CN118127056A

CN118127056A - Recombinant vector of fungal immunomodulatory protein and protein preparation method

Info

Publication number: CN118127056A
Application number: CN202410470791.8A
Authority: CN
Inventors: 李洪波; 李许; 张树琴; 张赛名
Original assignee: Hunan University of Medicine
Current assignee: Hunan University of Medicine
Filing date: 2024-04-18
Publication date: 2024-06-04

Abstract

The invention relates to a recombinant vector of fungal immunomodulatory protein, which comprises a target gene with a nucleotide sequence shown as SEQ ID No.1, or a sequence which has more than 90% homology with the nucleotide sequence shown as SEQ ID No.1 and codes the same biological functional protein; or a sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO.1 and encodes the same biologically functional protein. The recombinant vector is transformed into an escherichia coli expression strain, fusion of an expression product and a solubilizing factor Trx is realized, a large amount of soluble form of tuckahoe immunoregulatory protein FIP fusion protein is obtained, and the high-purity and high-activity tuckahoe immunoregulatory protein FIP is obtained through a simple affinity chromatography purification method, and has the activity of enhancing phagocytic activity of macrophages and inhibiting proliferation of tumor cells.

Description

Recombinant vector of fungal immunomodulatory protein and protein preparation method

Technical Field

The invention belongs to the technical field of biological genetic engineering, and relates to an immunoregulatory protein FIP gene of poria cocos, and a protein coded by the immunoregulatory protein FIP gene and application of the immunoregulatory protein FIP gene.

Background

The large edible fungi can be used as natural raw materials of immunomodulating and anticancer agents, have important edible and medicinal values, and are often used for treating human diseases in traditional eastern medical treatment. Several large fungi such as ganoderma lucidum have been used for several hundred years in China, japan, korea and other countries to treat various diseases. In vitro and in vivo tests, large fungi have shown immunomodulatory, antiviral and cholesterol lowering activities, which act by modulating the immune system. The active substances can be extracted from fruiting bodies, mycelia, spores and culture solution of large fungi. Biologically active compounds of the macro fungi mainly include polysaccharides, polysaccharide peptides, polysaccharide proteins, proteoglycans, proteins and the like. At present, about 350 medicinal fungus lectin is separated and purified in the whole world, and has great research value.

The fungus immunoregulatory protein (fungal immunomodulatory protein, FIP) is a kind of small molecular protein with biological activity function which is contained in edible and medicinal fungus fruiting bodies after lectin, has great similarity to immunoglobulin superfamily in structural function, has antitumor activity, antiallergic activity, inhibiting tumor growth, inducing cells to produce various cytokines such as interleukin, tumor necrosis factor, interferon and the like, can play a certain role in immunoregulation, and has good clinical application prospect and medicinal health care value. The proteins act on immune effector cells to excite the innate or acquired immune response of the body and induce the synthesis of biological effectors to perform the functions of immunoregulation and anti-tumor. FIPs is divided into a new family based on FIPs having similar amino acid sequence properties, spatial structure and identical immune response behavior. The FIPs family members exhibit varying degrees of immunomodulatory activity in both ex vivo and in vitro assays: FIPs can coagulate red blood cells, stimulate proliferation of human peripheral blood lymphocytes and mouse spleen cells, increase cytokine transcription, and have antitumor activity. At present, most of the fungus immunomodulators are found in ganoderma lucidum, ganoderma tsugae, microsporidianum, ganoderma sinensis, flammulina velutipes, straw mushroom, sweet ganoderma lucidum, red shell, ganoderma atrum and antrodia camphorata, namely LZ-8, FIP-gts, FIP-gmi, FIP-gja, FIP-fve, FIP-vvo, FIP-gsi, FIP-nha, FIP-gas and FIP-aca. Poria cocos FIP has also been recently and gradually studied.

Poria is a crude drug of the genuine and large quantities of China. Poria cocos is one of four raw materials called China because of its flat nature, bitter taste and non-toxicity, has the effects of excreting dampness and promoting diuresis, preventing cancer and resisting aging and enhancing organism immunity, and 65% of the clinical traditional Chinese medicine prescription contains Poria cocos, and is one of the most potential natural medicines in the current international medicine market. Meanwhile, the Chinese medicine is listed by the national health committee and the national Chinese medicine administration as a variety list of foods and medicines. The main component in the poria cocos is dietary fiber, and the proportion of the dietary fiber accounts for more than 80% of the dry weight of the poria cocos; and secondly, protein accounting for about 1.5% of the dry weight of the poria cocos. Purifying Poria dried sclerotium to obtain an immunomodulatory protein FIP. In vitro experiments show that the poria cocos protein FIP can stimulate RAW 264.7 macrophages to produce TNF-alpha and IL-1 beta, and can regulate and control the expression level of NF- κB related genes. Therefore, the fungal immunomodulatory protein FIP has important application and development values.

Natural FIPs is extracted from edible fungi, which is expensive, time-consuming and low in yield, and mg-grade protein can be extracted from each kilogram of mushrooms. Hsu et al in 1997 purified FIP-vvo from 3kg of volvariella volvacea fruiting bodies to obtain only 94mg of protein. A large amount of FIPs is difficult to obtain by a direct extraction method, and the clinical and medical application of FIPs is severely restricted. Therefore, research and establishment of a high-yield stable FIPs recombinant expression system become necessary choices. The rapid development of modern molecular biology and bioengineering technology provides a technical basis for obtaining a large number of recombinant immunoregulatory proteins by using engineering strains. In prokaryotic expression, the selection of the vector has direct influence on the expression of exogenous genes and the expression level. FIPs in the prior art is used for constructing an expression vector, then the expression vector is transformed into E.coli BL21 (DE 3) for expression, but all the obtained inclusion bodies are inactive, and only trace soluble proteins can be obtained from each liter of culture medium through dissolution, denaturation, renaturation and purification under proper conditions in vitro, so that the production amount is low; at present, a mode of modifying the DNA sequence of the poria cocos FIP and optimizing the expression and purification method of the poria cocos FIP to efficiently produce recombinant proteins does not exist, so that the application of the FIP in improving the cellular immunity of organisms is greatly influenced, and a method capable of reducing the cost, realizing mass production and having the biological activity of the poria cocos FIP is required to be developed.

Disclosure of Invention

In view of the above, the present invention is directed to a recombinant vector of fungal immunomodulatory protein and a method for preparing fungal immunomodulatory protein.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. a recombinant vector comprising an empty vector and a target gene inserted into the empty vector, wherein the target gene has a nucleotide sequence selected from the group consisting of a DNA fragment of one of the following nucleotide sequences:

1) The nucleotide sequence of SEQ ID NO.1 in the sequence table;

2) A nucleotide sequence which has more than 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the same biological functional protein;

3) A nucleotide sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO.1 and encodes the same biologically functional protein.

In the recombinant vector, the empty vector is a pET32 vector.

Further, the recombinant vector is obtained by inserting the gene shown in SEQ ID No.1 between BamHI and HindIII cleavage sites of the expression vector pET 32.

2. A recombinant bacterium transformed with any one of the above recombinant vectors.

3. A preparation method of a poria cocos immunoregulatory protein FIP comprises the following steps:

a. recombinant the gene shown in SEQ ID No.1 into a vector; then the strain is transformed into an escherichia coli strain to obtain an expression strain;

b. Culturing the expression strain in the step a in an LB liquid culture medium, adding 0.1-0.5 mM IPTG for induction, performing ultrasonic crushing after fermentation, and centrifuging to obtain supernatant to obtain the soluble recombinant poria immunoregulatory protein FIP.

In the further preparation method of the poria cocos immunoregulatory protein FIP, the vector is a pET32 vector.

Further, the E.coli is E.coli TOP10 strain.

The preparation method further comprises the following steps of: purifying the supernatant obtained in the step b) by using a nickel affinity chromatography column, balancing the chromatography column by using a balancing buffer solution, passing the supernatant through the column, pre-washing the column by using a pH 8.0 buffer solution containing 10-20 mM imidazole, and eluting the fusion protein by using a pH 8.0 buffer solution gradient containing 50-400 mM imidazole.

Further, the method comprises the steps of dialyzing under the condition that the pH value is 6.0-6.5, and ultrafiltering and concentrating the dialyzed substance.

Purified proteins prepared according to the methods of preparing proteins described above are also within the scope of the present invention.

4. The application of the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacterium in the preparation of immune products is provided.

5. The application of the protein obtained by the preparation method of the poria cocos immunoregulatory protein FIP in the preparation of immune products.

The invention has the beneficial effects that: the invention discloses a nucleotide sequence shown as SEQ ID NO.1 in a sequence table by modifying and testing a large number of nucleotide sequences, which can realize the fusion of an expression product and a solubilization factor Trx by utilizing a bioengineering means and obtain the soluble expression of a large number of Poria cocos immunomodulatory proteins FIP; further simply through affinity purification, the active protein with the purity higher than 95% and the concentration higher can be obtained. The poria cocos immunoregulatory protein FIP obtained by the invention can obviously activate macrophage RAW 264.7 to secrete and generate cytokines TNF-alpha, IL-1 beta, IL-6 and IL-12 under low concentration (10 ng/mL), and can stimulate TNF-alpha expression along with the increase of protein concentration, so that the recombinant FIP protein prepared by the invention has the activity of activating an immune system, and has wide market prospect in the application of preparing immune products.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 is a schematic diagram of pET32/FIP vector construction in an embodiment of the invention.

FIG. 2 is a SDS-PAGE detection result of target protein expressed by pET32/FIP recombinant vector in the embodiment of the invention.

FIG. 3 is a diagram showing SDS-PAGE detection of proteins before and after purification in the example of the present invention.

FIG. 4 is a diagram showing SDS-PAGE detection result of recombinant proteins after purification and concentration in the example of the present invention.

FIG. 5 is a diagram showing the result of SDS-PAGE detection of expressed total proteins in comparative example of the present invention.

FIG. 6 shows the ability of FIP protein to activate macrophages to secrete TNF- α in an embodiment of the invention.

FIG. 7 shows the increase of immunoglobulin content in serum by FIP protein in examples of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods for which specific conditions are not specified in the examples are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Materials, reagents and the like used in the examples are commercially available unless otherwise specified. In the examples, the percentages are mass percentages unless otherwise indicated. The quantitative tests in the following examples were all set up with three replicates, and the data are the mean or mean ± standard deviation of the three replicates.

The invention selects colibacillus expression bacteria, a vector amplification strain TOP10 and an expression vector pET32 which are all purchased from Invritrogen company in the United states.

The medium formulation used was as follows:

1) LB liquid medium: 10g of NaCl, 10g of peptone, 5g of yeast extract, 1L of distilled water, and performing high-pressure sterilization and room-temperature storage;

2) LB/Amp plate: 10g of NaCl, 10g of peptone, 5g of yeast extract, 1L of distilled water, 15g of agar powder, sterilizing under high pressure, cooling to below 70 ℃, adding 1mL of Ampicillin (AMPICILLIN) with the concentration of 100mg/mL, fully mixing, pouring into a plate, and preserving at 4 ℃ in a dark place;

3) LB/Amp Medium: 10g of NaCl, 10g of peptone, 5g of yeast extract, 1L of distilled water, sterilizing under high pressure, cooling to below 70 ℃, adding 1MLAMPICILLIN (100 mg/mL), fully mixing uniformly, and preserving at 4 ℃; LB liquid medium: 10g of NaCl, 10g of peptone, 5g of yeast extract, 1L of distilled water, and preserving at room temperature.

4) 50×TAE agarose gel electrophoresis buffer: 121g of Tris base, 28.6mL of glacial acetic acid, 50mL of 0.5mol/L EDTA (pH 8.0), adding distilled water to a volume of 500mL and preserving at room temperature;

5) 50mg/mL ampicillin preservative solution: ampicillin 0.5g, adding distilled water to dissolve and fix volume to 10mL, subpackaging, and preserving at-20deg.C;

6) 5 XSDS-PAGE loading buffer: 1M Tris-HCl (pH 6.8) 1.25mL,SDS 0.5g,BPB 25mg, glycerin 2.5mL, adding deionized water for dissolution, then fixing the volume to 5mL, subpackaging (about 500 mu L each part), then preserving at room temperature, and uniformly mixing by adding 25 mu L beta-mercaptoethanol into each part;

7) 5 XSDS-PAGE running buffer: 15.1g of Tris, 94g of glycine and 5.0g of SDS are added with about 800mL of deionized water, and after fully stirring and dissolving, the volume is fixed to 1L, and the mixture is preserved at room temperature;

8) Coomassie brilliant blue R-250 staining solution: coomassie brilliant blue R-250.25 g, adding 225mL of methanol, 46mL of glacial acetic acid and 225mL of deionized water, uniformly stirring, removing particulate matters by filter paper, and preserving at room temperature;

9) Coomassie brilliant blue decolorized solution: 50mL of glacial acetic acid, 150mL of methanol and 300mL of deionized water are mixed thoroughly and stored at room temperature.

Example 1

The embodiment provides an optimized poria cocos immunoregulatory protein FIP gene, and the specific nucleotide sequence is shown in SEQ ID NO:1, the amino acid sequence of the protein corresponding to the gene is shown as SEQ ID NO in a sequence table: 2. The optimized DNA is optimized and screened by referring to the characteristics of the E.coli expression system and the accumulated experience of the inventor on expressing the exogenous protein by using the E.coli expression system for a long time.

Connecting the optimized gene into an escherichia coli expression vector pET32 to obtain a recombinant vector, thermally converting the recombinant vector subjected to sequencing verification into competent cells of an escherichia coli expression strain, coating a corresponding resistant LB plate, culturing in a constant-temperature incubator at 37 ℃ for 12 hours, and screening transformants, wherein the construction of the pET32/FIP vector is shown in figure 1, and figure 1 is a schematic diagram of the construction of the pET32/FIP vector in the embodiment of the invention.

The method uses the pET32 recombinant vector of the optimized poria cocos immune regulation protein FIP gene sequence as an expression vector, the corresponding expression bacterial transformant is induced by 0.1-0.5mM IPTG at 18 ℃ to detect the expression of target protein, the result of SDS-PAGE of the bacterial total protein is shown as figure 2, the molecular weight of the poria cocos immune regulation protein FIP protein is about 25kDa, the pET32 vector is fused with a Trx fragment with the size of about 20kDa at the N-end of the target protein, the Trx-FIP size is about 45kDa, and the expressed target protein is shown as an arrow.

S1: optimizing genes, constructing prokaryotic expression vectors and transforming: the sequence set forth in SEQ ID NO:1, connecting the obtained product to a pUC universal vector to obtain pUC/FIP, carrying out double enzyme digestion on the pUC/FIP by utilizing BamH I and Hind III, and subcloning the obtained FIP fragment into an expression vector pET32 to obtain a recombinant expression vector pET32/FIP, wherein the main vector construction steps are as follows:

(1) The recombinant vector pUC/FIP was digested with BamH I and Hind III to obtain the desired fragment FIP, and the reaction system was as follows (the endonucleases and buffers used were purchased from Takara Co., ltd.):

(2) pET32 was digested with BamH I and Hind III to obtain a vector fragment, and the reaction system was as follows (endonuclease and buffer used were purchased from Dalian TAKARA):

(3) The target fragment and the vector fragment obtained in the steps (1) and (2) are recovered by using a DNA gel recovery kit, which is purchased from Dalian TAKARA company, and the specific operation is performed according to the kit instruction.

(4) The target fragment recovered in the step (3) and the vector are subjected to ligation reaction by using T4DNA ligase (purchased from Dalian TAKARA company), and the target gene is accurately inserted into the expression vector reading frame, wherein the reaction system is as follows:

transforming the recombinant vector pET32/FIP obtained in the above into an escherichia coli TOP10 strain, and extracting the recombinant vector pET32/FIP from TOP 10; transferring the recombinant vector pET32/FIP into a host cell escherichia coli expression strain by a heat shock method, and screening by an LB plate containing Amp resistance to obtain an escherichia coli expression strain transformant containing the recombinant vector pET 32/FIP.

S2: expression and extraction of soluble Poria immunoregulatory protein FIP fusion protein: culturing the escherichia coli expression strain transformant containing the recombinant vector pET32/FIP obtained in the step S1 in a liquid LB culture medium at 37 ℃ until the OD600 is 0.6, then respectively adding IPTG with the final concentration of 0, 0.1, 0.2 and 0.5mM, inducing for 24 hours at 18 ℃, collecting the thallus after induction, carrying out ultrasonic crushing with the crushing power of 300W, crushing for 2S, carrying out clearance 6S, recycling for 90 times, centrifuging, and taking the supernatant to obtain the recombinant soluble fusion protein poria immunoregulatory protein FIP, wherein the SDS-PAGE result under the denaturation and reduction conditions is shown in figure 2.

Example 2

The embodiment provides a method for preparing Poria cocos immunoregulatory protein FIP, which specifically comprises the following steps:

1) Purification of the fusion protein Poria immunoregulatory protein FIP: screening the step S1 to obtain a transformant of the escherichia coli expression strain containing the recombinant vector pET32/FIP, performing amplification culture (100 mL) on the transformant in a liquid LB culture medium at 37 ℃ until the OD ₆₀₀ is 0.6, inducing the transformant with 0.1mM IPTG for 20-24 hours at 18 ℃, collecting thalli of the expressed bacteria after the induction of the IPTG, suspending the thalli in 40mL of buffer A (containing 20mM Na ₂HPO₄, 200mM NaCl,10mM imidazole and 1mM protease inhibitor PMSF and pH 8.0), crushing the thalli by using an ultrasonic crusher, crushing the substrate with the crushing power of 300W, crushing the substrate for 2 seconds and the gap of 6 seconds, and circulating the substrate for 90 times; centrifuging 30000g of the crushed bacterial liquid for 15min at 4 ℃; adding the supernatant obtained by centrifugation into a nickel affinity chromatography column pre-balanced by a buffer solution A; after the protein purification column was rinsed with 100mL of buffer B (20 mM Na ₂HPO₄, 200mM NaCl,10mM imidazole pH 8.0), the protein was eluted by adding buffer C (20 mM Na ₂HPO₄, 200mM NaCl,pH 8.0) containing imidazole at concentrations of 50, 100, 200 and 400mM, respectively, and the 200mM imidazole eluted protein was Poria immunoregulatory protein FIP with a purity of 98% or more, and SDS-PAGE under denaturing and reducing conditions was as shown in FIG. 3.

S4: concentration of the fusion protein Poria immunoregulatory protein FIP: dialyzing the protein sample under 20mM NaH ₂PO₄ and citric acid buffer solution with pH of 7.0, and performing ultrafiltration concentration by using ultrafiltration tube with molecular weight cut-off of 3k Da to obtain high concentration recombinant Poria immunoregulatory protein FIP with purity of more than 95%, and performing SDS-PAGE under denaturing and reducing conditions to obtain the final product shown in figure 4. The concentration of the target protein was detected by using a gel scan in combination with Bradford method, and table 1 shows the results of the yield and purity of the soluble poria FIP fusion protein in the supernatant obtained after centrifugation through each purification step by collecting the cells after centrifugation and re-suspending the cells with 40mL of lysate followed by ultrasonication.

TABLE 1 protein purification results

The supernatant obtained in step S2 was added with SDS-PAGE sample buffer, and the soluble protein was analyzed. Soluble Trx-FIP fusion proteins can be obtained at 18℃at IPTG concentrations of 0.1-0.5 mM. To save the cost and shorten the production period, IPTG with the induction temperature of 18 ℃ and 0.1mM can be preferably adopted for induction expression.

Comparative example

Using the data obtained from the transcriptome, primers were designed, the target gene was amplified by RT-PCR, and after correct sequencing, the native FIP gene was digested with BamHI and HindIII, and ligated into the pET32 expression vector, which was also digested with BamHI and HindIII, with the sequence shown in SEQ ID No. 3. The recombinant vector is transformed into competent cells of an escherichia coli expression strain through heat shock, a corresponding resistant LB plate is coated, and the recombinant vector is cultured in a constant temperature incubator at 37 ℃ for 12 hours, and transformants are selected. Culturing an escherichia coli recombinant transformant containing a pre-optimized gene pET32/FIP vector in a liquid LB culture medium at 37 ℃ until the OD600 is 0.4, then respectively adding IPTG with the final concentration of 0.1mM, inducing for 24 hours at 18 ℃, performing ultrasonic crushing on the thallus collected after induction, crushing with the crushing power of 300W, crushing for 2s, forming a gap of 6s, circulating for 90 times, centrifuging, and taking supernatant to obtain the soluble recombinant FIP protein; in addition, 3 DNA sequences, SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, were designed and synthesized based on the codon preference of E.coli alone, and these 3 DNAs were ligated into pET32 expression vectors digested with BamHI and HindIII, respectively. The recombinant vector is transformed into competent cells of an escherichia coli expression strain through heat shock, a corresponding resistant LB plate is coated, and the recombinant vector is cultured in a constant temperature incubator at 37 ℃ for 12 hours, and transformants are selected. E.coli recombinant transformants containing the pET32/FIP vector of the pre-optimized gene were cultured in liquid LB medium at 37℃until OD600 was 0.4, then IPTG was added to a final concentration of 0.1mM, induction was carried out at 18℃for 24 hours, the cells collected after induction were sonicated, the disruption power was 300W, disruption was 2s, gap was 6s, and after 90 cycles, the supernatant was centrifuged, and the soluble recombinant FIP protein was not obtained either. The SDS-PAGE results of the native sequence and 3 gene sequences synthesized according to E.coli codon preference alone for soluble FIP proteins in the pET32 vector are shown in FIG. 5. The result of the comparative example shows that only the poria cocos immunoregulatory protein FIP gene provided by the invention can realize high-level soluble expression in escherichia coli, and the expression level of the soluble target protein of a natural sequence or other sequences is extremely low or not.

Example 3

Macrophages RAW 264.7 were subcultured in RPMI-1640 containing 10% FBS, and when the cell growth density reached about 80-90%, cells were adjusted to 1X 10 ⁵ cells/mL cell suspension by pancreatin digestion in RPMI-1640 medium, and inoculated into 96-well plates, 100. Mu.L each well was inoculated, and cultured in a 37℃carbon dioxide incubator for 36 hours. Adding recombinant Trx-FIP with final concentrations of 0, 1, 10, 50 and 100ng/mL into the 96-well plate, respectively making 3 repeated wells for each concentration, and culturing in a carbon dioxide incubator for 12 hours; ELISA detection kits detect TNF- α, IL-1β, IL-6 and IL-12 concentrations in cell culture supernatants. The results are shown in Table 2, and the results show that recombinant Trx-FIP has biological activity and can significantly activate macrophage RAW 264.7 to secrete cytokines TNF-alpha, IL-1 beta, IL-6 and IL-12 at low concentration (10 ng/mL) (the results are shown in Table 2). In addition, the detection of TNF-. Alpha.concentration in the supernatant of the cell culture broth stimulated with recombinant Trx-FIP at final concentrations of 0, 1, 10, 50 and 100ng/mL was performed using Western blotting with anti-TNF-. Alpha.antibody as primary antibody, and the results of the detection are shown in FIG. 6. Western blotting results also show that recombinant Trx-FIP can stimulate macrophages to secrete TNF-alpha, and the concentration relationship is positive.

TABLE 2 results of cytokine secretion

To further determine whether recombinant Poria Trx-FIP was biologically active at the animal level, trx-FIP was gavaged at doses of 1, 5 and 25mg/kg body weight for 2 weeks in 8 week old Kunming mice 1 time a day, and finally the change in immunoglobulin content in the blood of the mice was measured. The measurement results of immunoglobulin concentration showed that: the mice were lavaged at a dose of 1mg/kg body weight, and the average concentration of immunoglobulin in the blood of the mice after two weeks was 29g/L; the mice were gavaged at doses of 5mg/kg and 25mg/kg body weight, 1 time per day, and the average concentration of immunoglobulin in the blood of the mice was measured to be more than 33g/L after two weeks, whereas the concentration of immunoglobulin in the blood of the Kunming mice of the control group (gavage equal volume of PBS) was 19g/L, and it was found that Trx-FIP at a low concentration, the concentration of immunoglobulin in the body of the mice was significantly increased by gavage, and the results are shown in FIG. 7. The concentration of Trx-FIP in different gastric lavages does not affect the concentration of total serum proteins and albumin in mice of each experimental group. Animal experiment results further prove that the recombinant poria Trx-FIP has the activity of activating an immune system.

Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A recombinant vector comprising an empty vector and a target gene inserted into the empty vector, wherein the target gene has a nucleotide sequence comprising a DNA fragment of one of the following nucleotide sequences:

1) The nucleotide sequence of SEQ ID NO.1 in the sequence table;

2. The recombinant vector according to claim 1, wherein the empty vector is pET32 vector.

3. A recombinant bacterium, characterized in that: the recombinant bacterium is a recombinant bacterium transformed by the recombinant vector of claim 1 or 2.

4. The preparation method of the poria cocos immunoregulatory protein FIP is characterized by comprising the following steps:

5. The method for preparing the poria cocos immunoregulatory protein FIP according to claim 4, wherein the vector is pET32 vector.

6. The method for preparing the poria immunoregulatory protein FIP according to claim 4, wherein the escherichia coli is escherichia coli TOP10 strain.

7. The method of claim 4, further comprising the step of purifying the protein: purifying the supernatant obtained in the step b) by using a nickel affinity chromatography column, balancing the chromatography column by using a balancing buffer solution, passing the supernatant through the column, pre-washing the column by using a pH 8.0 buffer solution containing 10-20 mM imidazole, and eluting the fusion protein by using a pH 8.0 buffer solution gradient containing 50-400 mM imidazole.

8. A protein obtainable by a process according to any one of claims 4 to 7 and the use of the protein in the preparation of an immune product.