An optimized method for enhancing the stability of NleC protein using site-directed mutagenesis

Abstract

The present invention relates to the field of biotechnology, and discloses an optimization method for enhancing the stability of NleC protein using site-directed mutagenesis technology, including constructing NleC mutants using site-directed mutagenesis technology, packaging virus-infected cells, and comparing the improvement of protein stability after mutating lysine with wild-type NleC. The present invention analyzes the sequence of HA-NleC, uses site-directed mutagenesis technology to mutate any one of K102, K109 and K123 on NleC into a mutant of arginine, and packages virus-infected cells respectively. The doxycycline-induced expression results show that compared with wild-type NleC, the mutated lysine has a significant improvement on protein stability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology. Background Art

[0002] Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+-ALL) is one of the most malignant types of leukemia. Although there are targeted drugs such as imatinib and dasatinib targeting its direct pathogenic gene BCR-ABL1, the control effect is far inferior to that of chronic myeloid leukemia. In clinical trials, the combination of targeted drugs with ubiquitin-proteasome inhibitors has shown certain efficacy for Ph+-ALL, especially for refractory cases. However, on the one hand, ubiquitin proteasome inhibitors represented by bortezomib are highly toxic, and on the other hand, the drug resistance of ubiquitin proteasome inhibitors is not optimistic. One of the key anti-tumor mechanisms of ubiquitin proteasome inhibitors is to inhibit the ubiquitination and degradation of IκB and thus inhibit the nuclear translocation of NF-κB. In view of this, exploring new anti-NF-κB methods that do not rely on ubiquitin proteasomes combined with targeted drugs is expected to bring a new strategy for the treatment of Ph+-ALL.

[0003] NleC is one of the virulence factors injected into infected host cells by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) through a needle-like protein complex called the type III secretion system (T3SS). NleC specifically cleaves the p65 subunit of NF-κB in the p65-p50 heterodimer complex, blocking the NF-κB signaling pathway, thereby inhibiting the host's inflammatory response. One of the outstanding advantages of NleC protein is that it can cross the cell membrane of human cells to reach the cytoplasm without special treatment, thus circumventing the difficulty of transmembrane delivery of ordinary protein drugs. Studies have found that the NleC recombinant protein with an HA tag retains the ability to penetrate the membrane and degrade NF-κB p65, and can significantly enhance the killing ability of imatinib on Ph+-ALL cell line SUP-B15 cells, but its stability needs to be further improved. Improving the stability of NleC protein and enhancing its anti-NF-κB ability combined with targeted drugs is expected to bring a new strategy for the treatment of Ph+-ALL.

[0004] Currently, no effective solution has been proposed for the problems in the related technologies. Summary of the invention

[0005] In view of the problems in the related art, the present invention proposes an optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology to overcome the above-mentioned technical problems existing in the existing related art.

[0006] To this end, the specific technical solution adopted by the present invention is as follows:

[0007] An optimized method for enhancing the stability of NleC protein using site-directed mutagenesis technology, comprising:

[0008] The NleC mutant was constructed using site-directed mutagenesis technology, and the virus was packaged to infect cells. The effect of mutating lysine on protein stability was compared with that of wild-type NleC.

[0009] The NleC mutant is a mutant in which any one of the lysines at the K102 site, the K109 site and the K123 site on NleC is mutated to arginine.

[0010] Furthermore, the in vitro construction of the wild-type NleC comprises the following steps:

[0011] HA-Nlec fragment was chemically synthesized by double digestion with EcoRI / BamHI and digested overnight;

[0012] The HA-Nlec fragment after enzyme digestion was recovered from the gel and purified;

[0013] T4 ligase ligates the purified Plvx-Tetone-Puro vector after EcoRI / BamHI digestion and leaves at room temperature;

[0014] Place the transferred Stabl3 competent cells on ice, heat shock, and cool on ice;

[0015] Add AMP-free LB medium and culture on a shaker, coat the plate, and culture in an inverted incubator;

[0016] Pick single clones, expand culture, extract plasmids, verify by ECORI / XhoI restriction digestion, and sequence;

[0017] Select plasmids with correct enzyme digestion and sequencing results, and use the kit to extract transfection-grade plasmids;

[0018] Package lentivirus and infect SUP-B15 and Z33 cells.

[0019] Furthermore, the temperature for overnight enzyme digestion is 37°C. The room temperature placement time is 30min-60min. The amount of Stabl3 competent cells transferred is 10ul, the ice placement time is 30min-60min, and the ice cooling time is 2-3min. The heat shock temperature is 42°C, and the heat shock time is 90s. The amount of AMP-free + LB medium added is 900ul, the shaking incubation temperature is 37°C, and the shaking incubation time is 20-40min. The inverted incubation temperature is 37°C, and the inverted incubation time is 12-16h.

[0020] Furthermore, the system of T4 ligase ligating the Plvx-Tetone-Puro vector purified by EcoRI / BamHI digestion is:

[0021] Plvx-Tetone-Puro-ECORI / XhoI(50ng): 4ul;

[0022] HA-Nlec ECORI / XhoI(100ng): 6ul;

[0023] 10xT4 DNA Ligase Buffer: 2ul;

[0024] T4 DNA Ligase: 1ul;

[0025] Water: 7ul.

[0026] Furthermore, the NleC mutant is used to prepare drugs for treating leukemia and to break through drug resistance in combination with TKI.

[0027] The beneficial effects of the present invention are as follows: by analyzing the sequence of HA-NleC, using site-directed mutagenesis technology to mutate any one of K102, K109 and K123 on NleC into an arginine mutant, and packaging the virus to infect cells respectively, and the doxycycline-induced expression results show that compared with the wild-type NleC, the mutated lysine has a significant improvement effect on protein stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 is an analysis diagram of the effect of doxycycline on SUP-B15 and Z33 cells in the examples of the present invention;

[0030] Figure 2 Schematic diagram of the effect of MG132 and CQ on the expression level of HA-NleC in the embodiment of the present invention;

[0031] Figure 3 It is an in-depth analysis diagram of the effect of doxycycline on SUP-B15 and Z33 NleC and their mutant cells in the examples of the present invention. DETAILED DESCRIPTION

[0032] To further illustrate each embodiment, the present invention provides drawings, which are part of the disclosure of the present invention and are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these contents, ordinary technicians in the field should be able to understand other possible implementations and advantages of the present invention. The components in the figures are not drawn to scale, and similar component symbols are generally used to represent similar components.

[0033] According to an embodiment of the present invention, there is provided an optimization method for enhancing the stability of NleC protein using site-directed mutagenesis technology.

[0034] Since previous studies have shown that neither CMV nor EF1α promoters can effectively drive NleC expression in leukemia cells, tetracycline regulatory elements are used in this example to induce NleC expression in eukaryotic cells. PGEX-4T-1-HA-NleC was used as a DNA template to PCR amplify the HA-NleC fragment and clone it into the Plvx-Tetone-puro vector, transform the Stabl3 competent cells, pick a single clone, sequence and verify, extract the correct clone plasmid, and then package the lentivirus to infect the cells. Doxycycline induces HA-NleC protein expression, Figure 1 The results showed that HA-NleC protein expression could be obtained, but the expression level was poor, which caused difficulties for the next step of the experiment.

[0035] There are two protein degradation pathways in cells: ubiquitin proteasome degradation system and lysosomal degradation system. To explore the degradation mechanism of NleC in cells, cells were treated with MG132 or chloroquine during doxycycline induction to observe the effect of blocking the ubiquitin proteasome or lysosomal degradation pathway on the stability of NleC. The results further verified that NleC was mainly degraded through the ubiquitin proteasome pathway.

[0036] Specifically, in this embodiment, by analyzing the sequence of HA-NleC, an optimization method for enhancing the stability of NleC protein using site-directed mutagenesis technology is proposed, including: constructing NleC mutants using site-directed mutagenesis technology, packaging viruses to infect cells, and comparing the effect of mutating lysine on the stability of the protein with that of wild-type NleC;

[0037] The NleC mutant is a mutant in which any one of the lysines at the K102 site, the K109 site and the K123 site on NleC is mutated to arginine.

[0038] In another embodiment, gene molecular cloning, construction of Plvx-Tetone-HA-NleC-Puro plasmid, virus packaging, cell infection, and functional verification experiments include:

[0039] 1. Double digestion of chemically synthesized HA-Nlec fragment with ECORI / BamHI at 37℃ overnight;

[0040] The HA-NleC protein sequence is as follows:

[0041] YPYDVPDYAGGMICRVLDINMKIPSLQSNFNFSAPAGYSAPIAPNRA

[0042] ENAYADYVLDIGKRIPLSAADLSNVYESVIRAVHDSRSRLIDQHTVDMIG

[0043] NTVLDALSRSQTFRDAVSYGIHNE K VHI K YRNEYELNEESSV K IDDI

[0044] QSLTCNELYEYDVGQEPIFPICEAGENDNEEPYVSFSVAPDTDSYEMPSW

[0045] QEGLIHEIIHHVTGSSDPSGDSNIELGPTEILARRVAQELGWSVPDFKGYA

[0046] EPEREAHLRLRNLNALRQAAMRHEENERAFFERLGTISDRYEASPDFTEY

[0047] SAVSNIGYGFIQQHDFPGLAINDNLQDANQIQLYHGAPYIFTFGDVDKHN

[0048] QQ*; the bold and underlined sites are K102, K109, and K123 from left to right;

[0049] 2. Recover the HA-Nlec fragment after enzyme digestion from gel and purify it;

[0050] 3. T4 ligase ligates the Plvx-Tetone-Puro vector after EcoRI / BamHI digestion and purification. The system is as follows:

[0051] Plvx-Tetone-Puro-ECORI / XhoI(50ng):4ul

[0052] HA-Nlec ECORI / XhoI(100ng):6ul

[0053] 10xT4 DNA Ligase Buffer: 2ul

[0054] T4 DNA Ligase: 1ul

[0055] Water: 7ul

[0056] 4. Leave at room temperature for 30-60 minutes;

[0057] 5. Take 10ul and transfer to Stabl3 competent cells (100ul), place on ice for 30min-60min, heat shock at 42℃ for 90s, cool on ice for 2-3min, add 900ul AMP-free + LB medium, culture at 37℃ shaking for 20-40min, plate, and culture at 37℃ upside down for 12-16h;

[0058] 6. Pick single clones, expand culture, extract plasmids, verify by ECORI / XhoI restriction enzyme digestion, and sequence;

[0059] 7. Select plasmids with correct enzyme digestion and sequencing results, and use the kit to extract transfection-grade plasmids;

[0060] 8. Packaged lentivirus and infected SUP-B15 and Z33 cells. The results showed that doxycycline successfully induced the expression of HA-NleC protein, degraded P65, and enhanced the killing ability of imatinib on cells. Figure 1 As shown, Figure A shows SUP-B15 and Z33 cells induced by different concentrations of doxycycline for 72 hours, and Western blot was used to detect the expression of HA tags and P65 and P50 / 105 proteins; Figure B shows the statistical analysis of cell survival rate detected by Annexin V-APC / 7-AAD double staining flow cytometry after NleC protein was induced by 500 ng / mL doxycycline (Dox) for 72 hours with or without imatinib;

[0061] 9. To explore the degradation pathway of NleC in cells, in this example, cells were treated with MG132 or chloroquine during doxycycline induction to observe the effect of blocking the ubiquitin proteasome or lysosome degradation pathway on the stability of NleC. The results showed that NleC was mainly degraded via the ubiquitin proteasome pathway (e.g. Figure 2 shown);

[0062] In another embodiment, the site-directed mutagenesis technique is used to construct three NleC mutants and the functional verification experimental steps include:

[0063] In view of the above results, in this example, the NleC sequence was analyzed, and the K102, K109 and K123 on NleC were mutated to arginine respectively using the site-directed mutagenesis technology, and mutants containing lysine mutations to arginine sites were constructed, and the viruses were packaged to infect cells respectively. The results showed that: doxycycline-induced expression results showed that: compared with the wild-type NleC, the mutation of lysine had a significant improvement on protein stability.

[0064] like Figure 3 As shown in the figure, A shows the induction of SUP-B15, Z33 NleC and their mutant cells with different concentrations of doxycycline for 72 hours, and the expression of HA tag and P65, P50 / 105 proteins was detected by Western blot; B shows the statistical analysis of cell survival rate by Annexin V-APC / 7-AAD double staining flow cytometry after induction of SUP-B15, Z33 NleC and their mutant cells with or without imatinib for 72 hours with 500 ng / mL doxycycline (Dox).

[0065] In another embodiment, the NleC mutant is used to prepare drugs for treating leukemia and to break through drug resistance in combination with TKI.

[0066] In summary, by means of the above technical scheme of the present invention, the sequence of HA-NleC is analyzed, and the site-directed mutagenesis technology is used to mutate any one of the lysines in K102, K109 and K123 on NleC into an arginine mutant, and the virus is packaged and infected in cells respectively. The doxycycline-induced expression results show that compared with the wild-type NleC, the mutation of lysine has a significant improvement effect on protein stability.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An optimization method for enhancing the stability of NleC protein using site-directed mutagenesis technology, characterized in that: The method includes: The NleC mutant was constructed using site-directed mutagenesis technology, and the virus was packaged to infect cells. The effect of mutating lysine on protein stability was compared with that of wild-type NleC. Wherein, the NleC mutant is a mutant in which any one of the lysines at the K102 site, the K109 site and the K123 site on NleC is mutated to arginine; The in vitro construction of the wild-type NleC comprises the following steps: HA-Nlec fragment was chemically synthesized by double digestion with EcoRI / BamHI and digested overnight; The HA-Nlec fragment after enzyme digestion was recovered from the gel and purified; T4 ligase ligates the purified Plvx-Tetone-Puro vector after EcoRI / BamHI digestion and leaves at room temperature; Place the transferred Stabl3 competent cells on ice, heat shock, and cool on ice; Add AMP-free LB medium and culture on a shaker, coat the plate, and culture in an inverted incubator; Pick single clones, expand culture, extract plasmids, verify by ECORI / XhoI restriction digestion, and sequence; Select plasmids with correct enzyme digestion and sequencing results, and use the kit to extract transfection-grade plasmids; Package lentivirus and infect SUP-B15 and Z33 cells; The HA-NleC protein sequence is as follows: YPYDVPDYAGGMICRVLDINMKIPSLQSNFNFSAPAGYSAPIAPNRAENAYADYVLDIGKRIPLSAADLSNVYESVIRAVHDSRSRLIDQHTVDMIGNTVLDALSRSQTFRDAVSYGIHNE K VHI K YRNEYELNEESSV K IDDIQSLTCNELYEYDVGQEPIFPICEAGENDNEEPYVSFSVAPDTDSYEMPSWQEGLIHEIIHHVTGSSDPSGDSNIELGPTEILARRVAQELGWSVPDFKGYAEPEREAHLRLRNLNALRQAAMRHEENERAFFERLGTISDRYEASPDFTEYSAVSNIGYGFIQQHDFPGLAINDNLQDANQIQLYHGAPYIFTFGDVDKHNQQ*, among which the underlined sites are K102, K109, and K123 from left to right.

2. The optimization method for enhancing the stability of NleC protein using site-directed mutagenesis technology according to claim 1, characterized in that: The overnight digestion temperature was 37°C.

3. The optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology according to claim 1, characterized in that: The system for T4 ligase to connect the Plvx-Tetone-Puro vector purified by EcoRI / BamHI digestion is: Plvx-Tetone-Puro-ECORI / XhoI: 4ul; HA-Nlec ECORI / XhoI: 6ul; 10xT4 DNA Ligase Buffer: 2ul; T4 DNA Ligase: 1ul; Water: 7ul.

4. The optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology according to claim 1, characterized in that: The storage time at room temperature is 30min-60min.

5. The optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology according to claim 1, characterized in that: The amount transferred to Stabl3 competent cells is 10ul, the time of keeping on ice is 30min-60min, and the time of cooling on ice is 2-3min.

6. The optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology according to claim 1, characterized in that: The heat shock temperature was 42°C and the heat shock time was 90 s.

7. The optimization method for enhancing the stability of NleC protein by using site-directed mutagenesis technology according to claim 1, characterized in that: The amount of AMP-free LB medium added was 900ul, the shaking incubation temperature was 37°C, and the shaking incubation time was 20-40min.

8. The optimization method for enhancing the stability of NleC protein using site-directed mutagenesis technology according to claim 1, characterized in that: The inverted culture temperature is 37°C and the inverted culture time is 12-16h.

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