Micromonas minuta gene knockout method and application thereof

Abstract

The invention provides a gene knockout method of micromonas microflora and application of the gene knockout method. The method comprises the following steps: firstly, obtaining an upstream homologous arm fragment and a downstream homologous arm fragment of a gene tmpc to be knocked out, and an ermB resistance gene fragment; connecting the three fragments to obtain a fused DNA fragment; and finally, gene knockout of the micromonas minuta is realized by adopting a natural transformation method. According to the invention, the problem that the micromonas minuta is difficult to transform and edit genes is effectively solved. And the provided micromonas tautomonas of which the gene tmpc is knocked out can be used for researching the mechanism that the micromonas tautomonas is attached to and invades the OSCC cells. According to the present invention, it is verified that the TmpC is the necessary effector of P.micra for promoting OSCC transfer in vivo through the micromonas sp of which the gene tmpc is knocked out, and the adhesion and invasion ability of the P.micra to OSCC cells in vitro is significantly reduced;

Description

Technical Field

[0001] The invention relates to a method for knocking out a gene of Micromonas minutissima and application thereof, and belongs to the technical field of molecular biology. Background Art

[0002] Parvimonas micra (P. micra) is a Gram-positive anaerobic bacterium that is now considered a periodontitis pathogen. It belongs to the subgingival bacterial orange complex and is one of the main pathogens of oral infectious diseases such as periodontitis, apical abscesses and pulpal infections. In the progressive stage of periodontitis, as tissue damage worsens and periodontal pockets deepen, P. micra can penetrate deeper into the surrounding periodontal tissues to exert destructive effects, and it also has the ability to adhere to host cells. Exploring the role and mechanism of Parvimonas micra in the occurrence and development of periodontitis is an important scientific proposition in the research and treatment of periodontitis.

[0003] Adhesion and invasion of host cells and tissues by pathogens are important mechanisms for their colonization, spread, escape from host immune defenses, and induction of host pathogenic responses. Oral pathogens have extremely strong tissue invasion capabilities, which can continuously stimulate and destroy gingival cells and are one of the main factors in gingival tissue decline.

[0004] Tmpc has been verified to be a membrane protein of P. micra. It interacts with surface proteins of human oral squamous cell carcinoma (OSCC) and may bind to OSCC surface receptors to mediate P. micra adhesion. Constructing gene mutants by gene knockout and then studying the properties of gene mutants is an important method to determine gene function or study metabolic pathways. However, Micromonas microti is sensitive to overtreatment, so it is difficult to treat with chemical treatment (CaCl 2 ) and electroporation for transformation and gene editing of Micromonas microti, which seriously limits the detailed mechanism research of P. microbiology pathobiology. It is urgent to develop a new method for gene knockout of Micromonas microti. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for knocking out a gene of Micromonas minutissima and its application.

[0006] The technical solution of the present invention is as follows:

[0007] A method for knocking out a gene of Micromonas minutissima, comprising the following steps:

[0008] (1) Using the Micromonas pumila genome as a template, primers were designed and PCR amplification was performed to obtain the upstream homology arm fragment and the downstream homology arm fragment of the gene to be knocked out tmpc;

[0009] (2) Artificially synthesizing the ermB resistance gene template, designing primers, and obtaining the ermB resistance gene sequence by PCR amplification;

[0010] (3) Using the overlap PCR method, the upstream homology arm fragment, the ermB resistance gene fragment, and the downstream homology arm fragment are sequentially connected to obtain a fusion DNA fragment;

[0011] (4) dissolving the fusion DNA fragment in double distilled water to obtain a fusion DNA fragment solution; then dropping the fusion DNA fragment solution onto the BHI solid culture medium and letting it stand for 12 to 24 hours to obtain a culture medium containing the fusion DNA fragment;

[0012] Cultivating Micromonas microti to obtain a suspension of Micromonas microti; then dropping the suspension of Micromonas microti onto a culture medium containing the fusion DNA fragment, and culturing at 37° C. under anaerobic conditions for 24 to 48 hours, so that the fusion DNA fragment is transformed into Micromonas microti;

[0013] (5) The transformed Micromonas microti was spread on a BHI solid medium containing erythromycin and cultured for 4 to 8 days. After screening, the Micromonas microti with the target gene tmpc knocked out was obtained.

[0014] Preferably according to the present invention, in step (1), the NCBI accession number of the gene to be knocked out tmpc is NCTC11808_01529; the length of the upstream homology arm fragment of the gene to be knocked out tmpc is 1479 bp, and the length of the downstream homology arm fragment of tmpc is 1308 bp.

[0015] Preferably according to the present invention, in step (1), the primer sequence for amplifying the upstream homology arm fragment is as follows:

[0016] 1F: 5'-CTAACGAAAGTCACAAAAATGCAAC-3',

[0017] 1R: 5'-CTTATCTCCATTATATCTTATAAAACCTCCTAAATTATTTAGTCTAGT-3';

[0018] The primer sequences for amplifying the downstream homology arm fragment are as follows:

[0019] 3F: 5'-GAAATAATTCTATGAGTCGCATTATTTAACAAAAGGAAGCGAAAGC-3',

[0020] 3R: 5'-TCTTATTCCCAAACCTGGTCCTG-3'.

[0021] Preferably according to the present invention, in step (2), the NCBI accession number of the ermB resistance gene is AB057644;

[0022] The primer sequences for amplifying the ermB resistance gene are as follows:

[0023] 2F: 5'-AAGATATAATGGGAGATAAGACGTTC-3',

[0024] 2R: 5'-GCGACTCATAGAATTATTTCCTCCC-3'.

[0025] Preferably according to the present invention, in step (3), the upstream homology arm fragment, the ermB resistance gene and the downstream homology arm fragment are connected using a Clon Express One Step Cloning Kit.

[0026] Preferably according to the present invention, in step (4), the concentration of the fusion DNA fragment solution is 0.01-0.1 μg / μL; preferably 0.05 μg / μL.

[0027] Preferably, according to the present invention, in step (4), the volume ratio of the fusion DNA fragment solution to the Micromonas minutissima bacterial solution is 1:1.

[0028] Preferably, according to the present invention, in step (5), the screening is to detect mutants using the homology arm inner primers 4F / 4R;

[0029] The sequence of the primer 4F / 4R is as follows:

[0030] 4F: 5'-TGATAATTATGCTTGGAATGTTTC-3',

[0031] 4R: 5'-ACATAATCCCAAAATTGGTA-3'.

[0032] A Micromonas microti with gene tmpc knocked out is constructed according to the above method.

[0033] The above-mentioned Micromonas microti with the tmpc gene knocked out was used to study the mechanism of Micromonas microti attachment and invasion of oral squamous cell carcinoma cells (OSCC).

[0034] Beneficial effects:

[0035] 1. The present invention uses the natural transformation method for the first time to achieve gene knockout of Micromonas microti, effectively overcoming the problem that it is difficult to transform and edit genes in Micromonas microti. In addition, the naturally transformed DNA enters Micromonas microti in the form of a single strand. Unlike the double-stranded DNA introduced artificially, the single-stranded DNA is largely resistant to degradation by most bacterial restriction endonucleases.

[0036] 2. The method for knocking out the gene of Micromonas minimus provided by the present invention does not require the construction and introduction of an expression vector, and can directly perform genetic complementation editing, thereby reducing the experimental steps for developing plasmids adapted to bacteria.

[0037] 3. The tmpc knockout gene Micromonas microti provided by the present invention can be used to study the mechanism of Micromonas microti adhesion and invasion of OSCC cells. The tmpc knockout gene Micromonas microti verified that TmpC is a necessary effector of P. micra in promoting OSCC metastasis in vivo. Compared with the wild-type P. micra (PmWT), the adhesion and invasion ability of P. micraΔtmpC on OSCC cells in vitro was significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of gene knockout in Micromonas pusillus.

[0039] Figure 2 This is the identification result of Micromonas minimus with the tmpc gene knocked out.

[0040] Figure 3 The results of Micromonas microti attachment and invasion to OSCC after knocking out the target gene tmpc. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0042] Micromonas microsporus (P. micra) ATCC 33270 is a standard type strain and can be purchased commercially.

[0043] Human OSCC cell lines CAL27 and SCC15 were purchased from Shanghai Basic Culture Technology Co., Ltd.

[0044] Example 1. Knockout of the tmpc gene in Micromonas pumila

[0045] like Figure 1 As shown, a method for knocking out a gene in Micromonas microti, comprising the following steps:

[0046] (1) After thawing, the Micromonas microsporus (P. micra) ATCC 33270 strain was inoculated on BHI solid medium containing 10% sterile defibrinated sheep blood and 80% N 2 , 10% CO 2 and 10% H 2 The cells were incubated at 37°C in an anaerobic incubator for 3 to 5 days until colonies grew on the BHI agar solid medium. A single colony was picked and inoculated into Schaedler broth medium and continued to be cultured for 3 to 5 days until the cells grew to the logarithmic phase. The bacterial culture was collected and the genome of Micromonas microti was extracted using a genome extraction kit.

[0047] Using the Micromonas pumila genome as a template, primers 1F / 1R and 3F / 3R of the homology arms of the gene to be knocked out tmpc were designed, and the upstream homology arm fragment and the downstream homology arm fragment of the gene to be knocked out tmpc were obtained by PCR amplification, wherein the NCBI accession number of tmpc is NCTC11808_01529;

[0048] The sequence of the primer 1F / 1R for amplifying the upstream homology arm fragment is as follows:

[0049] 1F: 5'-CTAACGAAAGTCACAAAAATGCAAC-3',

[0050] 1R: 5'-CTTATCTCCATTATATCTTATAAAACCTCCTAAATTATTTAGTCTAGT-3';

[0051] The sequence of the primer 3F / 3R for amplifying the downstream homology arm fragment is as follows:

[0052] 3F: 5'-GAAATAATTCTATGAGTCGCATTATTTAACAAAAGGAAGCGAAAGC-3',

[0053] 3R: 5'-TCTTATTCCCAAACCTGGTCCTG-3';

[0054] The PCR amplification system was: 50 μl system, including 200 ng template, 0.4 μM upstream primer and downstream primer respectively, 25 μL 2×Phanta Flash Master Mix, and the rest was made up with water.

[0055] The PCR amplification program was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 58°C for 5 s, extension at 72°C, 5-10 s / kb, 55 sec (30 cycles), termination extension for 1 min, and finally incubation at 4°C.

[0056] (2) Commissioning BGI to artificially synthesize the ermB resistance gene template, designing primers 2F / 2R, and obtaining the ermB resistance gene fragment through PCR amplification;

[0057] The sequence of the primers 2F / 2R for amplifying the ermB resistance gene is as follows:

[0058] 2F: 5'-AAGATATAATGGGAGATAAGACGTTC-3',

[0059] 2R: 5'-GCGACTCATAGAATTATTTCCTCCC-3';

[0060] The PCR amplification system was: 50 μl system, including 200 ng template, 0.4 μM upstream primer and downstream primer respectively, 25 μL 2×Phanta Flash Master Mix, and the rest was made up with water.

[0061] The PCR amplification program was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 58°C for 5 s, extension at 72°C, 5-10 s / kb, 55 sec (30 cycles), termination extension for 1 min, and finally incubation at 4°C.

[0062] (3) Using the Clon Express One Step Cloning Kit, the upstream homology arm fragment, the ermB resistance gene fragment, and the downstream homology arm fragment were sequentially connected by overlap PCR to obtain a fusion DNA fragment;

[0063] (4) Dissolving 1 μg of the fusion DNA fragment in 20 μL of double distilled water to obtain a fusion DNA fragment solution with a concentration of 0.05 μg / μL; then adding 20 μL of the fusion DNA fragment solution dropwise to the BHI solid culture medium and letting it stand for 24 hours to obtain a culture medium containing the fusion DNA fragment;

[0064] streak inoculate a bacterial liquid of Micromonas microcarpa (P. micra) ATCC 33270 on a BHI solid culture medium, and culture in an anaerobic incubator for 3 to 5 days according to the conditions in step (1) until a single colony grows; collect Micromonas microcarpa from the BHI solid culture medium using a disposable inoculation loop, inoculate into 1 mL of BHI liquid culture medium, rotate the quantitative loop to promote the release of bacteria into the culture medium, adjust the concentration of the cell suspension to OD600 = 0.4, and obtain a Micromonas microcarpa suspension; then drop 20 μL of the Micromonas microcarpa suspension (OD600 = 0.4) onto the culture medium containing the fusion DNA fragment, and culture under anaerobic conditions at 37° C. for 36 h, so that the fusion DNA fragment is transformed into Micromonas microcarpa;

[0065] (5) Spreading the transformed Micromonas microti on a BHI solid medium containing erythromycin and continuing to culture for 5 days to obtain transformants; then using primers 4F / 4R to screen and identify the transformants to obtain Micromonas microti with the target gene tmpc knocked out (PmΔtmpc);

[0066] The sequence of the primer 4F / 4R is as follows:

[0067] 4F: 5'-TGATAATTATGCTTGGAATGTTTC-3',

[0068] 4R: 5'-ACATAATCCCAAAATTGGTA-3'.

[0069] The results of knockout strain identification using primers 1F / 2R, 1F / 3R, 2F / 3R and 4F / 4R are shown in Figure 2 shown. Figure 2 WT in the middle is wild-type P. micra, which serves as the control group.

[0070] Depend on Figure 2 It can be seen that compared with the wild-type Micromonas minimus, the tmpc gene in the screened strain has been knocked out from the Micromonas minimus genome, and the ermB gene has successfully replaced the tmpc gene. The present invention successfully achieves gene knockout of Micromonas minimus through a natural transformation method, and constructs Micromonas minimus with the target gene tmpc knocked out.

[0071] Example 2: Application of Micromonas minimus with knockout target gene tmpc in studying the mechanism of Micromonas minimus attachment and invasion of OSCC cells

[0072] 1. OSCC cell culture

[0073] Human OSCC cell lines CAL27 and SCC15 were cultured in a medium containing α-MEM (BI, Israel), 10% FBS (Gibco, USA) and 1% penicillin / streptomycin (Sigma-Aldrich, USA) at 37°C and 5% CO. 2 cultured in an incubator.

[0074] 2. Cultivation of target gene knockout strains

[0075] The wild-type P. micra (PmWT) and the Micromonas microti with the target gene tmpc knocked out (PmΔtmpc) constructed in Example 1 were respectively cultured at 37°C and anaerobic environment (80% N 2 , 10% CO 2 and 10% H 2) in SchaEdler anaerobic medium under the condition of 4% paraformaldehyde and 1% paraformaldehyde for 3 to 4 days. When PmWT and PmΔtmpc grew to the logarithmic growth phase, the supernatant was discarded after centrifugation, the bacteria were washed twice with PBS buffer, and the OD600 was adjusted to 1 using a microspectrophotometer to obtain PmWT bacterial suspension and PmΔtmpc bacterial suspension, respectively.

[0076] 3. Flow cytometry was used to detect the adhesion and invasion of Micromonas microti in OSCC cells with tmpc knockout target gene. 3×10 5 OSCC cells were seeded into 6-well plates containing the culture medium described in step 1 and cultured overnight.

[0077] For the analysis of bacterial attachment: PmWT bacterial suspension and PmΔtmpc bacterial suspension were labeled with a final concentration of 20 μM fluorescent dye CFSE for 30 min at 37°C in the dark, centrifuged, and the precipitate was washed twice with PBS buffer after centrifugation, and resuspended with PBS buffer to obtain CFSE-labeled PmWT bacteria and PmΔtmpc bacteria. At MOI = 100, CFSE-labeled PmWT bacteria and PmΔtmpc bacteria were added to OSCC cells after overnight culture, respectively, and co-cultured at 37°C for 2 h. The culture medium was discarded, and the OSCC cells were gently rinsed 3 times with PBS buffer, and the cells were collected by trypsin digestion. CFSE fluorescence was detected by flow cytometry, and the bacterial attachment rate was analyzed and calculated using Flowjo software.

[0078] For the analysis of bacterial invasion: CFSE-labeled PmWT and PmΔtmpc were added to OSCC cells cultured overnight at MOI = 100, and then 100 μg / ml gentamicin was added for 20 min to kill extracellular P. micra bacteria. The culture medium was discarded, and the OSCC cells were gently washed 3 times with PBS buffer. The cells were collected by trypsin digestion, and the intracellular CFSE fluorescence was detected by flow cytometry. The bacterial invasion rate was analyzed and calculated using Flowjo software.

[0079] Using the human OSCC cell line SCC15, the experiment of Micromonas microti with knockout target gene tmpc on OSCC cells was carried out in the same way. The final results are as follows Figure 3 shown.

[0080] Depend on Figure 3 It can be seen that compared with PmWT, PmΔtmpC has significantly reduced adhesion and invasion ability to OSCC cells in vitro, which verifies that TmpC is a necessary effector that mediates P. micra adhesion and invasion of OSCC cells and promotes OSCC metastasis. Therefore, the micromonas microti with knockout gene tmpc constructed by the present invention can be used in the study of the mechanism of micromonas microti adhesion and invasion of OSCC cells.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for knocking out a gene in Micromonas minutissima, characterized in that: The steps include: (1) Using the Micromonas pumila genome as a template, primers were designed and PCR amplification was performed to obtain the upstream homology arm fragment and the downstream homology arm fragment of the gene to be knocked out tmpc; (2) Artificially synthesizing the ermB resistance gene template, designing primers, and obtaining the ermB resistance gene sequence by PCR amplification; (3) Using the overlap PCR method, the upstream homology arm fragment, the ermB resistance gene, and the downstream homology arm fragment are sequentially connected to obtain a fusion DNA fragment; (4) dissolving the fusion DNA fragment in double distilled water to obtain a fusion DNA fragment solution; then dropping the fusion DNA fragment solution onto the BHI solid culture medium and letting it stand for 12 to 24 hours to obtain a culture medium containing the fusion DNA fragment; Cultivating Micromonas microti to obtain a suspension of Micromonas microti; then dropping the suspension of Micromonas microti onto a culture medium containing the fusion DNA fragment, and culturing at 37° C. under anaerobic conditions for 24 to 48 hours, so that the fusion DNA fragment is transformed into Micromonas microti; (5) The transformed Micromonas microti was spread on a BHI solid medium containing erythromycin and cultured for 4 to 8 days. After screening, the Micromonas microti with the target gene tmpc knocked out was obtained.

2. The method for knocking out a gene of Micromonas minutissima according to claim 1, characterized in that: In step (1), the NCBI accession number of the gene to be knocked out tmpc is NCTC11808_01529; the length of the upstream homology arm fragment of the gene to be knocked out tmpc is 1479 bp, and the length of the downstream homology arm fragment of tmpc is 1308 bp.

3. The method for knocking out a gene of Micromonas minutissima according to claim 1, characterized in that: In step (1), the primer sequence for amplifying the upstream homology arm fragment is as follows: 1F: 5'-CTAACGAAAGTCACAAAAATGCAAC-3', 1R: 5'-CTTATCTCCATTATATCTTATAAAACCTCCTAAATTATTTAGTCTAGT-3'; The primer sequences for amplifying the downstream homology arm fragment are as follows: 3F: 5'-GAAATAATTCTATGAGTCGCATTATTTAACAAAAGGAAGCGAAAGC-3', 3R: 5'-TCTTATTCCCAAACCTGGTCCTG-3'.

4. The method for knocking out a gene of Micromonas minutissima according to claim 1, wherein: In step (2), the NCBI accession number of the ermB resistance gene is AB057644; The primer sequences for amplifying the ermB resistance gene are as follows: 2F: 5'-AAGATATAATGGGAGATAAGACGTTC-3', 2R: 5'-GCGACTCATAGAATTATTTCCTCCC-3'.

5. The method for knocking out a gene of Micromonas minutissima according to claim 1, wherein: In step (3), the upstream homology arm fragment, the ermB resistance gene and the downstream homology arm fragment are connected using the Clon Express One Step Cloning Kit.

6. The method for knocking out a gene of Micromonas minutissima according to claim 1, wherein: In step (4), the concentration of the fusion DNA fragment solution is 0.01-0.1 μg / μL, preferably 0.05 μg / μL.

7. The method for knocking out a gene of Micromonas minutissima according to claim 1, wherein: In step (4), the volume ratio of the fusion DNA fragment solution to the Micromonas microti bacterial solution is 1:

1.

8. The method for knocking out a gene in Micromonas minutissima according to claim 1, wherein: In step (5), the screening is to detect mutants using the homology arm inner primers 4F / 4R; The sequence of the primer 4F / 4R is as follows: 4F: 5'-TGATAATTATGCTTGGAATGTTTC-3', 4R: 5'-ACATAATCCCAAAATTGGTA-3'.

9. A Micromonas microti with a knockout gene tmpc, characterized in that: The method according to claim 1 is used to construct the 10. Use of the Micromonas parvum with the tmpc gene knocked out according to claim 9 in studying the mechanism of Micromonas parvum attachment and invasion of OSCC cells.

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