretical size of the empty vector pSmart-I is 5428bp, and the theoretical size is consistent with the actual size;

and the result shows that the first lane from left to right is the size of the recombinant plasmid before enzyme digestion and is 7303bp, the second lane is the result after enzyme digestion, 4195bp and 3108bp are obtained after enzyme digestion, and the enzyme digestion results are consistent before and after the enzyme digestion results.

In conclusion, the successful construction of the recombinant plasmid pSmart-I-CSA is demonstrated.

Example 2

This example shows the transformation of recombinant plasmids into chemically competent cells. This example is based on example 1.

The basic process of the embodiment includes: chemically competent cells prepared from escherichia coli are adopted; the recombinant plasmid is transformed into chemically competent cells, and then single colonies are obtained by culturing.

Wherein the Escherichia coli is BL21(DE3) Escherichia coli strain; the inducer is IPTG.

The specific process of transforming the recombinant plasmid into chemically competent cells is as follows: respectively placing the prefreezed chemical competent cells and the prefreezed recombinant plasmids on ice for thawing; then adding the recombinant plasmid into the chemically competent cells and uniformly mixing; firstly placing the mixture in an ice bath, then carrying out heat shock, and then placing the mixture in the ice bath for standing; adding LB broth without antibiotic to culture, and recovering thallus. Wherein, the mixture is firstly placed in an ice bath for 30min, then is thermally shocked for 45s at 42 ℃, and then is placed in the ice bath for standing for 2 min; the culture conditions were 37 ℃ and 220rpm, and the culture was carried out for 60 min.

The specific process of obtaining single colony through culture is as follows: the recovered cells were cultured on an LB plate containing kanamycin to obtain white single colonies. Wherein the culture condition is that the culture is carried out for 16h at 37 ℃ in an inverted way.

The following is a specific procedure by way of example:

chemically competent cells BL21(DE3) and recombinant plasmid pSmart-I-CSA were thawed on ice. And (3) sucking the recombinant plasmid into the chemically competent cells, gently mixing the recombinant plasmid and the chemically competent cells uniformly, carrying out ice bath for 30min, carrying out heat shock for 45s at 42 ℃, quickly transferring the centrifuge tube into the ice bath, and standing the centrifuge tube for 2 min. Then, 450. mu.L of sterile antibiotic-free broth culture medium (LB) was added to each centrifuge tube, mixed well, and then cultured on a shaker at 37 ℃ and 220rpm for 60min to recover the cells. Centrifuging the recovered thallus at room temperature at 4000rpm for 2min, discarding part of supernatant, mixing the rest supernatant with thallus, and spreading on LB plate containing kanamycin. The cells were cultured in an inverted state at 37 ℃ for 16 hours to grow white single colonies. As shown in FIG. 2, white colonies on LB solid plates were transformed into competent cells.

Example 3

This example is an expression test of a target protein. This example is based on example 2.

The basic process of the embodiment includes: and performing liquid culture by taking the single colony as a seed, and then adding an inducer to induce and express the target protein to obtain the target protein mainly existing in an inclusion body form.

Wherein, the single colony is used as a seed to carry out liquid culture, and then an inducer is added into the liquid cultureThe process is as follows: picking single bacterial colony and inoculating the single bacterial colony in LB liquid culture medium containing kanamycin for recovery culture; inoculating the recovered bacterial liquid into a fresh LB liquid culture medium containing kanamycin, performing formal culture, and culturing to OD₆₀₀When the preset value is reached, adding an inducer and carrying out induction culture. Wherein the recovery culture conditions are 37 ℃, 220rpm, and the shaking culture is carried out overnight; the formal culture conditions were 37 ℃ and 220 rpm; the final concentration of the inducer added was 1mM, and the induction culture was carried out at 15 ℃ and 220rpm for 16 hours.

The following is a specific procedure by way of example:

a single colony was picked on an LB plate, inoculated in 5mL of LB medium containing 50. mu.g/mL of kanamycin, cultured at 37 ℃ at 220rpm, and shaken overnight. The next day, 500. mu.L of the recovered bacterial suspension was aspirated and inoculated into 50mL of fresh LB medium containing 50. mu.g/mL kanamycin, cultured at 37 ℃ and 220rpm with shaking until OD₆₀₀Pipette 1mL as uninduced control; 100mM isopropyl-beta-D-thiogalactoside (IPTG) was added to a final concentration of 1mM, 15 ℃, 220rpm, and induction was carried out for 16h to induce expression of the fusion protein CSA. 1mL of bacterial liquid for inducing for 2, 4, 6, 8 and 16 hours is collected respectively, and is subjected to ultrasound for 10s by using a diagenod non-contact ultrasonicator, the interval is 30s, and the circulation is carried out for 80 times. 13000rpm, at 4 ℃ for 30min, and collecting the supernatant and the precipitate respectively.

(1) Polyacrylamide gel (SDS-PAGE) identifies protein expression and protein solubility. Aspirate 120. mu.L of the supernatant, add 30. mu.L of SDS protein loading buffer (5X), and mix well. The precipitate was added 120. mu.L of phosphate buffer (PBS, pH 8.0), and 30. mu.L of SDS protein loading buffer (5X) was added and mixed well. The solution was boiled at 95 ℃ for 5 min. 10% Tris-Glycine polyacrylamide gel was prepared, and the loading was 20. mu.L. Electrophoresis conditions are 90V and 30 min; 120V, 70 min. After dyeing with Coomassie brilliant blue for 40min, the solution is heated and decolorized three times in a microwave oven for 5min each time. And (3) analyzing the expression condition and the solubility of the protein after decolorization.

The results are shown in FIG. 3. Panel A shows protein expression of expression strain BL21(DE3) after 2, 4, 6, 8 hours of IPTG induction, respectively, wherein 1: not inducing; 2: supernatant protein after 2h of induction; 3: precipitating the protein after 2h of induction; 4: supernatant protein after 4h of induction; 5: precipitating the protein after 4h of induction; 6: supernatant protein after 6h of induction; 7: precipitating the protein after 6h of induction; 8: supernatant protein after 8h of induction; 9: protein precipitation after 8h of induction.

Panel B shows protein expression of the expression strain BL21(DE3) 16 hours after IPTG induction (equivalent to overnight induction), wherein 1: not inducing; 2: total protein induced overnight with IPTG; 3: IPTG-induced overnight supernatant protein; 4: IPTG induced overnight precipitation of the protein.

Therefore, the theoretical molecular weight of the target protein is 78.7kD, and the actual protein is between 70 and 100kD, which is in line with the expected size. The expression level of the protein is in positive correlation with time (i.e., time-dependent), the longer the time is, the more the target protein is expressed, and most of the protein exists in the form of inclusion bodies.

(2) And (3) identifying the coverage rate of the peptide fragment and analyzing the N-terminal amino acid sequence. Carrying out enzymolysis on the inclusion body by using Trypsin, Chymotrypsin and Glu-C enzymes respectively, and detecting by LC-MS/MS to obtain the peptide segment after enzymolysis, wherein the proportion of the total number of amino acids in the peptide segment to the total number of amino acids in the protein is the coverage rate of the peptide segment. Carrying out enzymolysis on SDS-PAGE gel, desalting peptide sections, and adding 80 mu L Nano-HPLC Buffer B into a sample containing the polypeptide for volatilizing. Finally, LC-MS/MS detection is carried out, and the chromatographic column is Acclaim PepMap^TMRSLC 75 μm 15cm, NanoViper C18, 2 μm, 100A. Mobile phase a phase: 0.1% FA in water, mobile phase B: 0.1%/80% FA/acetonitrile, gradient elution procedure as shown in Table 1, mobile phase flow rate of 300 nL/min. The mass spectrum conditions are as follows: primary MS mass resolution was set to 70000, automatic gain control value (AGC) was set to 1e6, maximum injection time 50 MS; the mass spectrometry scanning is set as a primary full-scan nucleus ratio m/z range 300-1600; all MS/MS spectrum collection is completed by high-energy collision cracking in a data-dependent positive ion mode, and the collision energy is set to be 28; the resolution of MS/MS was set to 17500, the automatic gain control was set to 1e5, and the maximum injection time was 50 MS; the dynamic exclusion time was set to 30 s. Mass spectral data were analyzed using Thermo BioPharma finder3.2 software.

TABLE 1 protease determination mobile phase gradient elution procedure

FIG. 4 shows the result of peptide coverage detection, wherein the A picture is the BPI map of the test sample after Trypsin enzymolysis; b is a BPI map of a sample subjected to zymohydrolysis by Chymotrypsin; and the C picture is a BPI map of the test sample subjected to Glu-C enzymolysis. The results show that the coverage rate of the test sample after the enzymolysis of the Trypsin is 93.5 percent, the coverage rate of the test sample after the enzymolysis of the Chymotrypsin is 100.0 percent, and the coverage rate of the test sample after the enzymolysis of the Glu-C is 59.9 percent. Comprehensively obtaining the peptide fragment coverage rate of the test sample to be 100.0%.

FIG. 5 shows the result of N-terminal amino acid sequence analysis, wherein Panel A is a BPI map of a sample subjected to zymolysis with Chymotrypsin; b is a primary mass spectrogram of an N-terminal sequence; the C picture is an N-terminal sequence secondary mass spectrogram; and the D picture is an N-terminal sequence b and a y ion picture. In conclusion, the N-terminal amino acid sequence of the test sample is DGIRIQADQTPEDLDMEDNDIIEAHREQIGGDTHKSEIAHRF, which corresponds to the target protein sequence.

(3) Western Blot (WB) identification analysis. A rabbit monoclonal antibody against his tag; the secondary antibody adopts goat anti-rabbit IgG-HRP. Since the expressed protein is fused with a histidine tag, an antibody against the histidine tag can specifically bind to the fusion protein. FIG. 6 is a graph showing the result of Western Blot identification, wherein the ratio of 1: 0.2mM IPTG induced total protein; 2: 0.2mM IPTG induced supernatant protein; 3: 0.2mM IPTG induced precipitated protein; 4: 1mM IPTG induced total protein; 5: 1mM IPTG induced supernatant protein; 6: 1mM IPTG induced precipitation of protein. The results showed that the histidine-fused protein was successfully expressed.

Example 4

This example shows protein expression and inclusion body solubilization, purification and renaturation. This example is based on example 2.

The basic process of the embodiment includes:

(1) and performing liquid culture by taking the single colony as a seed, and then adding an inducer to induce and express the target protein to obtain the target protein mainly existing in an inclusion body form.

Wherein, the specific process of taking the single colony as the seed to carry out liquid culture and then adding the inducer comprises the following steps: picking single bacterial colony and inoculating the single bacterial colony in LB liquid culture medium containing kanamycin for recovery culture; inoculating the recovered bacterial liquid into a fresh LB liquid culture medium containing kanamycin, performing formal culture, and culturing to OD₆₀₀When the preset value is reached, adding an inducer and carrying out induction culture. Wherein the recovery culture conditions are 37 ℃, 220rpm, and the shaking culture is carried out overnight; the formal culture conditions were 37 ℃ and 220 rpm; the final concentration of the inducer added was 1mM, and the induction culture was carried out at 15 ℃ and 220rpm for 16 hours.

(2) Carrying out ultrasonic crushing on thalli, centrifuging and collecting precipitate; then, the precipitate is washed with a washing buffer solution and dissolved in a dissolving buffer solution to obtain a dissolving solution containing the target protein.

Wherein the centrifugation conditions after the ultrasonic disruption of the thalli are 12500rpm, 4 ℃ and 30 min.

The specific process of washing with the washing buffer is as follows:

resuspending the precipitate with a first wash buffer, then centrifuging and collecting the precipitate, repeating for a predetermined number of times;

then resuspending the precipitate with a second washing buffer solution, then centrifugally collecting the precipitate, and repeating the steps for a preset number of times;

centrifuging at 12500rpm and 4 deg.C for 30 min; the predetermined number of times is 3.

The first wash buffer was: a buffer containing 5 + -0.05 mM DTT, 50 + -0.5 mM Tris-HCl, pH 8.0 + -0.1 mM NaCl, 500 + -5 mM NaCl, 1 + -0.01% Triton X-100, 1 + -0.01 mM EDTA;

the second wash buffer was: a buffer containing 5 + -0.05 mM DTT, 50 + -0.5 mM Tris-HCl, pH 8.0 + -0.1 mM NaCl, 500 + -5 mM NaCl, 1 + -0.01 mM EDTA;

the specific process of dissolution with the dissolution buffer is as follows:

resuspending the precipitate with a lysis buffer solution, stirring for a predetermined time, and centrifuging to collect supernatant to obtain a lysis solution containing the target protein; the preset time is 1 h; the centrifugation conditions were 12500rpm, 4 ℃ and 15 min.

The lysis buffer was: a buffer solution containing 50 + -0.5 mM of Tris, pH 6.0-6.3, 8 + -0.08M of urea, 1 + -0.01 mM of EDTA and 5 + -0.05 mM of DTT.

(3) Purifying the dissolved solution to obtain the target protein, wherein the specific process comprises the following steps: the dissolving solution is firstly subjected to crude extraction and purification of target protein by a nickel column affinity chromatography method, and then a band containing the target protein is identified, separated and collected by an SDS-PAGE method;

wherein the specific model of the nickel column is GE 5mL Histrap; the nickel column affinity chromatography adopts a binding buffer solution, a washing buffer solution and an elution buffer solution, wherein the binding buffer solution is a pH7.4 buffer solution containing 20mM sodium phosphate, 500mM sodium chloride and 10mM imidazole, the washing buffer solution is a pH7.4 buffer solution containing 20mM sodium phosphate, 500mM sodium chloride and 40mM imidazole, and the elution buffer solution is a pH7.4 buffer solution containing 20mM sodium phosphate, 500mM sodium chloride and 250mM imidazole.

(4) The target protein in the solution is refolded into a physiological state, namely renaturation, by adopting a gradual gradient dialysis mode.

Wherein, the specific process of the step-by-step gradient dialysis is as follows:

respectively dialyzing for a preset time in a buffer solution R1, a buffer solution R2, a buffer solution R3, a buffer solution R4 and a buffer solution R5 in sequence, and then performing ultrafiltration concentration; the dialysis temperature is 4 ℃ and the preset time is 8 h; the ultrafiltration concentration condition is 5000g, and the centrifugation is carried out for 25 min.

The buffer solution R1 is a buffer solution containing 50 + -0.5 mM of Tris-HCl with the pH value of 8.0 + -0.1, 100 + -1 mM of NaCl, 10 + -0.1% of glycerol, 1 + -0.01 mM of DTT and 4 + -0.04 mM of urea;

the buffer solution R2 is a buffer solution containing 50 + -0.5 mM of Tris-HCl with the pH value of 8.0 + -0.1, 100 + -1 mM of NaCl, 10 + -0.1% of glycerol, 1 + -0.01 mM of DTT and 2 + -0.02 mM of urea;

the buffer solution R3 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT, 1 plus or minus 0.01mM of urea and 600 plus or minus 6mM of L-arginine;

the buffer solution R4 is a buffer solution containing 50 + -0.5 mM of Tris-HCl with the pH value of 8.0 + -0.1, 100 + -1 mM of NaCl, 10 + -0.1% of glycerol, 1 + -0.01 mM of DTT, 0.5 + -0.005 mM of urea and 600 + -6 mM of L-arginine;

buffer R5 was a buffer containing 50. + -. 0.5mM Tris-HCl pH 8.0. + -. 0.1, 100. + -. 1mM NaCl, 10. + -. 0.1% glycerol, 1. + -. 0.01mM DTT, 600. + -. 6mM L-arginine.

The following is a specific procedure by way of example:

two single colonies were picked on LB plates and inoculated in 5mL of LB medium containing 50. mu.g/mL of kanamycin, respectively, and cultured at 37 ℃ and 220rpm with shaking overnight. The next day, 10mL of the recovered bacterial solution was aspirated and inoculated into 1L of fresh LB medium containing 50. mu.g/mL kanamycin, cultured at 37 ℃ and 220rpm with shaking until OD₆₀₀Pipette 1mL as uninduced control; 100mM isopropyl-. beta. -D-thiogalactoside (IPTG) was added to a final concentration of 1mM, 15 ℃ at 220rpm, and induced for 16 h.

And (3) crushing the thalli by using a SONICS VCX750 type ultrasonic crusher, working for 4s, performing intermittent working for 8s, and working for 25min, and collecting 120 mu L of the thalli to perform total protein identification.

Centrifuging at 12500rpm and 4 ℃ for 30min after ultrasonic treatment, collecting precipitate, sucking 1mL of supernatant for identification of supernatant protein, and discarding the residual supernatant. The cells were collected, resuspended in washing buffer 1, centrifuged at 12500rpm for 30min at 4 ℃ and repeated three times, resuspended in washing buffer 2, centrifuged at 12500rpm for 30min at 4 ℃ and repeated three times. The pellet was resuspended by adding 20mL of lysis buffer and stirred at room temperature for 1 h. 12500rpm, 4 deg.C, centrifuging for 15min, collecting supernatant, and collecting 1mL for identifying dissolved protein.

SDS-PAGE gel electrophoresis verifies the protein expression and the inclusion body dissolution. 10% Tris-Glycine polyacrylamide gel was prepared, and the loading was 20. mu.L. Electrophoresis conditions are 90V and 30 min; 120V, 70 min. After dyeing with Coomassie brilliant blue for 40min, the solution is heated and decolorized three times in a microwave oven for 5min each time. FIG. 7 is a graph showing the results of SDS-PAGE identification of protein expression and inclusion body solubilization, in which 1: solubilized inclusion bodies; 2: not inducing; 3: total protein 16 hours after IPTG induction; 4: the supernatant after ultrasonication; 5: precipitation after ultrasonication; 6: solubilized inclusion bodies; m: marker, from top to bottom, is 180, 130, 100, 70, 55, 40, 35 kDa. The results showed that the molecular size of the solubilized protein was as expected, and nickel column purification was performed using the solubilized protein sample.

The nickel column (i.e., the equilibrated nickel column) was rinsed slowly with 5 Column Volumes (CV) of binding buffer. Centrifuging the dissolved solution, taking a supernatant containing the target protein, injecting the supernatant into a nickel column by using an injector, and collecting the flow-through solution at the same time for subsequent SDS-PAGE identification; incubating the nickel column with the target protein at 4 ℃ overnight (12h), washing the nickel column with 10-15 CV impurity washing buffer solution for the next day, and collecting the impurity washing solution for subsequent SDS-PAGE identification; and finally eluting the target protein by using 5CV elution buffer solution, and collecting the target protein for subsequent SDS-PAGE identification.

In FIG. 8, panel A shows the flow-through of SDS-PAGE to identify nickel column purification, showing that there are many hetero-proteins; and the B picture is a result of the SDS-PAGE identification nickel column purification, wherein lanes 1, 2 and 3 are impurity washing liquid, more impurity proteins can be seen, lanes 4-9 are eluent, the target protein can be gradually shown and thickened, and the lane 9 is high in protein purity. The molecular mass of the protein is measured by using a high-resolution mass spectrometer (Q active mass spectrometer), and a C picture shows that the detected relative molecular mass is 78744.58Da and is matched with the theoretical molecular mass, so that the 9 th lane protein is selected for renaturation and further purification.

The protein is refolded to a physiological state, i.e., renaturation, in the buffer by stepwise gradient dialysis. Dialyzed in the following five buffers (R1, R2, R3, R4, R5) for 8h at 4 ℃. Finally, the target protein is centrifugally concentrated by an ultrafiltration tube (5000g, centrifugation for 25 min).

R1:50mM Tris-HCl(pH 8.0),100mM NaCl,10％Glycerol,1mM DTT,4mM Urea；

R2:50mM Tris-HCl(pH 8.0),100mM NaCl,10％Glycerol,1mM DTT,2mM Urea；

R3:50mM Tris-HCl(pH 8.0),100mM NaCl,10％Glycerol,1mM DTT,1mM Urea,600mM L-arginine；

R4:50mM Tris-HCl(pH 8.0),100mM NaCl,10％Glycerol,1mM DTT,0.5mM Urea,600mM L-arginine；

R5:50mM Tris-HCl(pH 8.0),100mM NaCl,10％Glycerol,1mM DTT,600mM L-arginine。

Example 5

This example shows the target protein after purification and renaturation. This example is based on example 4.

The basic process of the embodiment includes:

and (3) adopting a gel size exclusion column to purify and renaturate the target protein to obtain the recombinant camel source serum albumin.

Wherein the specific model of the gel size exclusion column is GE Superdex 7510/300 incrasase; the elution buffer used was 50. + -. 0.5mM sodium phosphate, 500. + -. 5mM sodium chloride at pH 7.0.

The following is a specific procedure by way of example:

the renatured protein solution of interest was centrifuged by ultrafiltration (5000g, 25min), concentrated to 600. mu.L, and eluted through size exclusion column Superdex 7510/300 increase (GE) in pH7.0 buffer containing 50mM sodium phosphate and 500mM sodium chloride.

And (4) generating a peak when the elution volume is 15-20 mL, collecting the target protein at the moment, and performing SDS-PAGE identification. FIG. 9 shows a peak diagram of 600uL of AKTA purifier in combination with Superdex 7510/300 analytical column, the first peak from left to right being the target protein; the SDS-PAGE patterns are 6 th, 5 th and 4 th from right to left, namely the target protein, the purity and the yield of the target protein accord with expectations, and the bactrian camel serum albumin is obtained by the step. The obtained albumin has good water solubility. The albumin has the advantages of high purity, high safety, high cost performance and the like, and is expected to become a substitute of the human serum albumin so as to meet the demand of patients on albumin.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Sequence listing

<110> Nanjing university of medical science

<120> preparation method of recombinant camel-derived serum albumin

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ttagatgccc tgaaagaacg cattctggca agtagcgccc gtcagcgtct gcgttgtacc 600

agcattcaga aatttggtga ccgcgccctg aaagcatgga gcgttggcca tctgagtcag 660

aaatttccga aagccgattt tgcagaaatt agtaaaatcg tgaccgatct gaccaaaatt 720

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gaaaaagatg ttttcctggg tatgtttctg catgaatatg cccgtcgtca tcctgaatat 1020

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Met Gly His His His His His His Met Ser Asp Ser Glu Val Asn Gln

1 5 10 15

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

20 25 30

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

35 40 45

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

50 55 60

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

65 70 75 80

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

85 90 95

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Asp Thr His Lys Ser Glu

100 105 110

Ile Ala His Arg Phe Lys Asp Leu Gly Glu Asp Asp Phe Lys Gly Leu

115 120 125

Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Asp

130 135 140

His Val Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val

145 150 155 160

Ala Asp Glu Ser Ala Ala Asp Cys Asp Lys Ser Leu His Thr Leu Phe

165 170 175

Gly Asp Lys Leu Cys Thr Val Ala Ser Leu Arg Glu Thr Tyr Gly Glu

180 185 190

Met Ala Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe

195 200 205

Leu Gln His Lys Ser Asp Asn Pro Asp Leu Pro Lys Leu Lys Pro Glu

210 215 220

Pro Glu Ala Leu Cys Thr Ala Phe Gln Glu Asn Glu Lys Arg Phe Gly

225 230 235 240

Gly Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala

245 250 255

Pro Glu Leu Leu Tyr Tyr Ala His Gln Tyr Lys His Val Phe Glu Glu

260 265 270

Cys Cys Lys Asp Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp

275 280 285

Ala Leu Lys Glu Arg Ile Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg

290 295 300

Cys Thr Ser Ile Gln Lys Phe Gly Asp Arg Ala Leu Lys Ala Trp Ser

305 310 315 320

Val Gly His Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Ile

325 330 335

Ser Lys Ile Val Thr Asp Leu Thr Lys Ile His Lys Glu Cys Cys Gln

340 345 350

Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr

355 360 365

Phe Cys Asp Asn Gln Glu Thr Ile Ser Ser Lys Leu Lys Glu Cys Cys

370 375 380

Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile His Glu Ala Glu Arg

385 390 395 400

Asp Glu Met Pro Glu Asn Leu Pro Ala Ile Thr Glu Gln Phe Ala Glu

405 410 415

Asp Lys Asp Val Cys Lys His Tyr Thr Glu Glu Lys Asp Val Phe Leu

420 425 430

Gly Met Phe Leu His Glu Tyr Ala Arg Arg His Pro Glu Tyr Ala Val

435 440 445

Ser Leu Leu Leu Arg Ile Ala Lys Glu Tyr Glu Ala Thr Leu Glu Asp

450 455 460

Cys Cys Ala Lys Asp Asp Pro His Ala Cys Tyr Ala Thr Val Phe Asp

465 470 475 480

Lys Leu Gln His Leu Ala Asp Glu Pro Gln Asn Leu Val Lys Gln Asn

485 490 495

Cys Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Asp Ile

500 505 510

Leu Val Arg Tyr Thr Lys Arg Leu Pro Gln Val Ser Thr Pro Thr Leu

515 520 525

Val Glu Val Ala Arg Gly Leu Gly Arg Val Gly Thr Lys Cys Cys Thr

530 535 540

Leu Pro Glu Ser Asn Arg Met Ser Cys Ala Glu Asp Tyr Leu Ser Leu

545 550 555 560

Ile Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Pro

565 570 575

Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys

580 585 590

Phe Ser Ser Leu Thr Ala Asp Glu Thr Tyr Glu Pro Lys Glu Phe Asp

595 600 605

Glu Lys Thr Phe Thr Phe His Ala Asp Leu Cys Ser Val Ser Glu Pro

610 615 620

Glu Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Leu Lys His

625 630 635 640

Lys Pro Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Glu Lys Phe

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Val Ala Phe Val Asp Lys Cys Cys Ala Ala Val Asp Lys Glu Ala Cys

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Phe Thr Val Glu Gly Pro Leu Leu Val Ala Ala Thr Arg Thr Ala Leu

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Ala

Claims (10)

1. A preparation method of recombinant camel serum albumin is characterized by comprising the following steps:

firstly, carrying out PCR amplification on a gene segment for coding recombinant camel source serum albumin, and then recombining the gene segment with a plasmid vector to construct a recombinant plasmid; the amino acid sequence of the recombinant camel source serum albumin is shown as SEQ ID No. 2;

secondly, adopting chemically competent cells prepared by using escherichia coli; transforming the recombinant plasmid into chemically competent cells, and culturing to obtain single colonies; carrying out liquid culture by taking a single colony as a seed, and then adding an inducer to induce and express a target protein to obtain the target protein mainly existing in an inclusion body form;

thirdly, centrifugally collecting precipitate after ultrasonically crushing thalli; then washing the precipitate with a washing buffer solution, and dissolving the precipitate with a dissolving buffer solution to obtain a dissolving solution containing the target protein; purifying the dissolved solution to obtain target protein; adopting a gradual gradient dialysis mode to refold the target protein into a physiological state, namely renaturation;

and fourthly, purifying the renatured target protein to obtain the recombinant camel source serum albumin.

2. The method for producing a recombinant camel serum albumin according to claim 1, wherein the recombinant camel serum albumin is produced by the method,

in the first step, the plasmid vector is pSmart-I; the enzyme cutting sites selected during the recombination construction are as follows: BamHI endonuclease site, and XhoI endonuclease site;

in the second step, the Escherichia coli is BL21(DE3) Escherichia coli strain; the inducer is IPTG;

in the third step, the specific process of washing with the washing buffer is as follows:

resuspending the precipitate with a first wash buffer, then centrifuging and collecting the precipitate, repeating for a predetermined number of times;

then resuspending the precipitate with a second washing buffer solution, then centrifugally collecting the precipitate, and repeating the steps for a preset number of times;

the first wash buffer is: a buffer containing 5 + -0.05 mM DTT, 50 + -0.5 mM Tris-HCl, pH 8.0 + -0.1 mM NaCl, 500 + -5 mM NaCl, 1 + -0.01% Triton X-100, 1 + -0.01 mM EDTA;

the second washing buffer solution is: a buffer containing 5 + -0.05 mM DTT, 50 + -0.5 mM Tris-HCl, pH 8.0 + -0.1 mM NaCl, 500 + -5 mM NaCl, 1 + -0.01 mM EDTA;

the specific process of dissolution with the dissolution buffer is as follows:

resuspending the precipitate with a lysis buffer solution, stirring for a predetermined time, and centrifuging to collect supernatant to obtain a lysis solution containing the target protein;

the lysis buffer is: a buffer solution containing 50 + -0.5 mM of Tris, pH 6.0-6.3, 8 + -0.08M of urea, 1 + -0.01 mM of EDTA and 5 + -0.05 mM of DTT;

in the fourth step, the renatured target protein is purified by using a gel size exclusion column.

3. The method for producing a recombinant camel serum albumin according to claim 2, wherein the recombinant camel serum albumin is produced by the method,

in the third step, the specific process of purifying the dissolving solution to obtain the target protein is as follows:

the dissolving solution is firstly subjected to crude extraction and purification of target protein by a nickel column affinity chromatography method, and then a band containing the target protein is identified, separated and collected by an SDS-PAGE method;

the specific process of gradual gradient dialysis is as follows:

respectively dialyzing for a preset time in a buffer solution R1, a buffer solution R2, a buffer solution R3, a buffer solution R4 and a buffer solution R5 in sequence, and then performing ultrafiltration concentration;

the buffer solution R1 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1 mM, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1% of glycerol, 1 plus or minus 0.01mM of DTT and 4 plus or minus 0.04mM of urea;

the buffer solution R2 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT and 2 plus or minus 0.02mM of urea;

the buffer solution R3 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT, 1 plus or minus 0.01mM of urea and 600 plus or minus 6mM of L-arginine;

the buffer solution R4 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1 percent of glycerol, 1 plus or minus 0.01mM of DTT, 0.5 plus or minus 0.005mM of urea and 600 plus or minus 6mM of L-arginine;

the buffer solution R5 is a buffer solution containing 50 plus or minus 0.5mM of Tris-HCl with the pH value of 8.0 plus or minus 0.1, 100 plus or minus 1mM of NaCl, 10 plus or minus 0.1% of glycerol, 1 plus or minus 0.01mM of DTT and 600 plus or minus 6mM of L-arginine.

4. The method for producing a recombinant camel serum albumin according to claim 3, wherein the recombinant camel serum albumin is produced by the method,

in the first step, the nucleotide sequence of the gene fragment is shown as SEQ ID No. 1;

in the second step, the specific process of transforming the recombinant plasmid into chemically competent cells is as follows: respectively placing the prefreezed chemical competent cells and the prefreezed recombinant plasmids on ice for thawing; then adding the recombinant plasmid into the chemically competent cells and uniformly mixing; firstly placing the mixture in an ice bath, then carrying out heat shock, and then placing the mixture in the ice bath for standing; adding LB broth culture medium without antibiotic to culture, and recovering thallus;

the specific process of obtaining single colony through culture is as follows: culturing the recovered thallus on an LB (lysogeny broth) flat plate containing kanamycin to obtain a white single colony;

the specific process of taking a single colony as a seed to carry out liquid culture and then adding an inducer comprises the following steps: picking single bacterial colony and inoculating the single bacterial colony in LB liquid culture medium containing kanamycin for recovery culture; inoculating the recovered bacterial liquid into a fresh LB liquid culture medium containing kanamycin, performing formal culture, and culturing to OD₆₀₀When the preset value is reached, adding an inducer and carrying out induction culture.

5. The method for producing a recombinant camel serum albumin according to claim 4, wherein the recombinant camel serum albumin is produced by the method,

second, in the specific process of transforming the recombinant plasmid into chemically competent cells: placing in ice bath for 30min, then performing heat shock at 42 deg.C for 45s, and then placing in ice bath for standing for 2 min; culturing at 37 deg.C and 220rpm for 60 min;

in the specific process of obtaining single colonies by culturing: the culture condition is that the inverted culture is carried out for 16h at 37 ℃;

in the specific process of taking a single colony as a seed to carry out liquid culture and then adding an inducer: resuscitating culture conditions at 37 deg.C and 220rpm, and shaking culture overnight; the formal culture conditions were 37 ℃ and 220 rpm; the final concentration of the inducer added was 1mM, and the induction culture was carried out at 15 ℃ and 220rpm for 16 hours.

6. The method for producing a recombinant camel serum albumin according to claim 3, wherein the recombinant camel serum albumin is produced by the method,

in the third step, the centrifugation conditions after the ultrasonic disruption of the thalli are 12500rpm and 4 ℃, and the centrifugation is carried out for 30 min;

during the specific course of washing with the wash buffer: centrifuging at 12500rpm and 4 deg.C for 30 min; the predetermined number of times is 3;

during the specific process of lysis with lysis buffer: the preset time is 1 h; centrifuging at 12500rpm and 4 deg.C for 15 min;

in the specific process of purifying the dissolving solution to obtain the target protein: the specific model of the nickel column is GE 5mL Histrap; the nickel column affinity chromatography adopts a binding buffer solution, a washing buffer solution and an elution buffer solution, wherein the binding buffer solution is a buffer solution which is pH7.4 and contains 20mM sodium phosphate, 500mM sodium chloride and 10mM imidazole, the washing buffer solution is a buffer solution which is pH7.4 and contains 20mM sodium phosphate, 500mM sodium chloride and 40mM imidazole, and the elution buffer solution is a buffer solution which is pH7.4 and contains 20mM sodium phosphate, 500mM sodium chloride and 250mM imidazole;

during the specific course of stepwise gradient dialysis: the dialysis temperature is 4 ℃ and the preset time is 8 h; centrifuging for 25min under the ultrafiltration concentration condition of 5000 g;

in the fourth step, the specific model of the gel size exclusion column is GE Superdex 7510/300 incrase; the elution buffer used was 50. + -. 0.5mM sodium phosphate, 500. + -. 5mM sodium chloride at pH 7.0.

7. The amino acid sequence of the recombinant camel source serum albumin is shown as SEQ ID No. 2.

8. A gene fragment encoding the recombinant camel-derived serum albumin of claim 7.

9. A recombinant plasmid having the gene fragment of claim 8.

10. An engineered bacterium comprising the recombinant plasmid of claim 9.

Images