Host cell capable of efficiently secreting and expressing totipotent nuclease as well as construction and application of host cell

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a host cell capable of efficiently secreting and expressing totipotent nuclease as well as construction and application of the host cell. A vector of a target totipotent nuclease gene containing a fusion signal peptide sequence is constructed and transformed into escherichia coli cells, so that the gene is carried and expressed by the escherichia coli cells. Under proper culture conditions, high-purity target protein with the purity of about 92% can be obtained in the culture medium, and the high-purity target protein has good degradation capacity on nucleic acid, has the advantages of high efficiency, simplicity, convenience, economy and the like, and is beneficial to industrial production and application of all-round nuclease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a host cell that efficiently secretes and expresses a universal nuclease, and the construction and application thereof. Background Art

[0002] As more and more biological products (recombinant protein vaccines, Vero cell vaccines, cell therapy / gene therapy drugs, etc.) enter the therapeutic field, the quality control of biological products has become increasingly stringent. Among them, nucleic acid residues are the top priority of various quality control standards due to their potential harmfulness (such as DNA residues in protein drugs may bring activated oncogenes into the patient's body, increasing the risk of cancer). Referring to the standards of WHO, FDA and EU, my country clearly stipulates in the pharmacopoeia that the amount of DNA residue in biological products expressed by yeast and Escherichia coli shall not exceed 10ng / dose. my country's 2020 Pharmacopoeia will update the DNA residue standard for human rabies vaccine (Vero cells) to ≤3ng / dose. Therefore, there must be a step to remove nucleic acid residues in the production process of biological products to ensure that the nucleic acid residues in biological products meet the requirements of relevant regulations.

[0003] The difficulty in removing nucleic acid residues is that DNA carries a large amount of charge and is easily combined with other biomacromolecules, resulting in aggregation (adsorption) and encapsulation, making it difficult to completely remove. Traditional methods have the defects of incomplete removal of low-content nucleic acid residues, large workload and long time consumption. Nuclease is a genetically engineered endonuclease derived from Serratia marcescens that can degrade double-stranded, single-stranded, linear, and circular DNA and RNA, and completely degrade nucleic acids into 5'-monophosphate oligonucleotides of 3 to 5 bases in length. The application scenarios of nucleases include: (1) Application in the production of viral vectors for vaccines, cells and gene therapy: removing exogenous nucleic acids from vaccine products, reducing the risk of residual nucleic acid toxicity and improving product safety; removing nucleic acids wrapped around the surface of particles (viruses, inclusion bodies, etc.), facilitating the release and purification of particles; (2) Improving recombinant protein processes: reducing the viscosity of the feed solution caused by nucleic acids, shortening the processing time and increasing protein yield; (3) Preparation of electrophoresis and chromatography samples: sample preparation for ELISA, column chromatography, two-dimensional electrophoresis and blotting analysis can improve resolution and recovery rate after nuclease treatment; (4) Preventing cell aggregation: adding universal nuclease to the peripheral blood mononuclear cell (PBMC) thawing buffer can effectively prevent cell aggregation, thereby achieving cryopreservation of PBMC.

[0004] The nuclease products currently sold on the market are mainly Benzonase endonuclease, a product of Merck in Germany, but its price is relatively high, which limits its large-scale use in the biopharmaceutical industry. In the 1990s, Biedermann K published the research work "Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli", selecting Escherichia coli as the protein expression system and selecting the upstream sequence of S. marcescens nuclease to regulate the secretory expression of the protein. Although this method can obtain active S. marcescens nuclease, its expression level is low and the expression time is difficult to control. It is only suitable for laboratory research and not suitable for industrial production. In 2011, Cai Junjie published the research work "Recombinant Expression, Purification and Identification of Extracellular Nuclease of Serratia marcescens", constructed a prokaryotic expression vector of S. marcescens nuclease, but expressed the target protein in the form of inclusion bodies, making the purification process more complicated. In 2011, Chen Peng et al. published the research work "Prokaryotic Expression, Purification and Activity Analysis of S. marcescens Non-specific Nuclease", which constructed a recombinant expression system of S. marcescens nuclease fused with maltose binding protein. The fusion protein was distributed in the supernatant of the bacterial cell disruption liquid, which was not conducive to the subsequent extraction, separation and purification of the fusion protein. Therefore, it is very necessary to develop a method for producing all-purpose nuclease suitable for industrial application to meet the needs of pharmaceutical product development. Summary of the invention

[0005] In order to solve the problems of low expression level and complicated purification process in the existing preparation process of omnipotent nuclease, which is not conducive to industrial production, the present invention provides a host cell for efficiently secreting and expressing omnipotent nuclease and its construction and application. The present invention utilizes the method of efficiently secreting and expressing omnipotent nuclease by Escherichia coli, and the omnipotent nuclease is directly secreted into the fermentation liquid, which simplifies the purification process and does not require denaturation and renaturation operations. The obtained omnipotent nuclease has good degradation ability for nucleic acids.

[0006] In a first aspect, the present invention provides a fusion signal peptide having an amino acid sequence as shown in SEQ ID NO.3.

[0007] In a second aspect, the present invention provides a gene encoding a fusion signal peptide, which has a nucleotide sequence as shown in SEQ ID NO.2.

[0008] In a third aspect, the present invention provides a nucleic acid construct comprising: i) a gene encoding the aforementioned fusion signal peptide, and ii) a gene encoding a full-potent nuclease operably linked to the gene encoding the fusion signal peptide.

[0009] In some embodiments, the gene encoding the universal nuclease has a nucleotide sequence as shown in SEQ ID NO.1.

[0010] In a fourth aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid construct.

[0011] In a fifth aspect, the present invention provides a host cell that efficiently secretes and expresses a universal nuclease, comprising the aforementioned nucleic acid construct or expression vector.

[0012] In some embodiments, the host cell is E. coli.

[0013] In a sixth aspect, the present invention provides a method for efficiently secreting and expressing a pluripotent nuclease, comprising: constructing the aforementioned host cell; fermenting and culturing the host cell to induce the expression of the pluripotent nuclease; and isolating and obtaining the pluripotent nuclease from the fermentation and culture product.

[0014] In some embodiments, the medium for fermentation culture is LB medium, the temperature of fermentation culture is 35-38° C., and the fermentation culture is performed until OD600=0.6-1.0.

[0015] In some embodiments, the induction agent is isopropyl-β-D-thiogalactoside with a final concentration of 0.02-0.1 mM, the induction temperature is 20-25° C., and the induction time is 16-40 h.

[0016] In some embodiments, the separation method comprises: centrifuging the fermentation culture product to obtain a supernatant.

[0017] Due to the simple gene manipulation of the E. coli expression system, the selectivity of multiple hosts / vectors, the fast growth rate, and the simple cultivation, it is often used for laboratory and industrial-scale production of exogenous proteins. However, heterologous proteins containing disulfide bonds are prone to form inclusion bodies and accumulate in the cytoplasm, and an inclusion body denaturation and renaturation purification process is required, but the purification cycle is long and the production cost is high. By constructing a vector containing a fusion signal peptide sequence of the target omnipotent nuclease gene and transforming it into E. coli cells, the omnipotent nuclease is fused with the fusion signal peptide and expressed, which allows the target omnipotent nuclease protein precursor to be transferred from the cytoplasm to the periplasm and / or the extracellular space. Compared with cytoplasmic accumulation, the secretory production of exogenous proteins has several advantages. First, after the fusion signal peptide is cleaved, the original N-terminal amino acid of the target omnipotent nuclease is retained; second, the oxidative environment and the enzymes in the exoplasm contribute to the correct formation of disulfide bonds; third, the concentration of endogenous proteins in the periplasm and / or the extracellular space is low, making it easier to separate heterologous proteins from host protein contaminants on a laboratory scale. Based on the above advantages, the present invention provides an expression system capable of efficiently secreting and expressing a universal nuclease, which is of great significance for increasing the expression amount of the universal nuclease to meet the needs of pharmaceutical product development.

[0018] The beneficial effects of the present invention are:

[0019] The fusion signal peptide provided by the present invention has the ability to guide the omnipotent nuclease into the cell secretion pathway. By constructing a vector of the target omnipotent nuclease gene containing the fusion signal peptide sequence, it is transformed into Escherichia coli cells to carry and express the gene. Under appropriate culture conditions, a high-purity target protein can be obtained in the culture medium with a purity of about 92%, and has good degradation ability for nucleic acids, has the advantages of efficient expression (expression amount of more than 31 mg / L), simple purification, economy, etc., which is conducive to the industrial production and application of omnipotent nuclease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the pET22b-SPpelB-nucA-nucA plasmid map;

[0021] Figure 2The results of SDS-PAGE of the supernatant samples of fermentation broth induced at 20℃ for different induction times; Lane 1: M; Lane 2: I0-M; Lane 3: 0.02mM-I16-M; Lane 4: 0.02mM-I24-M; Lane 5: 0.02mM-I40-M; Lane 6: 0.06mM-I16-M; Lane 7: 0.06mM-I24-M; Lane 8: 0.06mM-I40-M; Lane 9: 0.1mM-I16-M; Lane 10: 0.1mM-I24-M; Lane 11: 0.1mM-I40-M; Lane 12: BCA (0.5μg); Lane 13: BCA (1μg); Lane 14: BCA (2 μg); M is the culture medium supernatant;

[0022] Figure 3 The results of SDS-PAGE of the supernatant samples of the fermentation broth induced at 25℃ for different induction times; Lane 1: M; Lane 2: I0-M; Lane 3: 0.02mM-I16-M; Lane 4: 0.02mM-I24-M; Lane 5: 0.02mM-I40-M; Lane 6: 0.06mM-I16-M; Lane 7: 0.06mM-I24-M; Lane 8: 0.06mM-I40-M; Lane 9: 0.1mM-I16-M; Lane 10: 0.1mM-I24-M; Lane 11: 0.1mM-I40-M; Lane 12: BCA (0.5μg); Lane 13: BCA (1μg); Lane 14: BCA (2 μg); M is the culture medium supernatant;

[0023] Figure 4 The SDS-PAGE results of samples at different positions under the optimal induction conditions; Lane 1: M; Lane 2: I0-S; Lane 3: I0-P; Lane 4: I0-PS; Lane 5: I0-M; Lane 6: I40-S; Lane 7: I40-P; Lane 8: I40-PS; Lane 9: I40-M; S is the supernatant after ultrasound, P is the precipitate after ultrasound, PS is the periplasmic space, and M is the culture medium supernatant;

[0024] Figure 5This is the electrophoresis result of nuclease degradation of plasmid DNA in the fermentation broth supernatant; among them: M: 10000bp Marker; 1: buffer (control); 2: I0-M; 3: I0-M / 10; 4: I0-M / 100; 5: I0-M / 1000; 6: I0-M / 10000; 7: I40-M; 8: I40-M / 10; 9: I40-M / 100; 10: I40-M / 1000; 11: I40-M / 10000. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1 Construction of recombinant expression plasmid

[0027] Codon optimization of universal nuclease gene

[0028] The wild-type gene sequence of the universal nuclease: Genbank: M19495.1, derived from Serratia marcescens, the gene coding region is 801 bp in length, of which 1 to 63 bp are the signal peptide part, and 64 to 801 bp are the mature region of the universal nuclease, that is, the length of the mature region is 738 bp. The gene is heterologously expressed in the prokaryotic system Escherichia coli, so the original sequence (mature region, length 738 bp) is optimized according to the codon preference of Escherichia coli. The specific sequence of the optimized universal nuclease nucA is as follows (SEQ ID NO.1):

[0029] GATACCCTGGAAAGCATTGATAACTGCGCGGTGGGCTGCCCGACCGGCGGCAGCAGCAACGTGAGCATTGTGCGCCATGCGTATACCCTGAACAATAACAGCACCACCAAATTTGCGAACTGGGTGGCGTATCATATTACCAAAGATACCCCGGCGAGCGGCAAAACCCGCAACTGGAAAACCG ATCCGGCGCTGAACCCGGCGGATACCCTGGCGCCGGCGGATTATACCGGCGCGAACGCGGCGCTGAAAGTGGATCGCGGCCATCAAGCGCCGCTGGCGAGCCTGGCGGGCGTGAGCGATTGGGAAAGCCTGAACTATCTGAGCAACATTACCCCGCAGAAAAGCGATCTGAACCAAGGCGCGTGG GCGCGCCTGGAAGATCAAGAACGCAAACTGATTGATCGCGCGGATATTAGCAGCGTGTATACCGTGACCGGCCCGCTGTATGAACGCGATATGGGCAAACTGCCGGGCACGCAGAAAGCGCATACCATTCCGAGCGCGTATTGGAAAGTGATTTTTATTAACAACAGCCCGGCGGTGAACCATT ATGCGGCGTTTCTGTTTGATCAGAACACCCCGAAAGGCGCGGATTTTTGTCAGTTTCCGTGACCGTGGATGAAATTGAAAAACGCACCGGCCTGATTATTTGGGCGGGCCTGCCGGATGATGTGCAAGCGAGCCTGAAAAGCAAACCGGGCGTGCTGCCGGAACTGATGGGCTGCAAAAACTAA

[0030] Design of signal peptide SP nucA (native signal peptide from nuclease nucA) and signal peptide SP pelB (Pectate lyase signal peptide) fused to obtain fusion signal peptide SP pelB-nucA , then the signal peptide SP pelB-nucA The plasmid pET22b-SP was synthesized by Nanjing GenScript Co., Ltd. after fusion expression with nucA, and a His purification tag and TEV restriction site were added between the fusion signal peptide and nucA, and the ampicillin resistance gene was replaced with a kanamycin resistance gene. pelB-nucA -nucA. Plasmid map pET22b-SPpelB-nucA -nucA see Figure 1 .

[0031] The fusion signal peptide SP pelB-nucA The specific nucleotide sequence is as follows (SEQ ID NO.2):

[0032] ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGCGCTTTAACAACAAGATGTTGGCCTTGGCCGCCCTGCTGTTCGCCGCGCAGGCGTCGGCC

[0033] Fusion signal peptide SP pelB-nucA The amino acid sequence is as follows (SEQ ID NO.3):

[0034] MKYLLPTAAAGLLLLAAQPAMAMRFNNKMLALAALLFAAQASA

[0035] The successfully synthesized plasmid was transformed into Escherichia coli BL21 (DE3) strain according to conventional methods.

[0036] Example 2 Optimization of induction conditions for recombinant strains

[0037] In order to improve the secretory expression of the omnipotent nuclease, the three conditions of the inducer IPTG concentration (set to 0.02mM, 0.06mM and 0.1mM), the induction temperature (set to 25℃ and 30℃) and the induction time (set to 16h, 24h and 40h) were optimized respectively.

[0038] The specific induction expression is as follows:

[0039] (1) E. coli BL21(DE3) / pET22b-SP pelB-nucA -nucA was activated on 50 μg / mL Kana resistance plates and cultured at 37°C overnight.

[0040] (2) Select a single colony and inoculate it into 50 mL of LB liquid medium (Kana) and culture it overnight at 37°C and 200 rpm.

[0041] (3) The next day, transfer the inoculum to another bottle of 100 mL of liquid LB medium (Kana) at a 2% inoculum volume and culture at 37°C and 200 rpm until OD600 = 0.6-1.0. Add an appropriate amount of IPTG and adjust the temperature to 20°C / 25°C for induction for 16-40 h.

[0042] After the induction expression was completed, the expression of the target protein in the fermentation broth was detected by SDS PAGE. Figure 2 , Figure 3 And as shown in Table 1.

[0043] Table 1 Protein expression under different induction conditions

[0044]

[0045]

[0046] Combining the SDS-PAGE results and the data in Table 2, it can be seen that the conditions for the highest expression of secretory proteins in shake flask culture are an induction temperature of 20°C, an inducer concentration of 100 μM, and an induction time of 40 h.

[0047] Example 3 Inducible expression of recombinant nuclease

[0048] The above-mentioned optimal induction conditions were selected, and the expression was induced according to the steps described in Example 2. After the induction expression was completed, the fermentation broth was first centrifuged to obtain the culture supernatant, and then the E. coli cells were collected and lysed, and the lysed supernatant and precipitate were obtained by centrifugation, and the recombinant nuclease expressed in the periplasmic space was extracted by osmotic shock method. The culture supernatant, the lysed supernatant, the precipitate and the samples extracted by osmotic shock method were respectively subjected to SDS-PAGE to detect the expression of the target protein, so as to compare the nuclease expression in cell secretion, cytoplasmic soluble expression, cytoplasmic inclusion body expression and periplasmic space. The results are shown as follows: Figure 4 As shown, a single band appeared in the culture supernatant with a purity of 92-93%, which was presumed to be the nuclease secreted from Escherichia coli into the culture medium.

[0049] Example 4 Verification of the degradation activity of the universal nuclease nucA on plasmids

[0050] Nuclease degradation nucleic acid reaction system:

[0051] The buffer is 50mM Tris-Hcl, pH 8.0, 1mM MgCl2.

[0052] The enzyme solution is buffer (control), I0-M, I0-M / 10, I0-M / 100, I0-M / 1000, I0-M / 10000, I40-M, I40-M / 10, I40-M / 100, I40-M / 1000, I40-M / 10000, wherein I0 represents 0 hour of induction, I40 represents 40 hours of induction, M represents fermentation broth supernatant, / 10 represents 10-fold dilution, / 100 represents 100-fold dilution, / 1000 represents 1000-fold dilution, / 10000 represents 10000-fold dilution, and illustratively, I40-M / 10000 represents 10000-fold dilution of the fermentation broth supernatant induced for 40 hours.

[0053] Table 2 Reaction system of plasmid DNA degradation by universal nuclease

[0054]

[0055] Reaction conditions: 37°C water bath for 30 min, add 10 μL 50 mM EDTA to terminate the reaction.

[0056] After the reaction was terminated, the reaction samples were subjected to agarose gel electrophoresis (120 V, 25 min). Figure 5 As shown, it can be seen that the crude nuclease solution in the culture medium has a good degradation effect on the plasmid DNA sample.

[0057] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A fusion signal peptide, characterized in that: It has an amino acid sequence as shown in SEQ ID NO.

3.

2. A gene encoding a fusion signal peptide, characterized in that: It has a nucleotide sequence as shown in SEQ ID NO.

2.

3. A nucleic acid construct, characterized in that It contains: i) a gene encoding the fusion signal peptide according to claim 2, and ii) a gene encoding a universal nuclease operably linked to the gene encoding the fusion signal peptide.

4. The nucleic acid construct according to claim 3, characterized in that The coding gene of the universal nuclease has a nucleotide sequence as shown in SEQ ID NO.

1.

5. An expression vector, characterized in that: It comprises the nucleic acid construct according to claim 3 or 4.

6. A host cell that efficiently secretes and expresses a universal nuclease, characterized in that: It comprises the nucleic acid construct according to claim 3 or 4, or the expression vector according to claim 5.

7. The host cell according to claim 6, characterized in that The host cell is Escherichia coli.

8. A method for efficiently secreting and expressing a universal nuclease, characterized in that: The method comprises: Constructing the host cell according to claim 6 or 7; Fermenting and culturing the host cells to induce the expression of the universal nuclease; and, The universal nuclease is isolated and obtained from the fermentation culture product.

9. The method according to claim 8, characterized in that The fermentation culture medium is LB medium, the fermentation culture temperature is 35-38°C, and the fermentation culture is carried out until OD600=0.6-1.0; And / or, the induction agent is isopropyl-β-D-thiogalactoside with a final concentration of 0.02-0.1 mM, the induction temperature is 20-25° C., and the induction time is 16-40 h.

10. The method according to claim 8, characterized in that The separation method comprises: centrifuging the fermentation culture product to obtain the supernatant.

Images