Method for prokaryotic expression of cat allergen Fel d 1 recombinant protein

Abstract

The invention provides a method for prokaryotically expressing cat allergen Fel d 1 recombinant protein, which comprises the following steps of: cloning a cat allergen Fel d 1 gene to multiple cloning sites of an expression vector pGEX-4T-1 to obtain a recombinant expression vector pGEX-4T-1-Fel d 1, transforming the recombinant expression vector into escherichia coli Rosetta (DE3) competent cells to construct recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), and carrying out prokaryotic expression on the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) to obtain the cat allergen Fel d 1 recombinant protein. IPTG (isopropyl-beta-d-thiogalactoside) is used for inducing protein expression, and a GST affinity chromatography medium is used for capturing high-purity soluble GST-Fel d 1 fusion protein. According to the invention, a pGEX-4T-1-GST label system is combined with Escherichia coli Rosetta (DE3), so that the problem of soluble expression of the cat allergen Fel d 1 protein is fundamentally solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a method for prokaryotic expression of cat allergen Fel d 1 recombinant protein. BACKGROUND

[0002] Cat allergen Fel d 1 is the main allergen protein causing human allergy to cats, accounting for about 95% of cat allergic patients. It has great application value in allergen-specific diagnosis (such as skin prick test, IgE detection) and immunotherapy (desensitization vaccine). At present, the commercial source of Fel d 1 is mainly natural extraction from cat dander or saliva, but this method has problems such as low yield, high cost, difficult to control purity, easy to be affected by batch difference and potential pathogen pollution, etc.

[0003] Recombinant expression of Fel d 1 protein in prokaryotic system (such as E. coli) by genetic engineering is an ideal way to solve the above problems. However, due to its specific disulfide bond structure and complex conformation, Fel d 1 protein is prone to form insoluble inclusion bodies in E. coli expression system, resulting in loss of natural conformation and biological activity of the protein, and the subsequent renaturation process is complex, inefficient and costly.

[0004] In the prior art, the skilled person in the art has tried various strategies to improve the solubility expression of Fel d 1, such as using common pET series vectors (such as pET-28a) and expressing in BL21(DE3) strain. However, the inventors found in the previous experiments that whether it is pET-28a-Fel d 1 vector or pGEX-4T-1-Fel d 1 vector transformed into BL21(DE3) strain, most of the expressed Fel d 1 protein still exists in the form of inclusion bodies, and the soluble part is very small, which cannot meet the needs of industrial production and clinical application for high-quality soluble protein.

[0005] Therefore, there is an urgent need in the art to develop a new method for efficiently producing high-solubility cat allergen Fel d 1 recombinant protein with natural conformation. SUMMARY

[0006] The technical problem to be solved: In view of the above technical problems, the present application provides a method for prokaryotic expression of cat allergen Fel d 1 recombinant protein, which constructs a recombinant plasmid and obtains a large amount of soluble expression of cat allergen Fel d 1 protein through purification.

[0007] Technical scheme: A method for prokaryotic expression of cat allergen Fel d 1 recombinant protein, comprising the following steps:

[0008] S1. Constructing a recombinant expression vector: cloning the cat allergen Fel d 1 gene into the multiple cloning site of the expression vector pGEX-4T-1 to obtain the recombinant expression vector pGEX-4T-1-Fel d 1;

[0009] S2. Transforming an expression host: transforming the recombinant expression vector pGEX-4T-1-Fel d 1 into E. coli Rosetta (DE3) competent cells to construct the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3);

[0010] S3. Inducing expression: activating and culturing the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) and adding IPTG to induce protein expression;

[0011] S4. Recovery and purification: collecting the bacteria after induction, centrifuging the broken bacteria to obtain the supernatant, and capturing the high-purity soluble GST-Fel d 1 fusion protein using GST affinity chromatography medium.

[0012] The nucleotide sequence of the Fel d 1 gene in the above step S1 is shown in SEQ ID NO. 1.

[0013] Preferably, the OD of the bacterial solution after the expansion culture in step S3 is 0.6-0.8. 600

[0014] Preferably, the final concentration of IPTG in step S3 is 0.1-1 mM.

[0015] Preferably, the temperature for IPTG induction in step S3 is 16-25℃, and the induction time is 15-20 h.

[0016] Further, the temperature for IPTG induction in step S3 is 25℃, and the induction time is 15 h.

[0017] The above cat allergen Fel d 1 recombinant protein is a fusion protein of Fel d 1 protein and GST tag.

[0018] The amino acid sequence of the above Fel d 1 protein is shown in SEQ ID NO. 2.

[0019] The nucleotide sequence of the above GST tag is shown in SEQ ID NO. 3.

[0020] Beneficial effects:

[0021] ​1. Carrier-host synergistic innovation: The present application combines pGEX-4T-1-GST tag system with E. coli Rosetta (DE3), and the GST tag as "soluble partner" can effectively promote the correct folding of Fel d 1, while E. coli Rosetta (DE3) solves the problems of low translation efficiency and incorrect folding caused by codon bias by supplementing the tRNA corresponding to the 6 rare codons (AUA, AGG, AGA, CUA, CCC, GGA) lacking in E. coli. The two have an unexpected synergistic effect, fundamentally solving the problem of soluble expression of Fel d 1.

[0022] 2. Process condition optimization: By adopting the strategy of low temperature and long time induction, the synthesis rate of protein is significantly reduced, which provides sufficient correct folding time for the newly born peptide chain and effectively inhibits the formation of inclusion bodies.

[0023] 3. Simple process, product can be directly applied: The GST-Fel d 1 fusion protein expressed by the present application directly exists in the supernatant, without the need for complex inclusion body denaturation and renaturation process, and high-purity GST-Fel d 1 fusion protein can be obtained in one step through conventional GST affinity chromatography. The fusion protein can be directly used for immunizing animals to prepare antibodies, or for screening molecules that bind to Fel d 1, greatly simplifying the process flow.

[0024] 4. Significant effect: Under the same conditions, the soluble expression amount of Fel d 1 protein of the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) constructed by the present application is much higher than that of pET-28a-Fel d 1 / BL21 (DE3), pET-28a-Fel d 1 / Rosetta (DE3) and pGEX-4T-1-Fel d 1 / BL21 (DE3). BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Plasmid map of recombinant expression vector pGEX-4T-1-Fel d 1;

[0026] Figure 2Figure 4 is a photograph of a SDS-PAGE gel showing the soluble expression of Fel d 1 under different induction conditions; in the figure: lane M: protein molecular weight marker; lane 1 (comparative example 1): recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosseta(DE3) (without IPTG induction); lane 2 (comparative example 2): empty strain pGEX-4T-1 / Rosseta(DE3) (IPTG induction); lane 3 (example 4): recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosseta(DE3) (induced at 16°C for 20 h); lane 4 (example 3): recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosseta(DE3) (induced at 25°C for 15 h); lane 5 (example 5): recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosseta(DE3) (induced at 30°C for 8 h); lane 6 (example 6): recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosseta(DE3) (induced at 37°C for 4 h);

[0027] Figure 3 Figure 5 is a photograph of a SDS-PAGE gel showing the soluble expression of Fel d 1 under different induction concentrations; in the figure: lane M: protein molecular weight marker; lane 1: 0.1 mM IPTG (example 7); lane 2: 0.2 mM IPTG (example 8); lane 3: 0.5 mM IPTG (example 3); lane 4: 0.6 mM IPTG (example 9); lane 5: 0.8 mM IPTG (example 10); lane 6: 1.0 mM IPTG (example 11);

[0028] Figure 4Figure 1 is a diagram of the soluble expression of Fel d 1 in different expression systems; in the figure: lane M: protein molecular weight marker; lane 1: supernatant after induction of recombinant engineering bacteria pET-28a-Fel d 1 / BL21(DE3) (comparative example 3); lane 2: supernatant after induction of recombinant engineering bacteria pGEX-4T-1-Fel d 1 / BL21(DE3) (comparative example 4); lane 3: supernatant after induction of recombinant engineering bacteria pET-28a-Fel d 1 / Rosetta(DE3) (comparative example 5); lane 4: supernatant after induction of recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta(DE3) (example 3); lane 5: precipitate after induction of recombinant engineering bacteria pET-28a-Fel d 1 / BL21(DE3) (comparative example 3); lane 6: precipitate after induction of recombinant engineering bacteria pGEX-4T-1-Fel d 1 / BL21(DE3) (comparative example 4); lane 7: precipitate after induction of recombinant engineering bacteria pET-28a-Fel d 1 / Rosetta(DE3) (comparative example 5); lane 8: precipitate after induction of recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta(DE3) (example 3);

[0029] Figure 5 Figure 2 is a diagram of the purification of GST-Fel d 1 fusion protein; in the figure: lane M: protein molecular weight marker; lane 1: supernatant before purification; FT: flow-through liquid of loading; W1 and W2: first and second step washing liquid, used to wash off non-specific or weakly bound impurities from the resin; E1-E5: eluent collected in steps (tube 1 to tube 5), using elution buffer containing reduced glutathione to specifically compete for the elution of GST fusion protein bound to the resin. DETAILED DESCRIPTION

[0030] The application will be further described in conjunction with the following examples, which are illustrative of the application but not limiting the application to the following examples:

[0031] Example 1

[0032] This example is a method for constructing a Fel d 1 recombinant expression vector pGEX-4T-1-Fel d 1, which comprises the following steps:

[0033] S1. According to the Fel d 1 crystal structure (id: 1PUO) published by the PDB protein structure database, it is confirmed that Fel d 1 is a heterodimer, each heterodimer is composed of chain 1 and chain 2, which are connected by a disulfide bond, and are encoded by genes CH1 and CH2, respectively, and the order of chain 1 and chain 2 on a single vector is CH2-CH1, and the cat allergen Fel d 1 gene is synthesized after codon optimization;

[0034] S2. The expression vector pGEX-4T-1 was double-digested with BamH I and Xho I restriction enzymes, and the double-digested system and conditions are shown in Table 1. The double-digested expression vector pGEX-4T-1 was mixed with the Fel d 1 gene, and homologous recombination was performed according to Table 2;

[0035] S3. The E. coli DH5α competent cells were taken out at -80℃, and placed on ice for 10 min. 8 μL of the ligation product was taken and added to the E. coli DH5α competent cells, which were placed on ice for 30 min without moving or shaking. After 90 s of 42℃ water bath heat shock, the cells were quickly placed on ice for 2 min. 800 μL of sterile and antibiotic-free fresh LB medium was added, and the cells were cultured at 37℃ and 220 rpm for 45 min. The cells were centrifuged at 5500 rpm for 5 min, and 800 μL of supernatant was discarded. The remaining bacterial cells were resuspended and evenly spread on LB plates containing ampicillin using a sterile spreader. The plates were incubated at 37℃ overnight until single colonies were formed.

[0036] S4. The positive clones were screened, and the plasmid was extracted for double digestion and DNA sequencing verification. After confirming that the sequence was correct, the recombinant expression vector pGEX-4T-1-Fel d 1 was obtained.

[0037] Table 1 Reaction system and conditions for double digestion

[0038]

[0039] Table 2 Reaction system and conditions for homologous recombination

[0040]

[0041] Example 2

[0042] This example is a method for constructing the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), which includes the following steps:

[0043] S1. The recombinant expression vector pGEX-4T-1-Fel d 1 constructed in Example 1 was transformed into E. coli Rosetta (DE3), and the E. coli Rosetta (DE3) competent cells were taken out at -80°C, placed on ice for 10 min, 8 μL of the recombinant expression vector plasmid pGEX-4T-1-Fel d 1 was added to the E. coli Rosetta (DE3) competent cells, placed on ice for 30 min, and do not move during vibration, 42°C water bath heat shock for 90 s, then placed on ice quickly and stood for 2 min, added 800 μL of sterile, non-antibiotic fresh LB medium, cultured at 37°C, 220 rpm on a shaker for 45 min, centrifuged at 5500 rpm for 5 min, discarded 800 μL of supernatant, resuspended the remaining bacteria, and evenly coated on LB plates containing chloramphenicol and ampicillin using a sterile coating rod, and cultured at 37°C overnight until single colonies were formed;

[0044] S2. The positive clones were screened, the plasmid was extracted for double enzyme digestion and DNA sequencing verification, and after confirming that the sequence was correct, the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) were obtained.

[0045] Example 3

[0046] This example is the induction expression and solubility analysis of the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3), which includes the following steps:

[0047] S1. The recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) constructed in Example 2 were inoculated in 10 mL of LB liquid medium containing chloramphenicol and ampicillin, and cultured at 37°C, 220 rpm on a shaker overnight.

[0048] S2. The overnight cultured bacteria were transferred to fresh LB liquid medium containing chloramphenicol and ampicillin at a ratio of 1%, and cultured at 37°C, 220 rpm on a shaker until the OD 600 of the bacteria was 0.6-0.8, and IPTG was added at a final concentration of 0.5 mM for induction expression, and the induction conditions were 25°C, 220 rpm, and induction for 15 h.

[0049] S3. The induced bacteria were collected, resuspended with PBS, and then broken using an ultrasonic disrupter, and the whole process was operated on ice, and the broken supernatant and precipitate were collected by centrifugation at 4°C, 12000 rpm for 2 min, and the supernatant and precipitate were mixed with 4×SDS loading, respectively, and boiled for 5 min to denature the proteins.

[0050] S4. The treated supernatant and precipitate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and stained using Coomassie brilliant blue without destaining.

[0051] Example 4

[0052] The difference between this example and Example 3 is that the induction condition in this example is 16℃ induction for 20 h, and the remaining steps are the same as in Example 3.

[0053] Example 5

[0054] The difference between this example and Example 3 is that the induction condition in this example is 30℃ induction for 8 h, and the remaining steps are the same as in Example 3.

[0055] Example 6

[0056] The difference between this example and Example 3 is that the induction condition in this example is 37℃ induction for 4 h, and the remaining steps are the same as in Example 3.

[0057] Comparative Example 1

[0058] The difference between this comparative example and Example 3 is that the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta(DE3) in this comparative example is not induced by IPTG, including the following steps:

[0059] S1. The recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta(DE3) constructed in Example 2 was inoculated into 10 mL of LB liquid medium containing chloramphenicol and ampicillin, and cultured overnight at 37℃ with 220 rpm shaking;

[0060] S2. The bacteria liquid after overnight culture was transferred to fresh LB liquid medium containing chloramphenicol and ampicillin at a ratio of 1%, and cultured at 37℃ with 220 rpm shaking until the OD 600 of the bacteria liquid was 0.6-0.8, then transferred to a 25℃ shaking bed and cultured at 220 rpm for 15 h;

[0061] S3. The bacteria liquid was collected, resuspended with PBS, and then subjected to crushing treatment using an ultrasonic crusher. The whole process was operated on ice at 4℃ and 12000 rpm for 2 min to collect the supernatant and precipitate after crushing. The supernatant and precipitate were mixed with 4×SDS loading, respectively, and boiled for 5 min to denature the proteins fully.

[0062] S4. The treated supernatant and precipitate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and stained using Coomassie brilliant blue without destaining.

[0063] Comparative Example 2

[0064] The difference between the present comparative example and Example 3 is that in the present comparative example, the PGEX-4T-1 empty vector is transformed into E. coli Rosetta (DE3) including the following steps:

[0065] S1. Transform the expression vector pGEX-4T-1 into E. coli Rosetta (DE3), take the E. coli Rosetta (DE3) competent cells out at -80°C, place on ice for 10 min, take 8 μL of the expression vector plasmid pGEX-4T-1 and add to the E. coli Rosetta (DE3) competent cells, place on ice for 30 min, do not move or shake during this period, after 90 s of 42°C water bath heat shock, quickly place on ice for 2 min, add 800 μL of sterile, antibiotic-free fresh LB medium, shake culture at 37°C, 220 rpm for 45 min, centrifuge at 5500 rpm for 5 min, discard 800 μL of supernatant, resuspend the remaining bacterial cells, and evenly spread on LB plates containing chloramphenicol and ampicillin using a sterile spreader, and incubate at 37°C overnight until single colonies grow;

[0066] S2. Screen positive clones, extract plasmids for double enzyme digestion and DNA sequencing verification, and obtain the engineering bacteria PGEX-4T-1 / Rosetta (DE3) after confirming that the sequence is correct;

[0067] S3. Inoculate the engineering bacteria PGEX-4T-1 / Rosetta (DE3) into 10 mL of LB liquid medium containing chloramphenicol and ampicillin, and shake culture overnight at 37°C, 220 rpm;

[0068] S2. Transfer the overnight cultured bacterial liquid into fresh LB liquid medium containing chloramphenicol and ampicillin at a ratio of 1%, and shake culture at 37°C, 220 rpm until the OD 600 of the bacterial liquid is 0.6-0.8; add IPTG to a final concentration of 0.5 mM for induction expression, and the induction conditions are 25°C, 220 rpm, and induction for 15 h;

[0069] S3. Collect the bacterial liquid, resuspend with PBS, and use an ultrasonic disrupter for crushing treatment until the bacterial liquid becomes clear and transparent, the whole process is operated on ice, centrifuge at 4°C, 12000 rpm for 2 min to collect the supernatant and precipitate after crushing, mix the supernatant and precipitate with 4xSDS loading, respectively, and boil for 5 min to denature the protein fully;

[0070] S4. The treated supernatant and precipitate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and stained using Coomassie brilliant blue R-250.

[0071] As shown in Figure 2 compared with Comparative Examples 1-2, the expression of the GST-Fel d 1 fusion protein can be detected under the four induction conditions of Examples 3-6, and the target protein band is the thickest in the soluble fraction (supernatant) of Example 3 (induction at 25°C for 15 h), and the expression amount is the highest, which is significantly higher than that of Examples 4-6. Therefore, it is determined that the induction at 25°C for 15 h is the optimal condition for inducing the expression of the GST-Fel d 1 fusion protein.

[0072] Example 7

[0073] The difference between this example and Example 3 is that the concentration of IPTG in this example is 0.1 mM, and the remaining steps are the same as those of Example 3.

[0074] Example 8

[0075] The difference between this example and Example 3 is that the concentration of IPTG in this example is 0.2 mM, and the remaining steps are the same as those of Example 3.

[0076] Example 9

[0077] The difference between this example and Example 3 is that the concentration of IPTG in this example is 0.6 mM, and the remaining steps are the same as those of Example 3.

[0078] Example 10

[0079] The difference between this example and Example 3 is that the concentration of IPTG in this example is 0.8 mM, and the remaining steps are the same as those of Example 3.

[0080] Example 11

[0081] The difference between this example and Example 3 is that the concentration of IPTG in this example is 1.0 mM, and the remaining steps are the same as those of Example 3.

[0082] Table 3 Quantification of protein band intensity

[0083]

[0084] As shown in Figure 3As shown in Table 3, the GST-Fel d 1 fusion proteins of Example 3 and Examples 7-11 can achieve soluble expression under IPTG induction at different concentrations (0.1-1 mM). When the IPTG concentration is in the range of 0.5-0.6 mM, the expression amount of soluble protein reaches a peak, and when the IPTG concentration is further increased, the expression amount of soluble protein does not increase significantly, but a slight downward trend in the proportion of soluble protein appears in some high concentration groups. Considering the production cost and induction efficiency, 0.5 mM (Example 3) is determined as the optimal IPTG induction concentration.

[0085] Comparative Example 3

[0086] The difference between the present comparative example and Example 3 is that the recombinant engineering bacteria in the present comparative example is pET-28a-Fel d 1 / BL21(DE3), which is the recombinant expression vector pET-28a-Fel d 1 transformed into Escherichia coli BL21(DE3), including the following steps:

[0087] S1. Constructing the recombinant expression vector pET-28a-Fel d 1: the specific steps are the same as those in Example 1;

[0088] S2. Transforming the expression host: the culture medium resistance is kanamycin, and the specific steps are the same as those in Example 2;

[0089] S3. Inducing expression: the culture medium resistance is kanamycin, and the specific steps are the same as those in Example 3.

[0090] Comparative Example 4

[0091] The difference between the present comparative example and Example 3 is that the recombinant engineering bacteria in the present comparative example is pGEX-4T-1-Fel d1 / BL21(DE3), which is the recombinant expression vector pGEX-4T-1-Fel d 1 transformed into Escherichia coli BL21(DE3), including the following steps:

[0092] S1. Transforming the expression host: the culture medium resistance is ampicillin, and the specific steps are the same as those in Example 2;

[0093] S2. Inducing expression: the culture medium resistance is ampicillin, and the specific steps are the same as those in Example 3.

[0094] Comparative Example 5

[0095] The difference between the present comparative example and Example 3 is that the recombinant engineering bacteria in the present comparative example is pET-28a-Fel d 1 / Rosetta(DE3), which is the recombinant expression vector pET-28a-Fel d 1 transformed into Escherichia coli Rosetta(DE3), including the following steps:

[0096] S1. Constructing recombinant expression vector pET-28a-Fel d 1: the specific steps are the same as those in Example 1.

[0097] S2. Transforming expression host: the culture medium resistance is kanamycin and ampicillin, and the specific steps are the same as those in Example 2.

[0098] S3. Inducing expression: the culture medium resistance is kanamycin and ampicillin, and the specific steps are the same as those in Example 3.

[0099] As shown in Figure 4 Example 3 (pGEX-4T-1-Fel d 1 / Rosetta (DE3)), after IPTG induction, a target band of about 44.3 kDa can be observed in the lanes of the soluble part (S) and the insoluble part (P). There is no such band in the corresponding positions of Comparative Example 1 (uninduced sample UI) and Comparative Example 2 (Rosetta (DE3) into which PGEX-4T-1 empty vector is transformed), proving that the induction is successful and that the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) express the GST-Fel d 1 fusion protein. In the lanes of the soluble part (S) and the insoluble part (P) of Comparative Example 3 (Rosetta (DE3) into which pET-28a-Fel d 1 is transformed) and Comparative Example 4 (BL21 (DE3) into which pGEX-4T-1-Fel d 1 is transformed), no target band is observed.

[0100] The above induction expression combined with SDS-PAGE analysis proves that the recombinant engineering bacteria pGEX-4T-1-Fel d 1 / Rosetta (DE3) constructed in the present application can efficiently express the GST-Fel d 1 fusion protein under the condition of 25℃ and 0.1 mM IPTG induction, and the protein mainly exists in a soluble form, fully embodying the unique advantages and outstanding effects of the combination of the vector and the host bacteria selected in the present application in solving the problem of Fel d 1 soluble expression.

[0101] Example 12

[0102] This example is the purification expression of Fel d 1 protein, including the following steps:

[0103] S1. The bacteria solution after induction of Example 3 was centrifuged to collect the bacteria, which was then ultrasonically broken and centrifuged. The supernatant containing the soluble GST-Fel d 1 fusion protein was transferred to a pre-equilibrated GST affinity chromatography column, which was then washed with equilibrium buffer (PBS, pH 7.4) to remove impurities. The column was then eluted with elution buffer (containing 50 mM Tris, 150 mM NaCl, 10 mM GSH, pH 8.0) containing 10 mM reduced glutathione, and the eluate was collected. The eluted components were analyzed by SDS-PAGE.

[0104] As shown in Figure 5 the expected molecular weight size (about 44.3 kDa, GST about 26 kDa + Fel d 1 about 18.3 kDa) near a single, clear main band, indicating that the high-purity soluble GST-Fel d 1 fusion protein was successfully obtained by the method of the present application. The purity was more than 90% by gel scanning density analysis.

[0105] The above description is only a preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not deviate from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.

Claims

1. A method for prokaryotic expression of recombinant feline allergen Fel d1 protein, characterized in that, Includes the following steps: S1. Construction of recombinant expression vector: The feline allergen Fel d 1 gene was cloned into the multiple cloning site of the expression vector pGEX-4T-1 to obtain the recombinant expression vector pGEX-4T-1-Fel d 1; S2. Transformation of expression host: The recombinant expression vector pGEX-4T-1-Fel d1 was transformed into Escherichia coli Rosetta(DE3) competent cells to construct the recombinant engineered strain pGEX-4T-1-Fel d1 / Rosetta(DE3); S3. Induction of expression: The recombinant engineered bacteria pGEX-4T-1-Fel d 1 / Rosetta(DE3) was activated, cultured in a large scale, and IPTG was added to induce protein expression; S4. Recovery and purification: After induction, the bacterial cells were collected, and after the bacterial cells were lysed, the supernatant was collected by centrifugation. The high-purity soluble GST-Fel d1 fusion protein was captured using GST affinity chromatography medium.

2. The method for prokaryotic expression of recombinant feline allergen Fel d1 protein according to claim 1, characterized in that: The nucleotide sequence of the Fel d 1 gene in step S1 is shown in SEQ ID NO.

1.

3. The method for prokaryotic expression of recombinant feline allergen Fel d1 protein according to claim 1, characterized in that: In step S3, the bacterial culture OD after expansion culture 600 The concentration of IPTG was 0.6–0.8 mM; the final concentration of IPTG was 0.1–1 mM; the induction temperature of IPTG was 16–25 °C; and the induction time was 15–20 h.

4. The method for prokaryotic expression of recombinant feline allergen Fel d1 protein according to claim 1, characterized in that: The feline allergen Fel d 1 recombinant protein is a fusion protein of Fel d 1 protein and GST tag.

5. A method for prokaryotic expression of recombinant feline allergen Fel d1 protein according to claim 4, wherein the amino acid sequence of Fel d1 protein is shown in SEQ ID NO.

2.

6. The method for prokaryotic expression of recombinant feline allergen Fel d 1 protein according to claim 4, wherein the nucleotide sequence of the GST tag is shown in SEQ ID NO.3.

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