Preparation method of soluble prokaryotic recombinant PRL

Abstract

The invention provides a preparation method of soluble prokaryotic recombinant PRL, the soluble prokaryotic recombinant PRL is obtained by deleting amino acids from the 80th site to the 88th site of a natural PRL amino acid sequence, and the amino acid sequence of the soluble prokaryotic recombinant PRL is as shown in SEQ ID NO: 1. The solubility of the soluble prokaryotic recombinant PRL is obviously higher than that of natural PRL, and the soluble prokaryotic recombinant PRL has higher antigen titer; according to the preparation method of the soluble prokaryotic recombinant PRL, a gene engineering means is adopted, a coding gene of the soluble prokaryotic recombinant PRL is cloned to an expression vector, host cells are transformed for prokaryotic expression, a large amount of soluble prokaryotic recombinant PRL can be obtained, a large amount of raw materials are provided for development of clinical diagnostic reagents, and the production cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a method for preparing soluble prokaryotic recombinant PRL. Background Art

[0002] Prolactin (PRL), also known as luteotropic hormone or luteotropin, is a protein hormone secreted by acidophilic cells in the anterior pituitary gland. Its main functions are to promote breast development and growth, stimulate and maintain lactation, and stimulate the generation of follicle LH receptors. The secretion of PRL shows pulsatile fluctuations and obvious circadian rhythm changes. The regulation of PRL secretion is mainly controlled by the prolactin release inhibitory hormone secreted by the hypothalamus, and it is the only adenohypophyseal hormone that is in an inhibitory state under normal physiological conditions. Thyrotropin-releasing hormone, estrogen, and factors such as stress and sleep can promote the secretion of PRL through different pathways. Large doses of estrogen, progesterone, and dopamine can inhibit the secretion of prolactin. Sucking can also increase the secretion of prolactin. The continuous sucking of the baby stimulates the anterior pituitary gland to secrete prolactin, so that lactation can be maintained for several months to several years. At present, the detection of serum prolactin is mainly used to assist in the diagnosis of diseases such as pituitary tumors, hyperprolactinemia, and infertility. In recent years, the scientific community has conducted more and more research on human prolactin, and has a deeper understanding of the diseases caused by its abnormalities.

[0003] There is extensive expression of PRL receptors in breast cancer tumor cells, and it is significantly correlated with tumor size, grade, and histological subtype. Detecting circulating tumor cells in the peripheral blood of cancer patients plays an important role in the diagnosis, prognosis evaluation, treatment plan selection, metastasis and recurrence prediction of breast cancer. Markers such as PRL have important value in the diagnosis and evaluation of metastasis after breast cancer surgery. PRL is also an important indicator for diagnosing infertility. The content of PRL in serum can be used to diagnose infertility and sterility, reflect the growth and development level, and is also an important parameter for indicating endocrine diseases, immune diseases (such as lupus erythematosus), and nervous system diseases. Precise quantitative detection of PRL is of great significance for clinical diagnosis. Abnormal manifestations of hyperprolactinemia suggest the possibility of multiple diseases. The clinical detection of PRL requires antibodies prepared by immunizing animals with highly pure soluble PRL, or obtaining B cells for hybridoma expression of PRL monoclonal antibodies. No matter which way, it is inseparable from soluble PRL prepared by in vitro expression. At present, most of the PRL proteins expressed by recombinant prokaryotic expression disclosed in the prior art are insoluble proteins. After expression, inclusion bodies need to be washed and purified and then renatured, resulting in a long production cycle and high cost, which is not conducive to the development and use of PRL detection reagents. Summary of the Invention

[0004] The present invention aims to provide a method for preparing soluble prokaryotic recombinant PRL. The soluble prokaryotic recombinant PRL has better solubility than natural PRL and higher antigen titer. The method for preparing the soluble prokaryotic recombinant PRL uses genetic engineering means to optimize the natural PRL protein sequence, clone the optimized PRL coding gene into an expression vector, transform host cells for prokaryotic expression, and a large amount of soluble prokaryotic recombinant PRL can be obtained, providing a large amount of raw materials for the development of clinical diagnostic reagents and reducing production costs.

[0005] To solve the above problems, on the one hand, the present invention provides a soluble prokaryotic recombinant PRL, which is obtained by deleting amino acids at positions 80-88 of the natural PRL amino acid sequence, and the amino acid sequence of the soluble prokaryotic recombinant PRL is shown as SEQ ID NO: 1. The present invention deletes amino acids at positions 80-88 of the amino acid sequence of natural PRL shown as SEQ ID NO: 3, and the obtained soluble prokaryotic recombinant PRL has better solubility and higher antigen titer compared with natural PRL.

[0006] The sequence of SEQ ID NO: 1 is as follows: MLPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMNQKLRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDSHKIDNYLKLLKCRIIHNNNC.

[0007] The sequence of SEQ ID NO: 3 is as follows: MLPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMNQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDSHKIDNYLKLLKCRIIHNNNC.

[0008] On the other hand, the present invention provides a coding gene for the soluble prokaryotic recombinant PRL, and the coding gene for the soluble prokaryotic recombinant PRL encodes the above-mentioned soluble prokaryotic recombinant PRL.

[0009] Optimize the coding gene of the above amino acid sequence according to the codon preference of Escherichia coli to obtain a gene sequence suitable for expression in Escherichia coli. Preferably, the nucleotide sequence of the coding gene of soluble prokaryotic recombinant PRL is as shown in SEQ ID NO: 2.

[0010] The sequence of SEQ ID NO: 2 is as follows: ATGCTGCCGATTTGCCCAGGTGGTGCCGCTCGCTGTCAAGTTACCCTGCGTGATCTGTTCGACCGTGCTGTTGTTCTGTCCCACTATATCCATAACCTGTCCAGCGAAATGTTCTCCGAGTTCGACAAACGCTACACTCATGGCCGTGGCTTCATTACGAAAGCCATTAACTCCTGCCACACCTCTTCCCTGGCTACTCCAGAAGACAAAGAACAGGCACAGCAAATGAACCAAAAACTGCGTTCCTGGAACGAACCGCTGTACCACCTGGTGACTGAGGTGCGTGGTATGCAGGAAGCACCGGAAGCTATCCTGAGCAAAGCGGTAGAGATCGAAGAACAGACCAAGCGTCTGCTGGAAGGTATGGAGCTGATCGTTTCTCAAGTTCATCCGGAGACTAAAGAAAACGAGATCTACCCAGTATGGTCTGGTCTGCCGAGCCTGCAAATGGCAGACGAGGAAAGCCGTCTGTCTGCTTACTATAACCTGCTGCATTGCCTGCGCCGTGATTCCCACAAAATCGACAACTACCTGAAACTGCTGAAATGTCGCATCATTCACAACAACAACTGC。

[0011] Another aspect of the present invention provides a prokaryotic recombinant vector, which contains the coding gene of the above soluble prokaryotic recombinant PRL. The prokaryotic recombinant vector can be a plasmid, a derivative of λ phage, or a plant or animal virus. Preferably, the prokaryotic recombinant vector is a plasmid. The plasmid is preferably pET28a.

[0012] Another aspect of the present invention provides a host cell, which contains the above prokaryotic recombinant vector. The host cell can be one of Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, yeast, and plant and animal cells. Preferably, the host cell is Escherichia coli.

[0013] Another aspect of the present invention provides a method for preparing soluble prokaryotic recombinant PRL, comprising the following steps: S1. Construct a soluble prokaryotic recombinant PRL genetic engineering strain, which contains the coding gene of the above-mentioned prokaryotic recombinant PRL; S2. Inoculate the soluble prokaryotic recombinant PRL genetic engineering strain into a seed medium for seed culture; S3. Inoculate the soluble prokaryotic recombinant PRL genetic engineering strain after seed culture into a fermentation medium and culture it to a specific concentration.

[0014] S4. Add an inducer for induction to obtain the soluble prokaryotic recombinant PRL.

[0015] The present invention uses genetic engineering methods to first construct a genetic engineering strain containing the gene of soluble prokaryotic recombinant PRL, and then culture and ferment the genetic engineering strain to achieve the prokaryotic expression of soluble prokaryotic recombinant PRL. A large amount of soluble prokaryotic recombinant PRL can be obtained through separation and purification. The soluble prokaryotic recombinant PRL has better solubility than natural PRL and higher antigen titer, and can be produced in large quantities by this method, providing a large amount of raw materials for the development of clinical diagnostic reagents and reducing production costs.

[0016] Preferably, S1 includes the following steps: S101. Synthesize the coding gene of the soluble prokaryotic recombinant PRL; S102. Clone the coding gene of the soluble prokaryotic recombinant PRL into an expression vector to obtain a recombinant vector, and the expression vector is pET28a; S103. Transfer the recombinant vector into competent cells, screen positive transformants to obtain the soluble prokaryotic recombinant PRL genetic engineering strain, and the competent cells are Escherichia coli competent cells.

[0017] More preferably, S1 includes the following steps: S101. Synthesize the coding gene of soluble prokaryotic recombinant PRL according to the sequence of SEQ ID NO: 2, and amplify the coding gene of soluble prokaryotic recombinant PRL using the coding gene of soluble prokaryotic recombinant PRL as a template with an upstream primer and a downstream primer. Among them, the upstream primer has the sequence shown in SEQ ID NO: 4, and the downstream primer has the sequence shown in SEQ ID NO: 5. The sequence of SEQ ID NO: 4 is as follows: 5’-GGATCCGAATTCATGCTGCCGATTTGCCC-3’.

[0018] The sequence of SEQ ID NO: 5 is as follows: 5’-GTGGTGCTCGAGGCAGTTGTTGTTGTGAATG-3’.

[0019] S102. After double digestion of the amplification product and the expression vector with EcoRI and XhoI respectively, ligate them with a ligase to clone the coding gene of the soluble prokaryotic recombinant PRL into the expression vector; S103. Transfer the expression vector into competent cells, screen for positive transformants to obtain the soluble prokaryotic recombinant PRL gene engineering strain, and the competent cells are Escherichia coli BL21(DE3) competent cells.

[0020] Preferably, S2 is specifically as follows: Inoculate the soluble prokaryotic recombinant PRL gene engineering strain into LB medium and culture it for 8 - 18 h under the conditions of 25 - 40°C and 80 - 220 rpm.

[0021] More preferably, S2 is specifically as follows: Inoculate the soluble prokaryotic recombinant PRL gene engineering strain into LB medium and culture it for 10 h under the stirring condition of 37°C and 200 rpm.

[0022] Preferably, S3 includes the following steps: Transfer the bacterial liquid obtained by seed culture in S2 into LB medium at an inoculation amount of 0.1 - 8%, and culture it at 25 - 40°C and 80 - 220 rpm until the OD600 is 0.6 - 0.8.

[0023] More preferably, step S3 includes the following steps: Transfer the bacterial liquid obtained by seed culture in S2 into 1 L of LB liquid medium at an inoculation amount of 1%, and culture it at 37°C and 200 rpm until the OD600 is 0.6 - 0.8.

[0024] Preferably, S4 includes the following steps: S401. Add IPTG with a final concentration of 0.5 - 5 mM to the bacterial liquid obtained in S3; S402. Culture the bacterial liquid obtained in S401 at 20 - 30°C and 80 - 220 rpm for 2 - 10 h to obtain a fermentation broth; S403. Centrifuge the fermentation broth, remove the supernatant, wash it twice with buffer, suspend the cells with a equilibration solution, and disrupt the cells; S404. Centrifuge the broken cells, filter the supernatant with a filter membrane, and then purify it by affinity chromatography to obtain the soluble prokaryotic recombinant PRL.

[0025] Further preferably, S4 includes the following steps: S401. Add IPTG with a final concentration of 1 mM to the bacterial liquid obtained in S3. S402. Culture the bacterial liquid obtained in S401 at 27 °C and 200 rpm for 5 h to obtain a fermentation broth. S403. Centrifuge the fermentation broth at 8000 rpm for 10 min, remove the supernatant, add 50 ml of PBS to resuspend and wash the precipitate, centrifuge at 8000 rpm for 10 min, discard the supernatant, and repeat the washing operation. After discarding the supernatant, add 50 ml of equilibration buffer (20 mM Tris-HCl, 300 mM NaCl) to suspend the cells, and ultrasonically disrupt the cells (300 W, working for 3 s, resting for 3 s). S404. Centrifuge the broken cells at 10000 rpm for 20 min, take the supernatant, filter it with a 0.45 μm filter membrane, and then purify it by affinity chromatography to obtain the soluble prokaryotic recombinant PRL.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The 80th to 88th amino acids of natural PRL are highly hydrophobic regions. Due to the lack of polar groups in this region, it is easy to drive the aggregation between protein molecules through hydrophobic interactions, forming insoluble aggregates. In the present invention, the 80th to 88th amino acids of natural PRL are deleted, eliminating the hydrophobic exposed region of the PRL protein, reducing the abnormal aggregation tendency caused by hydrophobic interactions between molecules. The obtained soluble prokaryotic recombinant PRL has better solubility than natural PRL, and its antigen titer is higher than that of natural PRL.

[0027] 2. The present invention uses genetic engineering methods to first construct a genetic engineering strain with the coding gene of soluble prokaryotic recombinant PRL, and then culture and ferment the genetic engineering strain to achieve the prokaryotic expression of soluble prokaryotic recombinant PRL. A large amount of soluble prokaryotic recombinant PRL can be obtained through separation and purification. The soluble prokaryotic recombinant PRL has better solubility than natural PRL, and its antigen titer is higher than that of natural PRL. It can be mass-produced by this method, providing a large amount of raw materials for the development of clinical diagnostic reagents and reducing production costs. Description of the Drawings

[0028] Figure 1 It is the electrophoresis diagram of the purified soluble prokaryotic recombinant PRL in Example 2 of the present invention. Detailed Embodiments

[0029] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0030] Example 1 Design the nucleotide sequence of the coding gene of soluble prokaryotic recombinant PRL, including the following steps: Based on the amino acid sequence of the soluble prokaryotic recombinant PRL of the present invention, this sequence is optimized according to the codon preference of Escherichia coli to obtain the coding gene of the soluble prokaryotic recombinant PRL. The amino acid sequence of the soluble prokaryotic recombinant PRL is shown in SEQ ID NO. 1, and the nucleotide sequence of the optimized coding gene of the soluble prokaryotic recombinant PRL is shown in SEQ ID NO. 2.

[0031] The sequence of SEQ ID NO. 1 is as follows: MLPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDQKDFLSLIVSILRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRRDSHKIDNYLKLLKCRIIHNNNC.

[0032] The sequence of SEQ ID NO. 2 is as follows: ATGCTGCCGATTTGCCCAGGTGGTGCCGCTCGCTGTCAAGTTACCCTGCGTGATCTGTTCGACCGTGCTGTTGTTCTGTCCCACTATATCCATAACCTGTCCAGCGAAATGTTCTCCGAGTTCGACAAACGCTACACTCATGGCCGTGGCTTCATTACGAAAGCCATTAACTCCTGCCACACCTCTTCCCTGGCTACTCCAGAAGACAAAGAACAGGCACAGCAAATGAACCAAAAACTGCGTTCCTGGAACGAACCGCTGTACCACCTGGTGACTGAGGTGCGTGGTATGCAGGAAGCACCGGAAGCTATCCTGAGCAAAGCGGTAGAGATCGAAGAACAGACCAAGCGTCTGCTGGAAGGTATGGAGCTGATCGTTTCTCAAGTTCATCCGGAGACTAAAGAAAACGAGATCTACCCAGTATGGTCTGGTCTGCCGAGCCTGCAAATGGCAGACGAGGAAAGCCGTCTGTCTGCTTACTATAACCTGCTGCATTGCCTGCGCCGTGATTCCCACAAAATCGACAACTACCTGAAACTGCTGAAATGTCGCATCATTCACAACAACAACTGC

[0033] Example 2 Construct a soluble prokaryotic recombinant PRL genetic engineering strain, comprising the following steps: S101. Synthesize the coding gene of soluble prokaryotic recombinant PRL according to the sequence of SEQ ID NO: 2, and amplify the coding gene of soluble prokaryotic recombinant PRL using the upstream primer and the downstream primer with the coding gene of soluble prokaryotic recombinant PRL as a template, wherein the upstream primer has the sequence shown in SEQ ID NO: 4, and the downstream primer has the sequence shown in SEQ ID NO: 5. The sequence of SEQ ID NO: 4 is as follows: 5’-GGATCCGAATTCATGCTGCCGATTTGCCC-3’.

[0034] The sequence of SEQ ID NO: 5 is as follows: 5’-GTGGTGCTCGAGGCAGTTGTTGTTGTGAATG-3’.

[0035] S102. After double digestion of the amplification product and the expression vector with EcoRI and XhoI respectively, they were ligated with a ligase, and the coding gene of soluble prokaryotic recombinant PRL was cloned onto the expression vector pET28a to obtain a recombinant plasmid, denoted as pET28a-PRL; S103. Take 100 μl of Escherichia coli competent cells, thaw them on ice, add 10 μg of pET28a-PRL plasmid, incubate on ice for 30 min, place the plasmid and competent cell mixture in a 42°C water bath for heat shock for 90 s, then place it in an ice bath for 2 min, add 900 μl of LB medium, and recover at 37°C and 200 rpm for 1 h. Then take 100 μl and spread it on an LB plate with kanamycin resistance, and culture overnight at 37°C to obtain a soluble prokaryotic recombinant PRL gene engineering strain.

[0036] Example 3 A preparation method of soluble prokaryotic recombinant PRL, comprising the following steps: S2. Seed culture of soluble prokaryotic recombinant PRL gene engineering strain: Pick a monoclonal colony of the soluble prokaryotic recombinant PRL gene engineering strain screened in Example 2 and inoculate it into 10 ml of LB medium, and culture it at 37°C and 200 rpm for 10 h.

[0037] S3. Transfer of soluble prokaryotic recombinant PRL gene engineering strain to fermentation medium: Transfer the bacterial liquid obtained from the seed culture in step S2 into 1 L of LB liquid medium at an inoculation amount of 1%, and culture it at 37°C and 200 rpm until the OD600 is 0.6 - 0.8.

[0038] S4. Fermentation culture of soluble prokaryotic recombinant PRL gene engineering strain: S401. Add IPTG with a final concentration of 1 mM to the bacterial liquid obtained in step S3; S402. Culture the bacterial liquid obtained in step S401 at 27°C and 200 rpm for 5 h to obtain a fermentation broth; S403. Centrifuge the fermentation broth at 8000 rpm for 10 min, remove the supernatant, add 50 ml of PBS to resuspend and wash the precipitate, centrifuge at 8000 rpm for 10 min, discard the supernatant, and repeat the washing operation. After discarding the supernatant, add 50 ml of equilibration buffer (20 mM Tris-HCl, 300 mM NaCl) to suspend the cells, and ultrasonically disrupt the cells (300 W, work for 3 s, rest for 3 s); S404. Centrifuge the broken cells at 10,000 rpm for 20 min. Take the supernatant, filter it through a 0.45-μm filter membrane, and then purify it by affinity chromatography to obtain the soluble prokaryotic recombinant PRL. The specific method of affinity chromatography is as follows: First, rinse and equilibrate the nickel column with the equilibration buffer (20 mM Tris-HCl, 300 mM NaCl), and load the sample at a flow rate of 5 ml / min. After loading, rinse with the equilibration buffer (20 mM Tris-HCl, 300 mM NaCl) for 10 column volumes to wash away the miscellaneous proteins. Then, elute PRL by adding imidazole with final concentrations of 50 mM and 100 mM to the equilibration buffer in sequence. Take samples of the supernatant, flow-through, wash solution, and eluate for electrophoresis. As Figure 1 shown, where 1 is the supernatant, 2 is the flow-through, 3 is the wash solution, 4 is the wash solution, 5 is the 50 mM eluate, 6 is the 100 mM eluate, and 7 is the standard protein molecular weight. The eluted PRL is desalted through a G25 gel column to obtain pure soluble prokaryotic recombinant PRL, and store it at -20 °C.

[0039] Comparative Example According to the methods of Examples 1 to 3, design and express the inclusion body of natural PRL for the amino acid sequence of natural PRL shown in SEQ ID NO: 3. Ferment the engineering bacteria containing the natural PRL-encoding gene, collect the bacterial cells, and after ultrasonic disruption, centrifuge to obtain the precipitate, which is the inclusion body of natural PRL. Wash the inclusion body three times with the inclusion body washing solution (50 mM Tris, 200 mM NaCl, 0.5% Triton X-100, 0.5 M urea, pH 9.0), wash it twice with pure water, centrifuge at 10,000 rpm for 20 min to obtain the precipitate, add the dissolution solution (50 mM Tris, 100 mM NaCl, 8 M urea, pH 8.0), mix well, stir and mix overnight at 4 °C. After dissolution, centrifuge at 10,000 rpm for 20 min, take the supernatant, and dialyze it successively against renaturation solution 1 (200 mM Tris, 500 mM NaCl, 5% glycerol, 5 μM EDTA, 4 M urea, pH 8.5), renaturation solution 2 (200 mM Tris, 500 mM NaCl, 5% glycerol, 5 μM EDTA, 2 M urea, pH 8.5), renaturation solution 3 (200 mM Tris, 500 mM NaCl, 5% glycerol, 5 μM EDTA, pH 8.5), and renaturation solution 4 (200 mM Tris, 500 mM NaCl, pH 8.0). After dialysis, obtain the natural PRL protein.

[0040] Experimental Example Potency Detection: (1) Coat the S-1PRL8 monoclonal antibody at a coating concentration of 1 μg / ml, add 100 μl / sample well to the enzyme-linked immunosorbent assay (ELISA) plate, and incubate overnight at 4 °C.

[0041] (2)Discard the liquid and add 200 μl of blocking solution (1% casein), and incubate at 37 °C for 2 h.

[0042] (3)Wash off the blocking solution (repeatedly 4 times), and add the natural PRL prepared in the comparative example and the soluble prokaryotic recombinant PRL antigen prepared in Example 3 respectively. The antigen concentrations are 1000 ng / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, 100 μl / well, and incubate at 37 °C for 40 min.

[0043] (4)Wash off the liquid (repeatedly 4 times), add S-1PRL5-HRP (diluted 1:2000), 100 μl / well, and incubate at 37 °C for 40 min.

[0044] (5)Wash off the liquid (repeatedly 4 times), add the chromogenic solution (100 μl / well), and incubate at 37 °C for 5 min.

[0045] (6)Add the stop solution (50 μl / well), and read the value in the microplate reader.

[0046] Analysis of experimental results: Figure 1 The soluble prokaryotic recombinant PRL shown was subjected to electrophoresis detection during the purification process. A clear PRL protein band was visible in the supernatant. At the position of the control standard protein molecular weight of about 35 kDa, the washing solution was collected twice for electrophoresis, and the target protein was not washed off. Elution was carried out with 50 mM and 100 mM imidazole respectively, and a large amount of soluble prokaryotic recombinant PRL was eluted with 100 mM imidazole.

[0047] The results of titer detection are shown in Table 1 below: Table 1 Comparison of titer detection of natural PRL and soluble prokaryotic recombinant PRL antigens

[0048] It can be seen from the detection data in the above table that at the same antigen concentration, the soluble prokaryotic recombinant PRL antigen has a higher absorbance in titer detection, indicating that the soluble prokaryotic recombinant PRL prepared by the method of the present invention has a higher antigen titer than natural PRL.

[0049] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A soluble prokaryotic recombinant PRL, characterized in that, The soluble prokaryotic recombinant PRL is obtained by deleting the amino acids at positions 80-88 of the natural PRL amino acid sequence, and the amino acid sequence of the soluble prokaryotic recombinant PRL is shown in SEQ ID NO:

1.

2. The coding gene of the soluble prokaryotic recombinant PRL as described in claim 1, characterized in that, The nucleotide sequence of the coding gene of the soluble prokaryotic recombinant PRL is shown in SEQ ID NO:

2.

3. A prokaryotic recombinant vector, characterized in that, The prokaryotic recombinant vector contains the coding gene of the soluble prokaryotic recombinant PRL as described in claim 2.

4. A host cell, characterized in that, The host cell contains the prokaryotic recombinant vector as described in claim 3.

5. A preparation method of soluble prokaryotic recombinant PRL, characterized in that, It includes the following steps: S1. Construct a soluble prokaryotic recombinant PRL genetic engineering strain, and the soluble prokaryotic recombinant PRL genetic engineering strain contains the coding gene of the prokaryotic recombinant PRL as described in claim 2; S2. Inoculate the soluble prokaryotic recombinant PRL genetic engineering strain into a seed culture medium for seed culture; S3. Inoculate the soluble prokaryotic recombinant PRL genetic engineering strain after seed culture into a fermentation medium for culture; S4. Add an inducer for induction to obtain the soluble prokaryotic recombinant PRL.

6. The preparation method of the soluble prokaryotic recombinant PRL according to claim 5, characterized in that, The S1 includes the following steps: S101. Synthesize the coding gene of the soluble prokaryotic recombinant PRL; S102. Clone the coding gene of the soluble prokaryotic recombinant PRL into an expression vector to obtain a recombinant vector; S103. Transfer the recombinant vector into competent cells, screen positive transformants to obtain the soluble prokaryotic recombinant PRL genetic engineering strain.

7. The preparation method of the soluble prokaryotic recombinant PRL according to claim 5, characterized in that, The S2 is specifically: Take the soluble prokaryotic recombinant PRL genetic engineering strain and inoculate it into an LB medium, and culture it at 25-40 °C and 80-220 rpm for 8-18 h.

8. The preparation method of the soluble prokaryotic recombinant PRL according to claim 5, characterized in that, The S3 includes the following steps: Transfer the bacterial liquid obtained by seed culture in step S2 into an LB medium at an inoculation amount of 0.1%-8%, and culture it at 25-40 °C and 80-220 rpm until the OD600 is 0.6-0.

8.

9. The preparation method of the soluble prokaryotic recombinant PRL according to claim 5, characterized in that, The S4 includes the following steps: S401. Add IPTG with a final concentration of 0.5-5 mM to the bacterial liquid obtained in S3; S402. Culture the bacterial liquid obtained in S401 at 20-30 °C and 80-220 rpm for 2-10 h to obtain a fermentation broth; S403. Centrifuge the fermentation broth described in S402, remove the supernatant, wash it twice with a buffer solution, add a equilibration solution to suspend the cells, and perform cell disruption treatment; S404. Centrifuge and separate the disrupted cells, filter the supernatant with a filter membrane, and purify it by affinity chromatography to obtain the soluble prokaryotic recombinant PRL.

Images