A method for targeted screening of teriparatide-producing bacteria using antibiotics

Abstract

This invention discloses a method for targeted screening of teriparatide-producing bacteria using antibiotics. Through two rounds of screening with different concentrations of antibiotics, E. coli strains capable of high teriparatide expression are obtained, and the plasmid stability is effectively maintained during strain passage without the addition of antibiotics. This targeted screening method not only effectively improves the screening efficiency of high teriparatide-producing bacteria but also overcomes the technical difficulty of adding antibiotics during the production of biopharmaceuticals.

Description

Technical Field

[0001] This invention relates to the field of bacterial strain screening, and more specifically to a method for targeted screening of bacterial strains using antibiotics. Background Technology

[0002] Genetically engineered bacteria are miniature bioreactors in modern bioengineering. Obtaining genetically engineered bacteria capable of efficiently and stably expressing exogenous genes is crucial for the fermentation industry. The main host expression systems used in genetically engineered bacteria include *Escherichia coli*, which can express and produce products such as peptides, amino acids, growth factors, and recombinant proteins through transfection with plasmid vectors. Plasmid vectors are generally used to identify positive transformants and maintain their segregation stability within the strain through antibiotic selection markers. Studies have shown that plasmid loss is significant in the late logarithmic growth phase, and the expression of exogenous genes further increases plasmid instability. Adding antibiotics to reduce plasmid loss is a common technique in fermentation, such as adding kanamycin, ampicillin, or tetracycline to the culture medium. Kanamycin is an aminoglycoside antibiotic isolated from *Streptomyces kanamyceticus*. As a protein synthesis inhibitor, its working concentration is generally 10–50 μg / ml.

[0003] While process development continues to advance, strain screening technology remains a crucial factor determining strain performance. Establishing stable, high-yielding strains is essential to maintain stability from seed bank establishment to the required number of generations for large-scale fermentation. The stability of each plasmid varies, influenced by factors such as host bacterial characteristics, plasmid copy number, inserted DNA size, and culture conditions. For specific production strains, it is necessary to determine the most suitable screening conditions to efficiently and directionally screen stable, high-yielding engineered strains. Secondly, the use of antibiotics should be avoided as much as possible in drug production. Antibiotics not only easily pollute the environment and generate drug-resistant bacteria, but also require removal from the finished product in subsequent processes, further increasing production costs and complicating processes. How to achieve stable plasmid existence in engineered bacteria and high expression of target proteins under antibiotic-free culture conditions is a pressing problem that needs to be solved in this field. Summary of the Invention

[0004] This invention overcomes the problems of existing technologies by proposing a method for targeted screening of high-yield teriparatide-producing bacteria using antibiotics. The method involves transfecting host bacteria with an expression plasmid containing both a kanamycin resistance gene and a teriparatide encoding gene. Through two rounds of screening with different concentrations of kanamycin and fermentation verification, genetically engineered bacteria capable of high teriparatide production are obtained, and the plasmid's stable inheritance is effectively maintained even under culture conditions without the addition of kanamycin. This screening method not only effectively improves the screening efficiency of high-yield teriparatide strains but also avoids antibiotic residue problems during production, further increasing teriparatide yield.

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

[0006] 1) Plasmid construction: Construct a teriparatide expression plasmid containing the kanamycin resistance gene and the teriparatide encoding gene;

[0007] 2) Transformation: The expression plasmid constructed in step 1) was transformed into competent E. coli cells, and the recombinant bacterial culture was obtained.

[0008] 3) Initial screening: Spread the bacterial culture from step 2) onto a solid medium containing 70-110 μg / ml kanamycin and incubate at a constant temperature until single colonies appear. Select well-grown single colonies and inoculate them into fermentation medium for protein expression. Select several strains with high expression levels of the target protein as initial screening strains.

[0009] 4) Secondary screening: Take the bacterial culture of each primary screening strain in step 3) and spread it on a solid medium containing 125-200 μg / ml kanamycin. Incubate at a constant temperature until a single colony appears. Select a single colony with good growth and inoculate it into a fermentation medium for protein expression. Select a number of strains with high expression levels of the target protein as secondary screening strains.

[0010] 5) Passage stability assessment: Passage each of the rescreened strains in step 4), and inoculate each generation of strains into fermentation medium for protein expression. Detect the plasmid retention rate and target protein expression level of each generation of strains. Select strains with plasmid retention rate ≥90% and high expression level after multiple passages as candidate high-yield strains.

[0011] In steps 3) to 5), no kanamycin is added during the process of inoculating the strain into the fermentation medium for protein expression and during the passage of the re-screened strain.

[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 multiple concentration gradients selected from the range of 125-200 μg / ml, such as at least two, at least three, at least four concentration gradients, or four concentration gradients. In one 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 implementations, the Escherichia coli is HB101.

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

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

[0017] In some embodiments, step 1) includes: 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 (SEQ ID NO.3-4) for PCR amplification to obtain a linear plasmid fragment with the ampicillin resistance gene deleted; finally, performing homologous recombination ligation of the linear plasmid fragment with the kanamycin resistance gene fragment, transforming the ligation product into Escherichia coli DH5α competent cells, picking positive single colonies 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 the pET28a(+) plasmid. In one specific embodiment, step 1) further includes: using the 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 implementations, selecting well-grown single colonies involves selecting single colonies with a diameter of approximately 1-2 mm from each plate.

[0021] In some implementations, inoculation into the fermentation medium for protein expression includes: inoculating the strain into LB liquid medium and incubating with constant temperature and shaking until OD... 600 The bacterial OD value reached 1.0 ± 0.2; then, it was transferred to TB liquid medium at a ratio of 2% and cultured under constant temperature and shaking until the bacterial OD value reached 1.0 ± 0.2. 600 When the concentration reached 1.0 ± 0.2, IPTG solution was added to a final concentration of 0.3 mM to induce protein expression. Fermentation was stopped after 3-4 hours, and total protein and target protein were detected by SDS-PAGE gel electrophoresis.

[0022] In some implementations, step 4) of passage includes: inoculating the screened strain into a culture medium and incubating with constant temperature and shaking until the cell density OD reaches a certain level. 6oo When cultured for 1-2 days, this is recorded as the P1 generation seed culture; then, take 2% of the P1 generation seed culture and inoculate it into the culture medium, and culture it at a constant temperature with shaking until OD. 6oo This is referred to as generation P2 seed culture medium; and so on, for multiple generations.

[0023] In some implementations, step 3) includes selecting several strains with a target protein expression level ≥20% as initial screening strains.

[0024] In some embodiments, step 4) includes: selecting several strains with a target protein expression level ≥25% as secondary screening strains. In some embodiments, step 4) includes: selecting strains capable of growing simultaneously at at least 3-4 kanamycin concentration gradients and exhibiting high target protein expression levels as secondary screening strains. In one specific embodiment, step 4) includes: selecting strains capable of growing simultaneously at kanamycin concentrations of 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml and exhibiting high target protein expression levels as secondary screening strains.

[0025] In some implementations, the number of generations is ≥10, ≥11, ≥12, ≥13, ≥14, or ≥15. In one specific implementation, the number of generations is 15.

[0026] In one specific implementation, the method includes:

[0027] 1) Plasmid construction: The DNA fragment shown in SEQ ID NO.2 was synthesized and inserted into the pThioHisA vector using EcoRI and SalI restriction sites to obtain plasmid A; then, using plasmid A as a template, primers (SEQ ID NO.3-4) were designed for PCR amplification to obtain a linear plasmid fragment with the ampicillin resistance gene deleted; finally, the linear plasmid fragment was homologously ligated with the kanamycin resistance gene fragment, and the ligation product was transformed into Escherichia coli DH5α competent cells. Positive single colonies were 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) was transformed into E. coli competent cells HB101, and the recombinant bacterial culture was obtained.

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

[0030] 4) Secondary screening: Take the bacterial culture of each strain from the initial screening in step 3), and spread each strain on solid medium containing 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin respectively. Incubate at constant temperature until single colonies appear. Pick single colonies with a diameter of about 1-2 mm from each plate and inoculate them into fermentation medium for protein expression. Select strains that can grow at 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin and have high expression levels of the target protein as secondary screening strains.

[0031] 5) Passage stability assessment: Passage each of the secondary screening strains in step 4), and inoculate each generation of strains into fermentation medium for protein expression. Detect the plasmid retention rate and target protein expression level of each generation of strains. Select strains with a plasmid retention rate ≥90% and high target protein expression level after 15 passages as candidate high-yield strains.

[0032] In steps 3) to 5), no kanamycin is added during the process of inoculating the strain into the fermentation medium for protein expression and during the passage of the re-screened strain.

[0033] The beneficial effects of this invention include:

[0034] 1. For Escherichia coli HB101 expressing teriparatide using the pThioHisA vector, appropriate resistance selection markers and selection concentrations were selected to ensure appropriate selection pressure, thereby more effectively and directionally selecting stable and high-yielding positive transformants.

[0035] 2. This invention explores for the first time the feasibility of using high concentrations of kanamycin for bacterial strain screening. In the initial screening, the kanamycin concentration is slightly higher than the conventional level. By appropriately increasing the screening pressure, the proportion of strains with high expression potential in positive transformants is increased, thereby improving the screening efficiency. In the secondary screening, by further increasing the kanamycin concentration, the strain production capacity is increased, and genetically stable engineered strains are obtained, reducing the probability of plasmid loss during passage.

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

[0037] 4. In large-scale fermentation experiments, this 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. This invention has determined suitable strain screening conditions and methods. Compared with conventional screening methods, the engineered bacteria obtained significantly increased the yield of teriparatide in Escherichia coli HB101 under culture conditions without the addition of kanamycin, and the plasmid remained stable during the fermentation process. When the strain was passaged to the 15th generation, the plasmid retention rate was ≥90%. Attached Figure Description

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

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

[0041] Figure 3 The results of the initial screening fermentation are shown; the dashed line represents 20%.

[0042] Figure 4 The results of the secondary screening fermentation are shown; the dashed line represents 25%.

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

[0044] Figure 6 The results of scale-up production in the fermenter are shown, where "-" indicates that no kanamycin was added during seed culture preparation and fermenter culture; "+" indicates that 70 μg / ml kanamycin was added during seed culture preparation and fermenter culture; the vertical axis represents the yield in mg / g inclusion bodies.

[0045] Figure 7 The results of SDS-PAGE electrophoresis are shown. Lane 1 is the protein molecular weight standard marker, and lanes 2 to 4 are the protein products of B70-02A (P15), B110-07C (P15), and B70-02A (fermenter-without kanamycin), respectively. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0047] The following examples were all performed under aseptic conditions, and the reagents, testing 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 15 g / L to 20 g / 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, dipotassium hydrogen phosphate 16.43 g / L, deionized water;

[0051] Target protein expression level % = Target protein / Total protein × 100%.

[0052] Example 1: Plasmid Construction and Host Bacterial Transformation

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

[0054] Using genetic engineering, the ampicillin resistance gene on 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, followed by agarose gel electrophoresis separation, gel extraction, and purification 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, followed by agarose gel electrophoresis separation, gel extraction, and purification using a gel extraction kit to obtain amplification product 2 (KanR fragment). Then, homologous recombination ligation of amplification product 1 and amplification product 2 was performed using a one-step directional cloning kit (NovoRec, NR005). The ligation product was transformed into *E. coli* DH5α competent cells, and positive single colonies were selected for plasmid extraction and further sequencing analysis to ensure that no unwanted mutations occurred in the vector elements, thus obtaining plasmid B. Its plasmid map is shown below. Figure 2 Thioredoxin, as a fusion partner of teriparatide PTH(1-34), can catalyze the refolding of the fusion protein, which is then removed via the thrombin cleavage site at the N-terminus of teriparatide.

[0055] Figure 1 The gel electrophoresis results of the amplification products are shown. The fragment size of amplification product 1 is approximately 3.7 kb, and the fragment size of amplification product 2 is approximately 0.8 kb, both as expected. The sequences of primers 1-4 are shown below:

[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 a heat shock method, expression plasmids A and B were transformed into *E. coli* HB101 competent cells to construct engineered bacteria A with ampicillin resistance and engineered bacteria B with kanamycin resistance, respectively. In short, plasmids (DNA content not exceeding 100 ng) were added to 100 μl of competent cells, gently shaken, and placed on ice for at least 15 min. After gentle mixing, the cells were placed in a 42 ℃ water bath for 1 min for heat shock, then quickly placed on ice for 2 min. Finally, 400 μl of LB liquid medium was added, gently mixed, and incubated at 37.0 ℃ and 220 rpm for 1–2 h.

[0061] Example 2: Screening for high-yield engineered bacteria using resistance markers

[0062] 2.1 Initial screening

[0063] Take 10 μl of each culture broth of engineered bacteria B from Example 1, and spread it onto LB solid medium plates containing 50 μg / ml, 70 μg / ml, 90 μg / ml, and 110 μg / ml kanamycin, respectively, and incubate at 37.0℃ for 18-20 h. After incubation, single colonies grown on each plate are picked for fermentation verification. Specifically, select single colonies with a diameter of approximately 1-2 mm from each plate, randomly pick 10 colonies / plate, and inoculate them onto LB liquid medium, and incubate at 37℃ with shaking until OD. 600 When the bacterial culture reaches 1.0 ± 0.2, transfer 1 ml to a shake flask containing 50 ml of TB liquid medium. Aliquot the remaining culture with glycerol and freeze. Incubate in the shake flasks at 37.0 °C and 220 rpm until the bacterial OD value reaches 1.0 ± 0.2. 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. Fermentation was stopped after 4 hours, and samples were taken to detect the cell OD. 600 The total protein and target protein were detected by SDS-PAGE gel electrophoresis, and the expression level of the target protein was calculated.

[0064] Figure 3The results of the initial screening fermentation are shown. 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 and strain names of B90 and B110 follow the same pattern.

[0065] Based on the initial fermentation results, it can be seen that the engineered strain B constructed in this invention has good tolerance to kanamycin and can be screened using high concentrations. Furthermore, increasing the kanamycin concentration is also beneficial for screening high-expression strains. Figure 3 It can be seen that under the initial screening condition of 50 μg / ml, only a small number of transformants could express 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 expression levels ≥20% increased from 30% at 50 μg / ml to 60% at 70 μg / ml. Furthermore, transformants with expression levels exceeding 30% were identified at screening concentrations of 70-110 μg / ml. This suggests that transformants that can tolerate high concentrations of kanamycin screening conditions may have stronger protein expression potential.

[0066] Under various screening conditions, several high-expression transformants were selected as candidate strains for secondary screening. Table 1 shows the fermentation results of the initial screening candidate strains. The expression level of each strain was at least 20%, and the cell density indicated that these strains grew well and did not show obvious growth inhibition.

[0067] Table 1

[0068] Strain No. Bacterial density OD 600 ]] Protein expression amount 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 Secondary screening

[0070] Resuscitate the candidate strains preserved from the initial screening and dilute them to 1×10⁻⁶. 4 CFU / mL. For each candidate strain, 50 μl was spread onto LB agar plates containing 125 μg / mL, 150 μg / mL, 175 μg / mL, and 200 μg / mL kanamycin, respectively, and incubated at 37.0 °C for 18–20 h. After incubation, single colonies approximately 1–2 mm in diameter were picked from each plate and inoculated onto LB liquid medium and cultured at 37 °C with shaking until OD (occurrence limit) was reached. 600 When the bacterial culture reaches 1.0 ± 0.2, transfer 1 ml to a shake flask containing 50 ml of TB liquid medium. Aliquot the remaining culture with glycerol and freeze. Incubate in the shake flasks at 37.0 °C and 220 rpm until the bacterial OD value reaches 1.0 ± 0.2. 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. Fermentation was stopped after 3 hours, and samples were taken to detect the cell OD. 600 The total protein and target protein were detected by SDS-PAGE gel electrophoresis, and the expression level of the target protein was calculated.

[0071] Figure 4 The results of the secondary screening fermentation are shown. B110-09 represents single colonies picked from kanamycin plates at concentrations of 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml. From left to right, these are single colonies selected under the 125 μg / ml screening condition and named B110-09A, B110-09B, B110-09C, and B110-09D. B110-08 represents single colonies picked from kanamycin plates at concentrations of 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml. From left to right, these are single colonies selected under the 125 μg / ml, 150 μg / ml, 175 μg / ml, and 200 μg / ml screening conditions and named B110-09D. Single colonies selected under the μg / ml screening condition are named B110-08A, single colonies selected under the 150 μg / ml screening condition are named B110-08B, single colonies selected under the 175 μg / ml screening condition are named B110-08C, and single colonies selected under the 200 μg / ml screening condition are named B110-08D; the meanings and names of other strains follow the same pattern.

[0072] It is evident that after secondary screening, only a small number of strains were able to stably retain high expression performance. Several high-expression secondary-screened strains were selected as candidate strains for passage stability assessment. Table 2 shows the fermentation results of the secondary-screened candidate strains. The expression level of each strain was at least 25%, and the cell density indicated that the growth of these strains was normal.

[0073] Table 2

[0074] Strain No. Fermentation broth cell density OD 600 ]] Protein expression amount 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 passage stability

[0076] The stability of the plasmid was assessed by continuous shake-flask subculturing. Each re-screened candidate strain was revived, and 1 ml of each strain was inoculated into 50 mL of LB broth and incubated at 37°C with shaking at 220 rpm. The culture was continued until the bacterial density OD reached a certain level. 6ooAfter 1-2 hours of cultivation, the culture is designated as P1 generation seed culture. A small amount of the seed culture is first frozen and then an appropriate concentration of IPTG solution is added to induce the expression of the target protein. Fermentation is stopped after 3 hours, and the fermentation broth is stored. 1 ml of P1 generation seed culture is inoculated into 50 ml of LB liquid medium and cultured at 37°C with shaking at 220 rpm until OD reaches the target protein expression level. 6oo The seed culture medium is designated as generation P2, with a small amount taken and frozen. Then, expression is induced and the fermentation broth is preserved following the procedures described above. This process of subculturing and fermentation is repeated until generation P15 is reached.

[0077] The plasmid retention rate of each generation of the strain was determined. Appropriate amounts of seed culture (cell density 1×10⁻⁶) were taken from each generation. 2 -1×10 3 The plasmid (cfu / mL) was spread onto LB agar plates and incubated overnight at 37°C. After colonies grew, 96 single colonies were randomly selected and inoculated into 96-well plates containing kanamycin (50 μg / mL) and LB agar without kanamycin, respectively. All plates were incubated overnight at 37°C. The number of wells with viable cells was counted. The plasmid retention rate was calculated using the following formula:

[0078] Plasmid retention rate = number of wells with viable cell counts containing kanamycin / number of wells without viable cell counts × 100%.

[0079] The results are shown in Table 3. Figure 5 As shown, most of the selected strains were able to be stably passaged multiple times. Among them, strains B70-02A and B70-02D maintained 100% plasmid retention after 15 generations, and their progeny still possessed high productivity. SDS-PAGE electrophoresis confirmed the induced expression product to be approximately 18.7 kDa, consistent with the theoretical value. Figure 7 (lanes 2 and 3).

[0080] Table 3

[0081] Strain No. Number of passages P with plasmid retention rate ≥ 90% P2 Protein expression amount P15 Protein expression amount 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: Scale-up production in fermentation tanks

[0083] 3.1 Preparation of Grade I Seed Liquid

[0084] Candidate high-yield strains B70-02A, B70-02D, and B110-07C, as well as some initial screening strains B70-02 and B50-08, were removed from a -80℃ freezer and thawed in a 37℃ water bath. 1 ml of each strain was inoculated into two shake flasks containing 50 ml of LB liquid medium each, and cultured at 37℃ and 220 rpm until OD (October Expiratory Time). 600 It reached 1.0±0.2.

[0085] 3.2 Preparation of Grade II Seed Liquid

[0086] The Grade I seed culture was transferred at a ratio of 3% to shake flasks containing 650 ml LB liquid medium (4 flasks / plant), and cultured at 37°C and 220 rpm until OD. 600 It reached 1.0±0.2.

[0087] 3.3 Fermenter Cultivation

[0088] Take 2.5L of Grade II seed culture 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, and when the OD... 600 When the pH reaches 12.00-14.00, add an appropriate concentration of IPTG to induce expression, and add feed at a uniform rate. Fermentation ends after 3-5 hours.

[0089] 3.4 Protein purification

[0090] After fermentation, the bacterial cells were collected by centrifugation. After the bacterial cells were broken down, the inclusion bodies were collected. Then, the teriparatide stock solution was obtained through steps such as chromatography and ultrafiltration (for specific methods, refer to CN102399285B). The purity of the stock solution was detected by HPLC, and the purity of teriparatide was as high as 98% or more.

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

[0092] Comparative Example 1

[0093] The culture medium of the ampicillin-resistant engineered bacterium A constructed in Example 1 was used for screening and fermentation according to the methods described in Examples 2-3, except that kanamycin was replaced with ampicillin. The results showed that the target protein expression level of the preferred engineered bacterium was only 20.6%, and the plasmid was stable up to the 10th generation in the passage stability assessment.

[0094] On the one hand, it is speculated that because the resistance gene fragment of ampicillin is larger than that of kanamycin, engineered strain A has a larger molecular weight recombinant plasmid, which makes the plasmid of engineered strain A more prone to division instability and plasmid loss during passage, resulting in lower yield. On the other hand, it is speculated that the above screening concentration is not applicable to ampicillin, resulting in the inability to obtain stable and high-yielding strains.

[0095] Comparative Example 2

[0096] Resuscitated strains B70-02A, B70-02D, B70-02, B110-08, and B50-08 were assessed for passage stability according to the method described in Example 2.3. It is evident that the screening method described in this invention is more effective than conventional single-round, low-antibiotic-concentration pressure screening in obtaining high-yielding and plasmid-stable engineered strains.

[0097] Table 4

[0098] Strain No. Number of passages P with plasmid retention rate ≥ 90% P15 Protein expression amount 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 embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for screening a high-yield bacteria of teriparatide by using antibiotics, characterized in that, The method comprises: 1) plasmid construction: construct a teriparatide expression plasmid selected from pThioHisA and containing a teriparatide coding gene, wherein an ampicillin resistance gene fragment on the pThioHisA plasmid is replaced with a kanamycin resistance gene fragment; 2) transformation: transform the expression plasmid constructed in step 1) into Escherichia coli competent cells HB101, and culture to obtain recombinant bacterial liquid; 3) preliminary screening: take the bacterial liquid of step 2) and spread on solid medium containing 70 μg / ml kanamycin for constant temperature culture until single colonies appear, and pick well-grown single colonies and inoculate into fermentation medium for protein expression, and select several strains with target protein expression amount ≥ 20% as preliminary screening strains; 4) rescreening: take the bacterial liquid of each preliminary screening strain of step 3), and spread each strain on solid medium containing 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin for constant temperature culture until single colonies appear, pick well-grown single colonies and inoculate into fermentation medium for protein expression, and select strains that can grow at four kanamycin concentration gradients and have target protein expression amount ≥ 25% as rescreening strains; 5) evaluation of passage stability: pass the rescreening strains of step 4), inoculate each generation of strains into fermentation medium for protein expression, detect the plasmid retention rate and target protein expression amount of each generation of strains, and select strains with plasmid retention rate ≥ 90% and high expression amount after multiple passages as candidate high-yield strains; In steps 3) to 5), no kanamycin is added during inoculation of the strains into fermentation medium for protein expression and passage of the rescreening strains; and the number of passages is ≥ 15.

2. The method of claim 1, wherein, The number of passages is 15.

3. The method of claim 2, wherein, The method comprises: 1) plasmid construction: synthesize a DNA fragment shown in SEQ ID NO. 2, and insert the DNA fragment into a pThioHisA vector by using EcoRI and SalI enzyme cutting sites to obtain plasmid A; then take plasmid A as a template, design primers for PCR amplification to obtain a linear plasmid fragment with an ampicillin resistance gene deleted; finally, perform homologous recombination and connection of the linear plasmid fragment and a kanamycin resistance gene fragment, transform the connection product into Escherichia coli DH5α competent cells, pick positive monoclonal colonies for plasmid extraction and sequencing identification, and obtain a teriparatide expression plasmid with a kanamycin resistance marker; 2) transformation: transform the expression plasmid constructed in step 1) into Escherichia coli competent cells HB101, and culture to obtain recombinant bacterial liquid; 3) preliminary screening: take the bacterial liquid of step 2) and spread on solid medium containing 70 μg / ml kanamycin for constant temperature culture until single colonies appear, pick single colonies with a diameter of 1-2 mm on the plate and inoculate into fermentation medium for protein expression, and select several strains with target protein expression amount ≥ 20% as preliminary screening strains; 4) rescreening: take the bacterial liquid of each preliminary screening strain of step 3), and spread each strain on solid medium containing 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin for constant temperature culture until single colonies appear, pick well-grown single colonies and inoculate into fermentation medium for protein expression, and select strains that can grow at four kanamycin concentration gradients and have target protein expression amount ≥ 25% as rescreening strains; 5) evaluation of passage stability: pass the rescreening strains of step 4), inoculate each generation of strains into fermentation medium for protein expression, detect the plasmid retention rate and target protein expression amount of each generation of strains, and select strains with plasmid retention rate ≥ 90% and high expression amount after multiple passages as candidate high-yield strains; In steps 3) to 5), no kanamycin is added during inoculation of the strains into fermentation medium for protein expression and passage of the rescreening strains; and the number of passages is ≥ 15. The number of passages is 15. 4) Rescreening: take the bacterial liquid of each primary screening strain in step 3), and inoculate each strain on solid medium containing 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin respectively for constant temperature culture until single colonies appear, and then pick up single colonies with a diameter of 1-2 mm on each plate to inoculate into fermentation medium for protein expression, and select strains that can grow in 125 μg / ml, 150 μg / ml, 175 μg / ml and 200 μg / ml kanamycin and the expression amount of the target protein is ≥25% as rescreening strains; 5) Passage stability evaluation: pass each rescreening strain in step 4), and inoculate each generation of strain into fermentation medium for protein expression, detect the plasmid retention rate and the expression amount of the target protein of each generation of strain, and select the strain with plasmid retention rate ≥90% and high expression amount of the target protein after 15 generations of passage as a candidate high-yield strain; In steps 3)-5), no kanamycin is added during the process of inoculating the strains into fermentation medium for protein expression and the passage of the rescreening strains.

4. The method according to any one of claims 1 to 3, characterized in that, The kanamycin resistance gene fragment is derived from pET28a(+) plasmid. The kanamycin resistance gene fragment is derived from pET28a(+) plasmid.

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