Method for directionally screening teriparatide high-yield strain by using antibiotics

Abstract

The invention discloses a method for directionally screening a teriparatide high-yield strain by using antibiotics, an escherichia coli production strain capable of highly expressing teriparatide is obtained through two rounds of screening of antibiotics with different concentrations, and the stability of plasmids in a strain passage process is still effectively maintained under the condition that no antibiotics are added. According to the directional screening method, the screening efficiency of the teriparatide high-yield strain can be effectively improved, and the technical difficulty that antibiotics need to be added in the production process of biological drugs is overcome.

Description

Technical Field

[0001] The invention relates to the field of bacterial strain screening, and in particular to a method for directed screening of bacterial strains using antibiotics. Background Art

[0002] Genetically engineered bacteria are micro-bioreactors in modern bioengineering. How to obtain genetically engineered bacteria that can efficiently and stably express exogenous genes is crucial to the fermentation industry. The main host expression systems used in genetically engineered bacteria include Escherichia coli, which can express and produce products such as polypeptides, amino acids, growth factors, and recombinant proteins by transfecting plasmid vectors. Plasmid vectors generally identify positive transformants and maintain their separation stability in strains through resistance screening markers. Studies have shown that plasmid loss is very obvious in the late logarithmic growth period, and the expression of exogenous genes will further increase the instability of plasmids. The addition of antibiotics to reduce the proportion of plasmid loss is one of the conventional technical means in the fermentation field, such as adding kanamycin, ampicillin, tetracycline, etc. to the culture medium. Kanamycin is an aminoglycoside antibiotic isolated from Streptomyces kanamyceticus. As an inhibitor of protein synthesis, its working concentration is generally 10 to 50 μg / ml.

[0003] As process development continues to advance, strain screening technology is an important factor in determining strain performance. It is necessary to establish stable and high-yield strains to maintain the stability of the generations required from seed bank establishment to large-scale fermentation. The stability of each plasmid is different, and the influencing factors include the characteristics of the host bacteria, the number of plasmid copies, the size of the inserted DNA, and the culture conditions. For a specific production strain, it is necessary to determine the most suitable screening conditions in order to efficiently and directional screen stable and high-yield engineered strains. Secondly, the use of antibiotics should be avoided as much as possible in drug production. Antibiotics are not only easy to pollute the environment and produce drug-resistant bacteria, but also need to be removed from the product in the subsequent process, which further increases the production cost and complicates the process. How to make the plasmid stably exist in the engineered bacteria and achieve high expression of the target protein under antibiotic-free culture conditions is an urgent problem to be solved in this field. Summary of the invention

[0004] The present invention overcomes the problems existing in the prior art and proposes a method for using antibiotics to screen high-yielding bacteria for teriparatide. The method transfects a host bacterium with an expression plasmid containing a kanamycin resistance gene and a teriparatide encoding gene, and obtains a genetically engineered bacterium capable of high-yielding teriparatide through two rounds of kanamycin screening and fermentation verification at different concentrations. The stable inheritance of the plasmid is still effectively maintained under the culture condition without adding kanamycin. The screening method of the present invention can not only effectively improve the screening efficiency of high-yielding strains of teriparatide, but also avoid the problem of antibiotic residues in the production process, and further improve the yield of teriparatide.

[0005] In one aspect, the present invention discloses a method for selectively screening teriparatide high-producing bacteria using antibiotics, the method comprising:

[0006] 1) Plasmid construction: constructing a teriparatide expression plasmid, wherein the plasmid contains a kanamycin resistance gene and a teriparatide encoding gene;

[0007] 2) Transformation: The expression plasmid constructed in step 1) is transformed into competent E. coli cells and cultured to obtain a recombinant bacterial solution;

[0008] 3) Primary screening: Spread the bacterial solution from step 2) on a solid medium containing 70-110 μg / ml kanamycin and culture at a constant temperature until single colonies appear. Pick out the single colonies with good growth and inoculate them into the fermentation medium for protein expression. Select several strains with high expression of the target protein as primary screening strains;

[0009] 4) Rescreening: The bacterial liquid of each primary screening strain in step 3) is spread on a solid culture medium containing 125-200 μg / ml kanamycin and cultured at a constant temperature until a single colony appears. A single colony with good growth is selected and inoculated into a fermentation medium for protein expression. Several strains with high expression of the target protein are selected as rescreening strains;

[0010] 5) Evaluation of the stability of the passage: The strains screened in step 4) were passaged, and each generation of strains was inoculated into the fermentation medium for protein expression. The plasmid retention rate and target protein expression level of each generation of strains were detected, and strains with a plasmid retention rate of ≥90% and high expression level for multiple passages were selected as candidate high-yield strains;

[0011] Among them, in step 3) to step 5), kanamycin is not added during the process of inoculating the strain into the fermentation medium for protein expression and rescreening the strain for subculture.

[0012] In some embodiments, the concentration of kanamycin in step 3) is selected from 70 μg / ml, 90 μg / ml or 110 μg / ml, preferably 70 μg / ml.

[0013] In some embodiments, the kanamycin concentration in step 4) includes a plurality of concentration gradients selected from the range of 125-200 μg / ml, such as at least 2, at least 3, at least 4 concentration gradients, or 4 concentration gradients. In a specific embodiment, the kanamycin concentration in step 4) includes 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml.

[0014] In some embodiments, the E. coli is HB101.

[0015] In some embodiments, the plasmid comprises an inducible plasmid. In some embodiments, the plasmid is selected from pThioHisA, and the ampicillin resistance gene segment on the pTDa plasmid is replaced with a kanamycin resistance gene segment.

[0016] In some embodiments, the teriparatide encoding gene comprises the nucleotide sequence shown below: TCTGTATCTGAAATCCAACTGATGCACAACCTGGGTAAACACCTGAACTCTATGGAACGTGTAGAATGGCTGCGTAAAAAACTGCAGGATGTACACAACTTC. The teriparatide encoding gene comprises the sequence shown in SEQ ID NO.2.

[0017] In some embodiments, the step 1) comprises: synthesizing the DNA fragment shown in SEQ ID NO.2, and inserting it into the pThioHisA vector using EcoRI and SalI restriction sites to obtain plasmid A; then using plasmid A as a template, designing primers (SEQID NO.3-4) for PCR amplification to obtain a linear plasmid fragment with the ampicillin resistance gene deleted; finally, the linear plasmid fragment is connected with the kanamycin resistance gene fragment by homologous recombination, and the connection product is transformed into Escherichia coli DH5α competent cells, and positive monoclonal colonies are picked for plasmid extraction and sequencing identification to obtain a teriparatide expression plasmid with a kanamycin resistance marker.

[0018] In some embodiments, the kanamycin resistance gene fragment is derived from pET28a(+) plasmid. In a specific embodiment, the step 1) further comprises: using pET28a(+) plasmid as a template, designing primers (SEQ ID NO.5-6) ​​for PCR amplification to obtain the kanamycin resistance gene fragment.

[0019] In some embodiments, the solid culture medium is LB solid culture medium. The fermentation medium includes LB liquid culture medium and TB liquid culture medium.

[0020] In some embodiments, picking up single colonies with good growth includes: selecting single colonies with a diameter of about 1-2 mm on each plate.

[0021] In some embodiments, inoculating the strain into a fermentation medium for protein expression comprises: inoculating the strain into LB liquid medium and culturing at a constant temperature until the OD 600 Reach 1.0±0.2; then transfer to TB liquid culture medium at a ratio of 2% and culture at constant temperature and shake until the bacterial OD 600 When it reached 1.0±0.2, IPTG solution was added to a final concentration of 0.3 mM to induce protein expression. The fermentation was terminated after 3-4 h, and the total protein and target protein were detected by SDS-PAGE gel electrophoresis.

[0022] In some embodiments, the subculture of step 4) includes: taking the re-screened strain and inoculating it in a culture medium for constant temperature shaking culture, and when the cell density OD 6oo The culture was recorded as P1 seed solution when 1-2 hours were reached. Then, the P1 seed solution was inoculated into the culture medium at a ratio of 2% and cultured at a constant temperature with shaking until OD 6oo is 1-2, recorded as P2 seed culture medium; and so on for multiple subcultures.

[0023] In some embodiments, step 3) includes selecting several strains with target protein expression levels ≥ 20% as primary screening strains.

[0024] In some embodiments, step 4) includes: selecting several strains with target protein expression ≥ 25% as rescreening strains. In some embodiments, step 4) includes: selecting strains that can grow simultaneously under at least 3-4 kanamycin concentration gradients and have high target protein expression as rescreening strains. In a specific embodiment, step 4) includes: selecting strains that can grow simultaneously under 125μg / ml, 150μg / ml, 175μg / ml and 200μg / ml kanamycin and have high target protein expression as rescreening strains.

[0025] In some embodiments, the passage number is ≥ 10, ≥ 11, ≥ 12, ≥ 13, ≥ 14, or ≥ 15. In a specific embodiment, the passage number is 15.

[0026] In a specific embodiment, the method comprises:

[0027] 1) Plasmid construction: synthesize the DNA fragment shown in SEQ ID NO.2, and insert it into the pThioHisA vector using the EcoRI and SalI restriction sites to obtain plasmid A; then use plasmid A as a template, design primers (SEQ ID NO.3-4) for PCR amplification, and obtain a linear plasmid fragment with the ampicillin resistance gene deleted; finally, the linear plasmid fragment is connected with the kanamycin resistance gene fragment by homologous recombination, and the connection product is transformed into Escherichia coli DH5α competent cells, and the positive monoclonal colonies are picked for plasmid extraction and sequencing identification to obtain a teriparatide expression plasmid with a kanamycin resistance marker;

[0028] 2) Transformation: The expression plasmid constructed in step 1) is transformed into competent E. coli cells HB101, and cultured to obtain a recombinant bacterial liquid;

[0029] 3) Primary screening: Spread the bacterial solution from step 2) on a solid culture medium containing 70 μg / ml kanamycin and culture at a constant temperature until single colonies appear. Pick single colonies with a diameter of about 1-2 mm on the plate and inoculate them into fermentation medium for protein expression. Select several strains with high expression of the target protein as primary screening strains;

[0030] 4) Rescreening: Take the bacterial liquid of each primary screening strain in step 3), and spread each strain on a solid culture medium containing 125μg / ml, 150μg / ml, 175μg / ml and 200μg / ml kanamycin for constant temperature culture until a single colony appears, pick a single colony with a diameter of about 1-2 mm on each plate and inoculate it into a fermentation medium for protein expression, and select the strain that can grow under 125μg / ml, 150μg / ml, 175μg / ml and 200μg / ml kanamycin at the same time and has a high expression of the target protein as the rescreening strain;

[0031] 5) Evaluation of the stability of the passage: The strains screened in step 4) were passaged, and each generation of strains was inoculated into the fermentation medium for protein expression. The plasmid retention rate and target protein expression level of each generation of strains were detected, and strains with a plasmid retention rate of ≥90% after 15 passages and a high target protein expression level were selected as candidate high-yield strains;

[0032] Among them, in step 3) to step 5), kanamycin is not added during the process of inoculating the strain into the fermentation medium for protein expression and rescreening the strain for subculture.

[0033] The beneficial effects of the present invention include:

[0034] 1. For E. coli HB101 expressing teriparatide with pThioHisA vector, appropriate resistance screening markers and screening concentrations were selected to ensure appropriate screening pressure, thereby more effectively selecting stable and high-yield positive transformants;

[0035] 2. The present invention explores for the first time the feasibility of using high-concentration kanamycin for strain screening. In the initial screening, kanamycin is slightly higher than the conventional level. By appropriately increasing the screening pressure, the proportion of strains with high expression potential in the positive transformants is increased, thereby improving the screening efficiency. In the rescreening, by further increasing the kanamycin concentration, the strain production capacity is increased while obtaining genetically stable engineered strains, thereby reducing the probability of plasmid loss during the subculture process.

[0036] 3. In the present invention, strains that can grow at 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin and have high expression of the target protein are selected for further screening in the rescreening. The high expression in the primary screening and the expression under multiple concentration gradients in the rescreening are mutually confirmed, which effectively ensures the reliability of the selected rescreening strains in terms of stability;

[0037] 4. In the process of large-scale fermentation experiments, the present invention effectively screens strains with high genetic stability by detecting the plasmid retention rate and expression level of the strains without adding antibiotics;

[0038] 5. The present invention determines suitable strain screening conditions and methods. Compared with conventional screening methods, the obtained engineered bacteria significantly increase the production of teriparatide in Escherichia coli HB101 under culture conditions without adding kanamycin, and keep the plasmid stable during the fermentation process. When it is passed to the 15th generation, the plasmid retention rate is ≥90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The gel electrophoresis results of the amplified bands are shown, wherein lane M and lane 6 are DNA molecular weight markers (DNA marker), lane 2 is amplified product 2, and lane 4 is amplified product 1.

[0040] Figure 2 A map of plasmid B is shown.

[0041] Figure 3 The results of the initial screening fermentation are shown, with the dotted line indicating 20%.

[0042] Figure 4 Results of repeated fermentations are shown, with the dotted line indicating 25%.

[0043] Figure 5 The plasmid retention rate of each generation of strains in subculture is shown.

[0044] Figure 6 The results of fermentation tank scale-up production are shown, where "-" represents no kanamycin added during seed solution preparation and fermentation tank culture; "+" represents the addition of 70 μg / ml kanamycin during seed solution preparation and fermentation tank culture; the vertical axis yield unit is mg / g inclusion body.

[0045] Figure 7 The results of SDS-PAGE electrophoresis are shown, wherein lane 1 is a protein molecular weight standard marker (Marker), and lanes 2 to 4 are protein products of B70-02A (P15), B110-07C (P15) and B70-02A (fermentation tank - without kanamycin), respectively. DETAILED DESCRIPTION

[0046] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0047] The following examples were all performed under a sterile environment, and the reagents, test methods, and calculation methods involved include:

[0048] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, deionized water;

[0049] LB solid medium: add 15g / L~20g / L agar powder to LB liquid medium;

[0050] TB liquid medium: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 ml / L, potassium dihydrogen phosphate 2.31 g / L, potassium hydrogen phosphate 16.43 g / L, deionized water;

[0051] Target protein expression % = target protein / total protein × 100%.

[0052] Example 1 Plasmid construction and host bacteria transformation

[0053] Based on the amino acid sequence of teriparatide (PTH 1-34) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO. 1), the preferred codons of Escherichia coli were selected to design the corresponding gene coding sequence: 5'- GAATTCCTGGTTCCGCGTTCTGTATCTGAAATCCAACTGATGCACAACCTGGGTAAACACCTGAACTCTATGGAACGTGTAGAATGGCTGCGTAAAAAAACTGCAGGATGTACACAACTTCTAA GTCGAC -3' (SEQ ID NO.2), wherein the underline is the restriction site, the italic is the thrombin cleavage site, and the 3' end contains a TAA terminator. Then, referring to the method described in Example 1 of patent document CN102399285B (CN102399285B is incorporated herein by reference in its entirety), the aforementioned coding gene was artificially synthesized and inserted into the expression vector pThioHisA using EcoRI and SalI restriction sites to construct an expression plasmid A of recombinant teriparatide, which has an ampicillin resistance gene (AmpR).

[0054] By means of genetic engineering, the ampicillin resistance gene on the expression plasmid A was replaced with the kanamycin resistance gene (KanR) to obtain another recombinant teriparatide expression plasmid B. In short, using plasmid A as a template, PCR amplification was performed with primers 1 and 2, and then separated by agarose gel electrophoresis, recovered by gel cutting, and purified using a gel extraction kit to obtain amplification product 1 (a linear fragment of plasmid A with AmpR deleted). Using pET28a (+) plasmid as a template, PCR amplification was performed with primers 3 and 4, and then separated by agarose gel electrophoresis, recovered by gel cutting, and purified using a gel extraction kit to obtain amplification product 2 (KanR fragment). Then, a one-step directional cloning kit (NovoRec, NR005) was used to homologously recombine amplification product 1 and amplification product 2, and the ligation product was transformed into Escherichia coli DH5α competent cells, and positive monoclonal colonies were picked for plasmid extraction, and further sequencing analysis was performed to ensure that no unexpected mutations occurred in the vector elements, thereby obtaining plasmid B, the plasmid map of which is shown in Figure 2 Thioredoxin, as a fusion partner of teriparatide PTH(1-34), can catalyze the renaturation of the fusion protein, which is subsequently removed through the thrombin cleavage site at the N-terminus of teriparatide.

[0055] Figure 1 The gel electrophoresis results of the amplified products are shown. The fragment size of amplified product 1 is about 3.7 kb, and the fragment size of amplified product 2 is about 0.8 kb, both of which are in line with expectations. The sequences of primers 1-4 are as follows:

[0056] Primer 1: 5'-CCGTTGAATATGGCTCATACTCTTCCTTTTTCAATA-3' (SEQ ID NO.3);

[0057] Primer 2: 5′-CTCGATGAGTTTTTCTAACTGTCAGACCAAGTTTAC-3′ (SEQ ID NO. 4);

[0058] Primer 3: 5′-GTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAG-3′ (SEQ ID NO. 5);

[0059] Primer 4: 5'-TATTGAAAAAGGAAGAGTATGAGCCATATTCAACGG-3' (SEQ ID NO. 6).

[0060] Using the heat shock method, expression plasmid A and expression plasmid B were transferred into Escherichia coli HB101 competent cells, respectively, to construct engineered bacteria A with ampicillin resistance and engineered bacteria B with kanamycin resistance. In short, add plasmids (DNA content does not exceed 100ng) to 100μl competent cells, shake gently, place on ice, and let stand for at least 15 min. After gently shaking, place in a 42 ℃ water bath for 1min for heat shock, then quickly put in ice, let stand for 2min, then add 400 μl LB liquid culture medium and mix gently, and culture at 37.0 ℃, 220 rpm for 1-2h.

[0061] Example 2 Screening of high-yield engineering bacteria using resistance markers

[0062] 2.1 Initial screening

[0063] Take 10 μl of the culture solution of the engineered bacteria B in Example 1, and spread them on LB solid culture medium plates containing 50 μg / ml, 70 μg / ml, 90 μg / ml and 110 μg / ml kanamycin, and culture them at 37.0°C for 18-20 hours. After the culture is completed, pick the single colonies grown on each plate for fermentation verification. Specifically, select single colonies with a diameter of about 1-2 mm on each plate, randomly pick 10 colonies / plate, and inoculate them in LB liquid culture medium at 37°C and shake and culture until OD 600 When the OD value reaches 1.0±0.2, transfer 1 ml to a shake flask containing 50 ml TB liquid medium, and freeze the remaining bacterial liquid in glycerol. Shake flask culture was carried out at 37.0℃ and 220rpm. 600 When the concentration reached 1.0±0.2, IPTG solution was added to a final concentration of 0.3 mM to induce the expression of the target protein. After 4 h, the fermentation was terminated and the bacterial OD was measured. 600 And use SDS-PAGE gel electrophoresis to detect total protein and target protein, and calculate the expression level of target protein.

[0064] Figure 3The results of the primary screening fermentation are shown, where B50 represents 10 single colonies picked from a 50 μg / ml kanamycin plate, named B50-01 to B50-10 from top to bottom; B70 represents 10 single colonies picked from a 70 μg / ml kanamycin plate, named B70-01 to B70-10 from top to bottom; the meanings of B90 and B110 and the strain naming are similar.

[0065] According to the initial screening fermentation results, it can be seen that the engineered bacteria B constructed by the present invention has good tolerance to kanamycin and can be screened at high concentrations. In addition, increasing the kanamycin concentration is also conducive to the screening of high-expressing strains. Figure 3 It can be seen that under the initial screening condition of 50 μg / ml, only a small number of transformants had an expression level of more than 20%, while increasing the screening concentration to at least 70 μg / ml significantly increased the proportion of high-expressing strains among the transformants. The proportion of strains with an expression level ≥ 20% increased from 30% at 50 μg / ml to 60% at 70 μg / ml, and transformants with an expression level of more than 30% were identified at screening concentrations of 70-110 μg / ml. It can be seen that transformants that can tolerate high-concentration kanamycin screening conditions may also have stronger protein expression potential.

[0066] From each screening condition, several highly expressed transformants were selected as candidate strains for rescreening. Table 1 shows the fermentation results of the primary screened candidate strains. The expression level of each strain was at least 20%, and the bacterial density showed that these strains grew well without showing obvious growth inhibition.

[0067] Table 1

[0068] Strain number <![CDATA[Cell density OD 600 > Target protein expression % B110-09 6.74 34.3 B110-08 6.73 42.4 B110-07 6.63 41.1 B110-06 8.03 35.3 B110-03 7.83 36.4 B110-01 8.09 38.6 B90-10 6.08 25.0 B90-07 6.34 24.5 B90-06 5.99 30.0 B70-08 6.87 30.3 B70-07 6.75 31.9 B70-03 6.59 26.3 B70-02 6.23 28.3 B50-08 6.00 24.1

[0069] 2.2 Rescreening

[0070] Resuscitate the candidate strains stored in the initial screening and dilute to 1×10 4 cfu / mL. For each candidate strain, 50 μl was spread on LB solid medium plates containing 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml kanamycin, and cultured at 37.0°C for 18-20 hours. After the culture was completed, single colonies with a diameter of about 1-2 mm on each plate were picked and inoculated into LB liquid medium at 37°C for shaking culture until OD 600 When the OD value reaches 1.0±0.2, transfer 1 ml to a shake flask containing 50 ml TB liquid medium, and freeze the remaining bacterial liquid in glycerol. Shake flask culture was carried out at 37.0℃ and 220 rpm. 600When the concentration reached 1.0±0.2, IPTG solution was added to a final concentration of 0.3 mM to induce the expression of the target protein. After 3 h, the fermentation was terminated and the bacterial OD was measured. 600 And use SDS-PAGE gel electrophoresis to detect total protein and target protein, and calculate the expression level of target protein.

[0071] Figure 4 The results of repeated fermentation are shown, among which B110-09 represents single colonies picked from 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml kanamycin plates, from left to right they are single colonies under 125 μg / ml screening conditions and named B110-09A, single colonies under 150 μg / ml screening conditions and named B110-09B, single colonies under 175 μg / ml screening conditions and named B110-09C, and single colonies under 200 μg / ml screening conditions and named B110-09D; B110-08 represents single colonies picked from 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml kanamycin plates, from left to right they are 125 The single colony under the screening conditions of 100 μg / ml was named B110-08A, the single colony under the screening conditions of 150 μg / ml was named B110-08B, the single colony under the screening conditions of 175 μg / ml was named B110-08C, and the single colony under the screening conditions of 200 μg / ml was named B110-08D; the meanings and names of other strains are deduced by analogy.

[0072] It can be seen that after rescreening, only a small number of strains can stably retain high expression performance. Several high-expression rescreened strains were selected as candidate strains for passage stability evaluation. Table 2 shows the fermentation results of the rescreened candidate strains. The expression level of each strain was at least 25%, and the bacterial density showed that the growth of these strains was normal.

[0073] Table 2

[0074] Strain number <![CDATA[Fermentation broth cell density OD 600 > Target protein expression % B110-07C 6.45 33.4 B110-07D 6.48 30.9 B110-06C 7.01 25.4 B110-01A 6.89 28.6 B110-01B 6.77 27.7 B70-08D 6.56 27.0 B70-02A 6.40 36.7 B70-02D 6.31 35.9

[0075] 2.3 Study on the stability of cell culture

[0076] Continuous shaking flask subculture was used to evaluate the stability of the plasmid. After reviving each candidate strain, 1 ml was taken and inoculated into 50 mL LB liquid medium. The culture was shaken at 37°C and 220 rpm. When the bacterial cell density OD 6ooWhen the culture reaches 1-2 hours, it is recorded as the P1 generation seed liquid. A small amount of seed liquid is first frozen, and then an appropriate concentration of IPTG solution is added to induce the expression of the target protein. After 3 hours, the fermentation is terminated and the fermentation liquid is stored. Take 1 ml of the P1 generation seed liquid and inoculate it into 50 ml of LB liquid medium. Cultivate at 37°C and 220 rpm with shaking until the OD reaches 0. 6oo 1-2, recorded as P2 generation seed culture solution, first take a small amount of seed solution and freeze it, then induce expression and store the fermentation solution according to the above operation. And so on, carry out subculture and fermentation until P15 generation.

[0077] Detect the plasmid retention rate of each generation of strains. Take appropriate amount of seed liquid (bacterial density 1×10 2 -1×10 3 cfu / mL) was spread on LB solid culture medium plates and cultured at 37°C overnight. When colonies grew on the plates, 96 single colonies were randomly picked and each single colony was inoculated into 96-well plates containing kanamycin (50 μg / ml) and LB medium without kanamycin, respectively. The number of surviving wells in the 96-well plates was counted after culturing at 37°C overnight. The plasmid retention rate was calculated as follows:

[0078] Plasmid retention rate = number of surviving wells containing kanamycin / number of surviving wells without kanamycin × 100%.

[0079] The results are shown in Table 3. Figure 5 As shown in the figure, most of the preferred strains can be stably subcultured for multiple times, among which B70-02A and B70-02D maintained a plasmid retention rate of 100% in the 15th generation strains, and their progeny still had a high production capacity. The induced expression product was identified by SDS-PAGE electrophoresis to be approximately 18.7 kDa, which is consistent with the theoretical value ( Figure 7 , lanes 2 and 3).

[0080] Table 3

[0081] Strain number The number of passages with a plasmid retention rate of ≥90% P2 Target protein expression % P15 Target protein expression % B110-07C 15 35.7 23.9 B110-07D 14 29.3 22.3 B110-06C 8 21.4 ＜15 B110-01A 15 22.5 ＜15 B110-01B 9 27.5 ＜15 B70-08D 12 22.7 ＜15 B70-02A 15 35.9 25.2 B70-02D 15 27.2 29.2

[0082] Example 3 Fermentation tank scale-up production

[0083] 3.1 Preparation of Grade I Seed Solution

[0084] The candidate high-yield strains B70-02A, B70-02D, B110-07C and some of the initial screening strains B70-02 and B50-08 were taken out from the -80℃ refrigerator, thawed in a 37℃ water bath, and 1 ml was inoculated into a shake flask containing 50 ml LB liquid culture medium (2 bottles / strain), and cultured at 37℃ and 220 rpm until OD 600 Reach 1.0±0.2.

[0085] 3.2 Preparation of Grade II Seed Solution

[0086] Transfer the Class I seed solution to a shake flask containing 650 ml LB liquid medium (4 flasks / plant) at a ratio of 3% and culture at 37°C and 220 rpm until the OD 600 Reach 1.0±0.2.

[0087] 3.3 Fermentation tank culture

[0088] Take 2.5L of the II seed solution and inoculate it into a 100L fermenter containing 22.5L of TB liquid medium. Control the temperature, dissolved oxygen, and pH within the appropriate range. 600 When the temperature reaches 12.00-14.00, add IPTG of appropriate concentration to induce expression, and add feed at a uniform rate, and end the fermentation after 3-5 hours.

[0089] 3.4 Protein purification

[0090] After the fermentation, the cells were collected by centrifugation, the cells were broken and the inclusion bodies were collected, and then the teriparatide stock solution was prepared by chromatography, ultrafiltration and other steps (for specific methods, refer to CN102399285B). The purity of the stock solution was tested by HPLC, and the purity of teriparatide was as high as more than 98%.

[0091] The yield of pure teriparatide was as follows Figure 6 As shown, it can be seen that the method for screening Escherichia coli strains producing teriparatide established in the present invention can effectively obtain stable and high-yield production strains, and the fermentation process is free from dependence on antibiotics. Further, the biological activity of the teriparatide stock solution prepared in this example was tested, and the results showed that it was consistent with the in vitro biological activity of the teriparatide national standard (activity test method reference CN102399285B).

[0092] Comparative Example 1

[0093] The culture solution of the ampicillin-resistant engineered bacteria A constructed in Example 1 was taken, and the engineered bacteria were screened and fermented according to the method described in Examples 2-3, except that kanamycin was replaced with ampicillin. The results showed that the expression level of the target protein of the preferred engineered bacteria was only 20.6%, and the plasmid could be stable to the 10th generation in the passage stability evaluation.

[0094] On the one hand, it is speculated that since the resistance gene fragment of ampicillin is larger than that of kanamycin, the engineered bacteria A has a recombinant plasmid with a larger molecular weight, which makes the plasmid of the engineered bacteria A more prone to division instability and plasmid loss during the subculture process, resulting in low yield; on the other hand, it is speculated that the above screening concentration is not suitable for ampicillin, resulting in the inability to obtain a stable and high-yield strain.

[0095] Comparative Example 2

[0096] The revived strains B70-02A, B70-02D, B70-02, B110-08, and B50-08 were evaluated for passage stability according to the method described in Example 2.3. It can be seen that the screening method of the present invention can more effectively obtain high-yield and plasmid-stable engineered strains compared to conventional single-round, low antibiotic concentration pressure screening.

[0097] Table 4

[0098] Strain number The number of passages with a plasmid retention rate of ≥90% P15 Target protein expression % B110-08 11 ＜15 B50-08 8 ＜15 B70-02 11 ＜15 B70-02A 15 ＞25 B70-02D 15 ＞25

[0099] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for screening teriparatide high-producing bacteria using antibiotics, characterized in that: The method comprises: 1) Plasmid construction: constructing a teriparatide expression plasmid, wherein the plasmid contains a kanamycin resistance gene and a teriparatide encoding gene; 2) Transformation: The expression plasmid constructed in step 1) is transformed into competent E. coli cells and cultured to obtain a recombinant bacterial solution; 3) Primary screening: Spread the bacterial solution from step 2) on a solid medium containing 70-110 μg / ml kanamycin and culture at a constant temperature until single colonies appear. Pick out the single colonies with good growth and inoculate them into the fermentation medium for protein expression. Select several strains with high expression of the target protein as primary screening strains; 4) Rescreening: The bacterial liquid of each primary screening strain in step 3) is spread on a solid culture medium containing 125-200 μg / ml kanamycin and cultured at a constant temperature until a single colony appears. A single colony with good growth is selected and inoculated into a fermentation medium for protein expression. Several strains with high expression of the target protein are selected as rescreening strains; 5) Evaluation of the stability of the passage: The strains screened in step 4) were passaged, and each generation of strains was inoculated into the fermentation medium for protein expression. The plasmid retention rate and target protein expression level of each generation of strains were detected, and strains with a plasmid retention rate of ≥90% and high expression level for multiple passages were selected as candidate high-yield strains; Among them, in step 3) to step 5), kanamycin is not added during the process of inoculating the strain into the fermentation medium for protein expression and rescreening the strain for subculture.

2. The method according to claim 1, characterized in that The kanamycin concentration in step 3) is selected from 70 μg / ml, 90 μg / ml or 110 μg / ml.

3. The method according to any one of claims 1 to 2, characterized in that: The kanamycin concentration in step 4) includes a plurality of concentration gradients selected from the range of 125-200 μg / ml.

4. The method according to claim 3, characterized in that The Escherichia coli is HB101.

5. The method according to claim 4, characterized in that The plasmid is selected from pThioHisA, and the ampicillin resistance gene fragment on the pThioHisA plasmid is replaced with a kanamycin resistance gene fragment.

6. The method according to claim 5, characterized in that Step 3) includes selecting several strains with target protein expression levels ≥ 20% as primary screening strains.

7. The method according to claim 6, characterized in that Step 4) includes: selecting several strains with target protein expression levels ≥ 25% as re-screening strains.

8. The method according to claim 7, characterized in that Step 4) includes: selecting a strain that can grow simultaneously under at least 3-4 kanamycin concentration gradients and has a high expression level of the target protein as a re-screening strain.

9. The method according to claim 8, characterized in that The passage number is ≥10.

10. The method according to claim 9, characterized in that The method comprises: 1) Plasmid construction: synthesize the DNA fragment shown in SEQ ID NO.2, and insert it into the pThioHisA vector using the EcoRI and SalI restriction sites to obtain plasmid A; then use plasmid A as a template, design primers for PCR amplification, and obtain a linear plasmid fragment with the ampicillin resistance gene deleted; finally, the linear plasmid fragment is homologously recombined with the kanamycin resistance gene fragment, and the ligation product is transformed into Escherichia coli DH5α competent cells, and the positive monoclonal colonies are picked for plasmid extraction and sequencing identification to obtain a teriparatide expression plasmid with a kanamycin resistance marker; 2) Transformation: The expression plasmid constructed in step 1) is transformed into competent E. coli cells HB101, and cultured to obtain a recombinant bacterial liquid; 3) Primary screening: Spread the bacterial solution from step 2) on a solid medium containing 70 μg / ml kanamycin and culture at a constant temperature until single colonies appear. Pick single colonies with a diameter of 1-2 mm on the plate and inoculate them into fermentation medium for protein expression. Select several strains with high expression of the target protein as primary screening strains; 4) Rescreening: Take the bacterial liquid of each primary screening strain in step 3), and spread each strain on a solid culture medium containing 125μg / ml, 150μg / ml, 175μg / ml and 200μg / ml kanamycin for constant temperature culture until a single colony appears, pick a single colony with a diameter of 1-2 mm on each plate and inoculate it into a fermentation medium for protein expression, and select the strain that can grow under 125μg / ml, 150μg / ml, 175μg / ml and 200μg / ml kanamycin at the same time and has a high expression of the target protein as the rescreening strain; 5) Evaluation of the stability of the passage: The strains screened in step 4) were passaged, and each generation of strains was inoculated into the fermentation medium for protein expression. The plasmid retention rate and target protein expression level of each generation of strains were detected, and strains with a plasmid retention rate of ≥90% after 15 passages and a high target protein expression level were selected as candidate high-yield strains; Among them, in step 3) to step 5), kanamycin is not added during the process of inoculating the strain into the fermentation medium for protein expression and rescreening the strain for subculture.

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