CN111961682A - Preparation method and application of recombinant protein HSP70_1-8 of Sporothrix globosum - Google Patents

Abstract

The invention discloses a preparation method and application of recombinant protein HSP70_1-8 of Sporothrix globosum, which comprises the steps of extracting total RNA of Sporothrix globosum, obtaining cDNA by reverse transcription PCR, obtaining the full-length sequence of the heat shock protein 70(HSP70) isomer 1-8 gene of Sporothrix globosum by using specific primer amplification, carrying out in-vitro recombination of gene segments by using a molecular biology method, preparing recombinant protein corresponding to the gene segments, applying the recombinant protein to rabbits to prepare corresponding antibody, wherein the antibody can be used for preparing products for quickly diagnosing the early stage of Sporothrix globosum infection. The invention constructs a recombinant protein prokaryotic expression system of Sporothrix sphaericus HSP70_1-8, and can express a large amount of proteins rapidly through induction; the purification is completed by one step by using a Ni affinity chromatographic column, and the purification process is simple and quick; the immune animal prepares high-titer polyclonal antibody, and the prepared antibody and the target protein have good reaction specificity.

Description

Preparation method and application of recombinant protein HSP70_1-8 of Sporothrix globosum

Technical Field

The present invention relates to the field of recombinant protein preparation. More specifically, the invention relates to a preparation method and application of recombinant protein HSP70_1-8 of Sporothrix globosum.

Background

Sporotrichosis (sportotrichosis) is the most common deep mycosis caused by infection with the diphase dark fungus schenkii sensu lato (sporothix schenckii sensu lato, hereinafter referred to as Ss). Ss. are mycelial phases in the environment, parasitic or saprophytic to plants or soil; after infecting the body, the focus is the yeast phase. Ss. infection can lead to chronic infection which is difficult to heal, and not only affects the skin, but also infects organs via lymphatic vessels, causing disseminated and systemic fatal infection. The three provinces in northeast China are one of the most serious areas with the global epidemic of the disease.

Early on, schencorhii was considered to be a single species, but with the progress of the research, Marimon et al identified Ss. as a complex of 6 species, including schencorhii (s.schenckii sensu stricto), sphaericella (s.globosa), brazilian sporothrix (s.brasiliensis), ruellinia (s.lurei, formly narrow s.var.lurei), sporothrix mexicana (s.mexicana), and globisporus xanthii (s.pallida, synonym s.albicans), wherein sporothrix globosa (in a global distribution), schencorhii (in principally the united states, argentina, brazil, peru et al america countries), and brazilian sporothrix (principally brazilian distribution) were common pathogenic bacteria. As for strains popular in China, most of genotyping researches find that the strains are basically Sporothrix globosum.

The diagnosis of the disease at present mainly depends on the combined application of pathological examination of skin tissues and culture of fungi. The pathological examination needs a plurality of time-consuming steps of specimen fixing, embedding, sheet making, dyeing and the like, a patient needs to wait for 5-7 days to obtain a result, however, if a pathological section does not show typical pathological changes such as a star body, a positive spore and the like, and the disease cannot be diagnosed, the diagnosis is carried out by combined fungus culture. The fungus culture also needs to take lesion tissues as samples, the culture result needs to wait for 7-14 days, the time consumption is longer, meanwhile, the fungus growth is greatly influenced by the environment, and when the environmental temperature, the humidity and the like are not suitable, the fungus growth is not easy to grow, so the positive rate cannot reach the ideal degree required by people. In addition, the two methods are invasive examination, scars are left after postoperative wound healing, and the method is not an ideal detection means for children patients and patients with facial infection. As described above, both the fungus culture and the pathological diagnosis take a long time, and medical institutions need to have high experimental conditions and professional technicians, which cannot be performed in primary hospitals, and can only treat the diseases by the clinical experience of doctors. The present research shows that after sporothrix infects human body or animal, its antigen can stimulate body to produce antibody, and this kind of antigen-antibody reaction can be used as the basis for developing in vitro diagnosis reagent.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a method for preparing recombinant protein of Sporothrix globosum HSP70_1-8 and use thereof. The invention aims to extract the total RNA of the Sporothrix globosum, obtain cDNA by reverse transcription PCR, obtain the full-length sequence of the 1-8 gene of the heat shock protein 70(HSP70) isomer of the Sporothrix globosum by using specific primer amplification, carry out in-vitro recombination of gene fragments by using a molecular biology method and prepare recombinant protein corresponding to the gene fragments, and apply the recombinant protein to rabbits to prepare corresponding antibodies which can be used for preparing products for quickly diagnosing the early infection of the Sporothrix globosum.

To achieve these objects and other advantages in accordance with the present invention, there is provided a method for preparing recombinant protein of Sporothrix globosum HSP70_1-8, which comprises the steps of:

s1: artificially synthesizing a gene sequence HSP70_1-8 of the Sporothrix globosum, and introducing an Nde I enzyme cutting site, a Hind III enzyme cutting site, a His label and a terminator into the gene sequence;

s2: verifying an artificially synthesized Sporothrix globosum HSP70_1-8 gene sequence in S1 to judge whether the synthesized gene sequence is an expected sequence;

s3: performing a connection reaction on the gene sequence HSP70_1-8 of the Sporothrix globosum and a pET-30a plasmid to construct an expression plasmid;

s4: amplifying the expression plasmid;

s5: carrying out induction expression on the plasmid amplified by the S4;

s6: purifying the product after the induced expression of S5 to obtain recombinant protein HSP70_1-8 of Sporothrix.

Preferably, the preparation method of the recombinant protein of Sporothrix sphaericus HSP70_1-8 comprises the following steps of:

using pUC57 plasmid as vector to prepare recombinant plasmid pUC57/HSP70_ 1-8;

on Amp (adenosine phosphate) LB medium agar plate picking plasmid pUC57/HSP70_1-8 containing DH5 alpha single colony, inoculated in Amp LB liquid medium, at 37 degrees C under 200rpm culture overnight; extracting plasmids according to the instruction of the whole gold plasmid extraction kit; adding TE buffer solution with the pH value of 8.0 to dissolve the precipitate; measuring an absorbance A value by using an ultraviolet spectrophotometer, and calculating the concentration of the plasmid; the obtained plasmid is subjected to double enzyme digestion, and the double enzyme digestion system is as follows:

carrying out enzyme digestion for 3h at 30 ℃, carrying out agarose gel electrophoresis identification, and recovering DNA fragments by using a full-scale gold gel recovery kit;

designing a primer according to an HSP70_1-8 gene sequence of Sporothrix globosum, wherein an upstream primer is 5'-CATATGGCACCCGCTGTAGGGA-3'; the downstream primer is 5'-AAGCTTTCATTAGTGGTGGTGGTG-3'; and amplifying the DNA fragment, sequencing the amplified product and determining whether the artificially synthesized Sporothrix globisporus HSP70_1-8 gene sequence is an expected sequence.

Preferably, the preparation method of the recombinant protein HSP70_1-8 of Sporothrix globosum is characterized in that the ligation reaction in S3 specifically comprises the following steps:

the pET-30a plasmid is subjected to Nde I and Hind III double digestion to recover a vector fragment, and then is subjected to a ligation reaction with the HSP70_1-8 gene sequence of Sporothrix globisporus under the following conditions:

adding water to a final volume of 25u1, mixing, reacting at 16 deg.C overnight, adding sodium acetate and cold anhydrous ethanol, and standing at-20 deg.C for 60 min; and (4) centrifuging, collecting the precipitate, precipitating with 70% cold ethanol, drying in vacuum, and dissolving in TE buffer solution to obtain the expression plasmid.

Preferably, the preparation method of the recombinant protein HSP70_1-8 of sporotrichosis sphaerica is that in S4, the expression plasmid is amplified, specifically:

picking a single colony of Escherichia coli E.coli DH5 alpha from an LB plate cultured for 20h at 37 ℃, transferring the single colony into a flask, and performing shake culture for 3h at 37 ℃ and 220 rpm;

then transferred to a centrifuge tube pre-cooled with ice and placed on ice until the culture is cooled to 0 ℃;

centrifuging at 4 deg.C and 4000rpm for 10min, and recovering cells;

CaCl precooled with ice₂Resuspending the pellet and placing on ice; adding the expression plasmid obtained in S3 into the above cells, stirring, and ice-cooling for 30 min; water bath at 42 deg.C for 90s, and placing on ice bath for 1 min; adding LB liquid culture medium, and performing shaking culture at 37 deg.C and 200rpm for 45 min; coating the culture solution on LB solid culture medium containing 100 mug/mL penicillin, and culturing at 37 ℃;

picking single colony to inoculate LB liquid culture medium containing 100 ug/mL penicillin, shaking at 37 deg.C and 200rpm overnight; extracting plasmid and carrying out double enzyme digestion identification to obtain recombinant plasmid.

Preferably, the preparation method of the recombinant protein HSP70_1-8 of Sporothrix globosum is characterized in that in S5, the plasmid amplified by S4 is induced to express, and specifically comprises the following steps:

transforming the recombinant plasmid identified by double enzyme digestion into a bacillus coli BL21 strain to prepare a host bacterium BL21 with the recombinant plasmid;

picking single colony, inoculating to 2 XYT liquid culture medium containing Amp with concentration of 100 μ g/mL, and culturing at 200rpm and 37 deg.C until OD600 is 0.6; then inoculating host bacteria BL21 with recombinant plasmids into a 2 XYT culture medium according to the concentration of 1 percent, wherein the Amp concentration is 100 mu g/mL; the culture was expanded to 1L as described above, and the cells were shake-cultured at 37 ℃ until the OD600 of the cells was 1.0, followed by IPTG to a final concentration of 0.8 mM.

Preferably, the preparation method of the recombinant protein HSP70_1-8 of sporotrichosis sphaerica is that in S6, a product after induced expression of S5 is purified, and specifically, the method comprises the following steps:

centrifuging the product after induction expression, collecting thalli, and adding 25 mu L of deionized water into each milliliter of culture to resuspend cells;

adding lysozyme into the cell suspension to a final concentration of 100 mug/mL, and standing at room temperature for 15 min;

adding pre-cooled 2 XPBS according to the proportion of 25 microliter per milliliter of culture, then carrying out ultrasonic crushing, adding 20 percent Triton X-100 to 1 percent of final concentration after crushing, mixing uniformly, and standing for 30 min; centrifuging at 12000rpm for 10min, and collecting supernatant;

and separating and purifying by using a Ni-Sepharose affinity chromatography column to obtain the recombinant protein HSP70_1-8 of the Sporothrix globisporus.

Use of a recombinant protein of Sporothrix sphaericus HSP70_1-8 as defined in any of the above in the preparation of antibodies.

The invention at least comprises the following beneficial effects:

1. according to the scheme, a Sporothrix globosum HSP70_1-8 protein prokaryotic expression system is constructed, and can be quickly expressed in large quantities through induction;

2. the fusion protein has a His label, can be purified by one step by using a Ni affinity chromatographic column, and has simple and rapid purification process;

3. the immune animal prepares high-titer polyclonal antibody, and the prepared antibody and the target protein have good reaction specificity.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a schematic diagram of plasmid construction.

FIG. 2 is a SDS-PAGE result of the expression and purification of recombinant protein HSP70_1-8 of Sporothrix globosum.

FIG. 3 is a result chart of Western blot expressed and purified by recombinant protein of Sporothrix globisporus HSP70_ 1-8.

FIG. 4 is a diagram of Western blot detection results of recombinant protein polyclonal antibodies of Sporothrix sphaericus HSP70_ 1-8.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1

First, obtaining the target Gene

According to the gene sequence of the Sporothrix sphaericus HSP70_1-8, entrusting a gene synthesis company (King Shi) to artificially synthesize the gene sequence of the Sporothrix sphaericus HSP70_1-8, and introducing a His label and a terminator at the downstream of the gene sequence; nde I restriction site-ATG-Heat shock 70kDa protein 1-8Sporothrix globosa pro-His tag-terminator (TAATGA) -Hind III restriction site are respectively introduced at the upstream and downstream; the resultant recombinant plasmid was designated pUC57/HSP70_ 1-8.

The artificially synthesized Sporothrix globisporus HSP70_1-8 gene sequence is verified:

a single colony of DH 5. alpha. containing plasmid pUC57/HSP 70-1-8 was picked up from an agar plate of LB (Amp 100. mu.g/mL), inoculated into a 5m LB (Amp 100. mu.g/mL) liquid medium, and cultured overnight at 37 ℃ at 200 rpm. Plasmids were extracted according to the instructions of the full-scale gold plasmid extraction kit. The precipitate was dissolved by adding 30. mu.L of TE (pH 8.0). And (4) measuring an absorbance A value by using an ultraviolet spectrophotometer, and calculating the plasmid concentration. The plasmid obtained was subjected to a double digestion (Nde I and Hind III) as follows:

and (3) carrying out enzyme digestion for 3h at 30 ℃, carrying out agarose gel electrophoresis identification, and recovering the DNA fragment by using a full-scale gold gel recovery kit for later use.

Designing a primer (an upstream primer 5'-CATATGGCACCCGCTGTAGGGA-3' and a downstream primer 5'-AAGCTTTCATTAGTGGTGGTGGTG-3') according to the HSP70_1-8 gene of the Sporothrix globiformis, amplifying a part of DNA fragments, sequencing the amplified product, determining that the artificially synthesized gene sequence is the heat shock protein 70 gene fragment of the Sporothrix globiformis, and later-stage standby.

Second, construction of expression plasmid

The expression plasmid pET-30a (+) recovers the heat shock protein 70 gene fragment by Nde I and Hind III double digestion (the reaction conditions are the same as above), and the heat shock protein 70 gene fragment is subjected to a ligation reaction with HSP70_1-8 gene, wherein the conditions are as follows:

water was added to a final volume of 25u1, the tube bottom was flicked and mixed, and the mixture was reacted at 16 ℃ overnight, and 2.5. mu.L (1/10) of 3M sodium acetate (pH5.2) and 2.5 times the volume of cold absolute ethanol were added thereto at the end of the reaction and left at-20 ℃ for 60 min. And centrifuging, collecting the precipitate, precipitating with 70% cold ethanol, vacuum drying, and dissolving with 25 μ L of TE.

Amplification of expression plasmids

E.coli DH 5. alpha. single colonies were picked from LB plates cultured at 37 ℃ for 20 hours, transferred to a 1L flask containing 100mL, and shake-cultured at 37 ℃ and 220rpm for 3 hours. Transfer to a 50mL centrifuge tube pre-cooled with ice and place on ice for 10min until the culture is cooled to 0 ℃. The cells were recovered by centrifugation at 4000rpm for 10min at 4 ℃. 0.1mol/L CaCl precooled with 10mL of ice₂Resuspend the pellet and place on ice. Add 200. mu.L of the above competent cells to 4. mu.L of the ligation product, stir gently and mix well, ice-wash for 30 min. Water bath at 42 deg.C for 90s, and rapidly placing on ice bath for 1 min. Adding 800 μ L liquid LB medium, shaking culture at 37 deg.C and 200rpmAnd culturing for 45 min. 200. mu.L of the culture broth was applied to LB solid medium (containing 100. mu.g/mL of penicillin), and cultured at 37 ℃.

Individual colonies were randomly picked and inoculated into 3mL LB liquid medium (containing 100. mu.g/mL penicillin) and shaken overnight at 200rpm at 37 ℃. Plasmids were extracted and identified by double digestion as described above.

Fourth, induced expression of recombinant strains

The plasmid identified by double enzyme digestion is transformed into Escherichia coli BL21 strain by the same method. Competent cells were detected under the same conditions with the empty plasmid pET-30a (+) as a control.

Single colonies were picked from the plate, inoculated into 2 XYT liquid medium containing 100mL of Amp at a concentration of 100. mu.g/mL, and cultured at 200rpm at 37 ℃ until the OD600 was about 0.6. Then, 3mL of 2 XYT medium was inoculated with the recombinant plasmid-carrying host bacterium BL21 at a concentration of 1%, and the Amp concentration was 100. mu.g/mL. The scale-up culture was carried out as described above to 1L. The cells were cultured with shaking at 37 ℃ until the OD600 of the cells was about 1.0, and IPTG was added to the cells to a final concentration of 0.8 mM.

Fifthly, purification of expression product

The culture after induction expression was centrifuged, the cells were collected, and the cells were resuspended in a ratio of 25. mu.L of deionized water per ml of culture. Lysozyme was added to the cell suspension to a final concentration of 100. mu.g/mL and left at room temperature for 15 min. Then 25. mu.L of pre-cooled 2 XPBS per ml of culture was added. And selecting a proper ultrasonic probe according to the crushing volume to perform ultrasonic crushing. Crushing, adding 20% Triton X-100 to 1% final concentration, mixing, and standing for 30 min. Centrifuging at 12000rpm for 10min, and collecting supernatant.

And separating and purifying the target protein by using a Ni-Sepharose affinity chromatography column, and collecting the eluent. The collected recombinant protein of Sporothrix sphaericus HSP70_1-8 was observed by SDS-PAGE and Western blot method. Referring specifically to fig. 2 and 3, in fig. 2, M1 is protein Marker; lane 1 bovine serum albumin (5.00. mu.g); lane 2: recombinant protein of Sporothrix sphaericus HSP70_1-8 (5.00. mu.g); in FIG. 3, M2 is protein Marker; lane 3, recombinant protein of Sporothrix asphericium HSP70_ 1-8; as can be seen from FIGS. 2 and 3, a single protein band was observed around 70kd, indicating that HSP70_1-8 recombinant protein could be successfully expressed by the above method and that the obtained protein was a single component.

The sequence of the recombinant protein HSP70_1-8 of the Sporothrix globisporus is as follows:

MAPAVGIDLGTTYSCVGIYRDDRIEIIANDQGNRTTPSFVAFTDTERLIGDAAKNQVAMNPQNTVFDAKRLIGRKFADPEVQADMKHFPFKVADRGGKPVIEVEFKGENKQFTPEEISSMVLIKMRETAESYLGGTINNAVVTVPAYFNDSQRQATKDAGLIAGLNVLRIINEPTAAAIAYGLDKKVEGERNVLIFDLGGGTFDVSLLTIEEGIFEVKSTAGDTHLGGEDFDNRLVNHFVSEFKRKNRKDVSTNARALRRLRTACERAKRTLSSSAQTSIEIDSLFEGIDFYTSITRARFEELCQDLFRSTLQPVDRVLTDAKIDKSQVHEIVLVGGSTRIPRIQKLITDYFNGKEPNKSINPDEAVAYGAAVQAAILSGDTSSKSTNEILLLDVAPLSLGIETAGGMMTKLIPRNTTIPTKKSEVFSTFSDNQPGVLIQVFEGERQRTKDNNLMGKFELTGIPPAPRGVPQIEVTFDLDANGIMNVSAVEKGTGKSNQIVITNDKGRLSKDDIERMLAEAEKFKEEDEAEGRRVAAKNGLESYAYSLRNTLSDSKVDEKLDAADKEKLKGEIDRVVSWLDESQQATREEYEEHQKELEAVANPIMMKFYGSGGPPGGAPDGAPGGFPGAAGGATHDDGPTVEEVDHHHHHH。

sixthly, obtaining animal immune serum and antibody

Mixing recombinant expressed Sporothrix globosum HSP70_1-8 recombinant protein and Freund's complete adjuvant in equal volume, emulsifying, performing subcutaneous multipoint injection on New Zealand rabbits, and immunizing again with Freund's incomplete adjuvant; after the third immunization, ear artery blood was taken every other week to determine the antiserum titer, and when the immunization titer did not increase any more, carotid artery was bled and serum was separated.

The antiserum is salted out by 50 percent and 33 percent saturated ammonium sulfate at one time, and then is subjected to agarose gel Protein A affinity chromatography to obtain a high-purity polyclonal antibody, and finally, the polyclonal antibody is identified by SDS-PAGE gel electrophoresis.

Seventhly, determination of immunogenicity and antibody titer of target protein

Antibody titers were determined by ELISA, by first dissolving the protein of interest in PBS (pH7.4), coating it in 96-well plates at a concentration of 4. mu.g/ml, 100. mu.L per well, and incubating overnight. The next day, purified antibody (antibody concentration: 1000ng/mL-1.95ng/mL) diluted in two times was added, respectively. Non-immune rabbit sera served as negative controls. The Secondary Antibody is Anti-Rabbit IgG Fc Monoclonal Secondary Antibody. The color was developed by HRP, and after the experiment was completed, OD450 was measured. Table 1 shows the results of ELISA detection of polyclonal antibody titer of recombinant protein HSP70_1-8 of Sporothrix globosum. As can be seen from Table 1, when the dilution factor of the antibody reaches 1:512000, the antigen can still be detected, indicating that the specific antibody of the HSP70_1-8 recombinant protein has higher titer.

TABLE 1

And (3) detecting the immunogenicity of the target protein by using a Western blot method. SDS-PAGE was performed on the target protein at final contents of 100ng, 50ng and 10ng, respectively. After the electrophoresis, the purified antibody was incubated at a concentration of 1ug/mL, and goat anti-rabbit IgG antibody was used as a secondary antibody. Referring to fig. 4, in fig. 4, lanes 1,4, 5: 100ng of recombinant protein HSP70_1-8 of Sporothrix globosum; lane 2: 50ng of recombinant protein HSP70_1-8 of Sporothrix globosum; lane 3: 10ng of recombinant protein HSP70_1-8 of Sporothrix globosum; lanes 1-3: the purified antibody is used as a primary antibody; lane 4: (ii) a non-immune serum; lane 5: PBS blank control. As can be seen from FIG. 4, the optical density decreased with the decrease of the concentration of HSP70_1-8 recombinant protein, and the antibody has a linear relationship with HSP70_1-8 recombinant protein, indicating that HSP70_1-8 recombinant protein has immunogenicity.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

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Claims (7)

1. A preparation method of recombinant protein HSP70_1-8 of Sporothrix globosum is characterized by comprising the following steps:

s1: artificially synthesizing a gene sequence HSP70_1-8 of the Sporothrix globosum, and introducing an Nde I enzyme cutting site, a Hind III enzyme cutting site, a His label and a terminator into the gene sequence;

s2: verifying an artificially synthesized Sporothrix globosum HSP70_1-8 gene sequence in S1 to judge whether the synthesized gene sequence is an expected sequence;

s3: performing a connection reaction on the gene sequence HSP70_1-8 of the Sporothrix globosum and a pET-30a plasmid to construct an expression plasmid;

s4: amplifying the expression plasmid;

s5: carrying out induction expression on the plasmid amplified by the S4;

s6: purifying the product after the induced expression of S5 to obtain recombinant protein HSP70_1-8 of Sporothrix.

2. The method for producing recombinant protein of Sporothrix sphaericus HSP70_1-8 in claim 1, wherein the method for verifying the artificially synthesized gene sequence of Sporothrix sphaericus HSP70_1-8 in S2 is:

using pUC57 plasmid as vector to prepare recombinant plasmid pUC57/HSP70_ 1-8;

picking single colony of DH5 alpha containing plasmid pUC57/HSP70_1-8 on agar plate of LB culture medium containing Amp, inoculating to LB liquid culture medium containing Amp, and culturing overnight at 37 deg.C and 200 rpm; extracting plasmids according to the instruction of the whole gold plasmid extraction kit; adding TE buffer solution with the pH value of 8.0 to dissolve the precipitate; measuring an absorbance A value by using an ultraviolet spectrophotometer, and calculating the concentration of the plasmid; the obtained plasmid is subjected to double enzyme digestion, and the double enzyme digestion system is as follows:

carrying out enzyme digestion for 3h at 30 ℃, carrying out agarose gel electrophoresis identification, and recovering DNA fragments by using a full-scale gold gel recovery kit;

designing a primer according to an HSP70_1-8 gene sequence of Sporothrix globosum, wherein an upstream primer is 5'-CATATGGCACCCGCTGTAGGGA-3'; the downstream primer is 5'-AAGCTTTCATTAGTGGTGGTGGTG-3'; and amplifying the DNA fragment, sequencing the amplified product and determining whether the artificially synthesized Sporothrix globisporus HSP70_1-8 gene sequence is an expected sequence.

3. The method for preparing recombinant protein HSP70_1-8 of Sporothrix globosum in claim 1, wherein the ligation reaction in S3 specifically comprises:

the pET-30a plasmid is subjected to Nde I and Hind III double digestion to recover a vector fragment, and then is subjected to a ligation reaction with the HSP70_1-8 gene sequence of Sporothrix globisporus under the following conditions:

adding water to a final volume of 25u1, mixing, reacting at 16 deg.C overnight, adding sodium acetate and cold anhydrous ethanol, and standing at-20 deg.C for 60 min; and (4) centrifuging, collecting the precipitate, precipitating with 70% cold ethanol, drying in vacuum, and dissolving in TE buffer solution to obtain the expression plasmid.

4. The method for preparing recombinant protein HSP70_1-8 from sporotrichosis sphaerica according to claim 1, wherein in S4 the expression plasmid is amplified, specifically:

picking a single colony of Escherichia coli E.coli DH5 alpha from an LB plate cultured for 20h at 37 ℃, transferring the single colony into a flask, and performing shake culture for 3h at 37 ℃ and 220 rpm;

then transferred to a centrifuge tube pre-cooled with ice and placed on ice until the culture is cooled to 0 ℃;

centrifuging at 4 deg.C and 4000rpm for 10min, and recovering cells;

CaCl precooled with ice₂Resuspending the pellet and placing on ice; adding the expression plasmid obtained in S3 into the above cells, stirring, and ice-cooling for 30 min; water bath at 42 deg.C for 90s, and placing on ice bath for 1 min; adding LB liquid culture medium, and performing shaking culture at 37 deg.C and 200rpm for 45 min; coating the culture solution on LB solid culture medium containing 100 mug/mL penicillin, and culturing at 37 ℃;

picking single colony to inoculate LB liquid culture medium containing 100 ug/mL penicillin, shaking at 37 deg.C and 200rpm overnight; extracting plasmid and carrying out double enzyme digestion identification to obtain recombinant plasmid.

5. The method for preparing recombinant protein HSP70_1-8 of claim 4 wherein in S5 the plasmid amplified in S4 is induced to express, specifically:

transforming the recombinant plasmid identified by double enzyme digestion into a bacillus coli BL21 strain to prepare a host bacterium BL21 with the recombinant plasmid;

picking single colony, inoculating to 2 XYT liquid culture medium containing Amp with concentration of 100 μ g/mL, and culturing at 200rpm and 37 deg.C until OD600 is 0.6; then inoculating host bacteria BL21 with recombinant plasmids into a 2 XYT culture medium according to the concentration of 1 percent, wherein the Amp concentration is 100 mu g/mL; the culture was expanded to 1L as described above, and the cells were shake-cultured at 37 ℃ until the OD600 of the cells was 1.0, followed by IPTG to a final concentration of 0.8 mM.

6. The method for preparing recombinant protein HSP70_1-8 from sporotrichosis sphaerica as claimed in claim 1, wherein in S6, the product after induced expression of S5 is purified, specifically:

centrifuging the product after induction expression, collecting thalli, and adding 25 mu L of deionized water into each milliliter of culture to resuspend cells;

adding lysozyme into the cell suspension to a final concentration of 100 mug/mL, and standing at room temperature for 15 min;

adding pre-cooled 2 XPBS according to the proportion of 25 microliter per milliliter of culture, then carrying out ultrasonic crushing, adding 20 percent Triton X-100 to 1 percent of final concentration after crushing, mixing uniformly, and standing for 30 min; centrifuging at 12000rpm for 10min, and collecting supernatant;

and separating and purifying by using a Ni-Sepharose affinity chromatography column to obtain the recombinant protein HSP70_1-8 of the Sporothrix globisporus.

7. Use of the recombinant protein of Sporothrix sphaericus HSP70_1-8 of any one of claims 1-6 for the preparation of antibodies.

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