CTSC overexpression plasmid construction method
By extracting RNA from mouse tail tissue and PCR amplification, the CTSC gene was obtained and cloned seamlessly onto the vector to form a CTSC overexpression plasmid, which solved the problems of low expression efficiency and unstable expression of CTSC protein, and achieved efficient and reliable CTSC protein expression.
Patent Information
- Application Number
- CN202510025070.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-04
AI Technical Summary
In the prior art, when constructing CTSC overexpression plasmids, the expression efficiency of CTSC protein is low and the expression amount is unstable, making it difficult to meet the needs of subsequent upstream and downstream CTSC research.
Total RNA was extracted based on mouse tail tissue, and PCR amplification was performed after reverse transcription. The CTSC gene was obtained and seamlessly cloned onto the penti-cmv-puro DEST vector to form a CTSC overexpression plasmid.
It improves the expression efficiency of exogenous proteins, increases the expression of CTSC protein by 4 times, and is reliable in expression, which can provide tool support for subsequent upstream and downstream research of CTSC.
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Figure CN119752969A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for constructing a CTSC overexpression plasmid. Background Art
[0002] Cathepsin C (CTSC), also known as dipeptidylpeptidase I (DPPI), is synthesized in the cell as a 60KDa zymogen, which is activated by hydrolysis of CTSL and CTSS into a 23KDa heavy chain and an 8KDa light chain, which then form a complex tetramer with catalytic activity. CTSC is an important cysteine protease in the papain family, and cysteine cathepsins are considered to be enzymes involved in the final protein degradation in lysosomes. Among lysosomal proteins, papain-like cysteine proteases are the largest family, with 11 proteases in humans [histones B, C, F, H, K, L, O, S, V, W, X] and another 8 members in mice (histones 1, 2, 3, 6, J, M, Q and R).
[0003] Studies have found that active CTSC hydrolyzes and activates a variety of serine proteases in immune cell lysosomes, such as CTSG, neutrophil elastase (NE) and proteinase 3 (PR3) in neutrophils, and chymotrypsin in mast cells, playing an important pro-inflammatory role in a variety of infections and inflammatory diseases. CTSC mutations can cause Papillon-Lefèvre syndrome, Haim-Munk syndrome and aggressive periodontitis; abnormal CTSC expression leads to a variety of inflammatory diseases such as arthritis and nephritis. CTSC is considered to be a new drug target for the treatment of inflammatory and immune diseases.
[0004] In the prior art, although the importance of CTSC as a new drug target for the treatment of inflammatory and immune diseases has been recognized, when constructing CTSC overexpression plasmids, the expression efficiency of CTSC protein is low and the expression level is unstable, which makes it difficult to meet the needs of subsequent CTSC upstream and downstream research. Summary of the invention
[0005] The purpose of the present invention is to provide a method for constructing a CTSC overexpression plasmid, aiming to solve the technical problems in the prior art that, when constructing a CTSC overexpression plasmid, the expression efficiency of CTSC protein is low and the expression level is unstable, making it difficult to meet the needs of subsequent CTSC upstream and downstream research.
[0006] To achieve the above purpose, the present invention adopts a method for constructing a CTSC overexpression plasmid.
[0007] The steps include:
[0008] Step 1: Extract total RNA from mouse tail tissue;
[0009] Step 2: Reverse transcription followed by PCR amplification to obtain the CTSC gene;
[0010] Step 3: digest the plenti-cmv-puro DEST vector with enzymes, and then connect the CTSC gene amplified in step 1 to the gap of the vector using seamless cloning to obtain a new circular plasmid DNA, which is the CTSC overexpression plasmid.
[0011] Wherein, in step 2, the primers for PCR amplification of CTSC gene are as follows:
[0012] The upstream primers were: (5′-3′): GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC ACC ATGGGT CCC TGG ACC CAC TC;
[0013] The downstream primers were: (5′-3′): GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA TAATTT AGG AAT CGG TAT.
[0014] Among them, in step 2, after obtaining the CTSC gene, it was identified by 0.6% agarose gel electrophoresis, electrophoresis at a constant voltage of 130V for 30 minutes, and ultraviolet development was performed to identify the bands of the CTSC gene. After development, the gene fragment was purified using a gel recovery and purification kit.
[0015] Among them, in step three, the plenti-cmv-puro DEST vector is digested with enzymes, and then the CTSC gene amplified in step two is connected to the gap of the vector by seamless cloning to obtain a new circular plasmid DNA, that is, a CTSC overexpression plasmid, including a Gateway reaction BP ligation reaction and a Gateway reaction LR ligation reaction.
[0016] The specific method of the BP connection reaction through the Gateway reaction is:
[0017] First, the amplified CTSC gene, pDONR, BP Clonase II Enzyme Mix (56481, Invitrigen) and TE Buffer were mixed in proportion to prepare 5 μl of the first reaction solution, and then allowed to stand at room temperature for 4 h;
[0018] Then, 0.7 μl of proteinase K was added to terminate the reaction, and the BP reaction solution was obtained at 37°C for 10 min;
[0019] Then, the BP reaction solution was introduced into E. coli, 1 μl of BP reaction solution was mixed with 50 μl of Stab13 (CD521, TRANS), placed on ice for 30 min, and heat-shocked at 42°C for 45-60 s; placed on ice for 2 min; a first mixed solution was obtained; 700 μl of LB (without antibiotics) was added to the first mixed solution, and the mixture was vigorously shaken at 37°C for 1 h; the first mixed solution was spread on an agar culture plate containing kanamycin (K8020-1g, Solarbio), and placed in a 37°C incubator overnight;
[0020] Finally, single colonies were picked from 2-3 agar culture plates, respectively transferred to 20 ml LB culture medium (containing kanamycin), cultured in a shaking incubator at 37° C. for 12-16 h, and plasmids were extracted to obtain the entry plasmid pDONR-CTSC-Flag.
[0021] The specific method of the LR connection reaction through the Gateway reaction is:
[0022] First, pDONR-CTSC-Flag, plenti-cmv-puro-DEST, LR Clonase II EnzymeMix (56484, Invitrigen) and TE Buffer were mixed in proportion to prepare 5 μl of the second reaction solution, and allowed to stand at room temperature for 2 h;
[0023] Then, 0.7 μl of proteinase K was added to terminate the reaction at 37°C for 10 min;
[0024] Then, the LR reaction solution was introduced into E. coli, 1 μl of the LR reaction solution was mixed with 50 μl of Stabl3, placed on ice for 30 min, and heat-shocked at 42°C for 45-60 s; placed on ice for 2 min to obtain a second mixed solution; 700 μl of LB (without antibiotics) was added to the second mixed solution, and vigorously shaken at 37°C for 1 h; the second mixed solution was spread on an agar culture plate containing Ampicilin (AC00339-0005, Diamond), and placed in a 37°C incubator overnight;
[0025] Then pick 2-3 single colonies on the agar culture plates, transfer them to 20 ml LB culture medium (containing Ampicilin), and culture them in a shaking incubator at 37°C for 12-16 hours;
[0026] Finally, gene sequencing and plasmid extraction (CW2105S, CWBIO) were performed to obtain the CTSC overexpression plasmid, and the plasmid was stored at -20°C.
[0027] A use of a CTSC overexpression plasmid constructed by the CTSC overexpression plasmid construction method as described above in preparing a product for detecting or studying CTSC targets.
[0028] A use of the CTSC overexpression plasmid constructed by the CTSC overexpression plasmid construction method as described above in the preparation of products for treating, detecting or studying diseases related to CTSC.
[0029] The diseases include but are not limited to Alzheimer's disease and mental illness.
[0030] The present invention discloses a method for constructing a CTSC overexpression plasmid. First, total RNA is extracted from mouse tail tissue, and PCR amplification is performed after reverse transcription to obtain the CTSC gene; the plenti-cmv-puro DEST vector is digested by enzymes, and then the CTSC gene amplified in step 1 is connected to the gap of the vector by seamless cloning to obtain a new circular plasmid DNA, which is the CTSC overexpression plasmid. The CTSC overexpression plasmid obtained by the present invention improves the expression efficiency of exogenous proteins and increases the expression amount of CTSC protein by 4 times. The expression of CTSC protein is reliable and can provide tool support for subsequent upstream and downstream research of CTSC. In this way, the technical problem in the prior art that, when constructing a CTSC overexpression plasmid, the expression efficiency of CTSC protein is low and the expression amount is unstable, which makes it difficult to meet the needs of subsequent upstream and downstream research of CTSC is solved. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] 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 or the description of the prior art 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.
[0032] Figure 1 It is a schematic diagram of the construction of the CTSC overexpression plasmid of the present invention.
[0033] Figure 2 It is a schematic diagram of the verification results of the mRNA level of CTSC after transfection of the present invention.
[0034] Figure 3 Figures a and b are the verification results of the protein levels of CTSCs after transfection. DETAILED DESCRIPTION
[0035] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.
[0036] See also Figure 1 to Figure 3 ,in Figure 1 It is a schematic diagram of the construction of the CTSC overexpression plasmid of the present invention. Figure 2 It is a schematic diagram of the verification results of the mRNA level of CTSC after transfection of the present invention. Figure 3 Figures a and b are the verification results of the protein levels of CTSCs after transfection.
[0037] The present invention provides a method for constructing a CTSC overexpression plasmid.
[0038] The steps include:
[0039] S101, total RNA extracted from mouse tail tissue;
[0040] S102, reverse transcription followed by PCR amplification to obtain the CTSC gene;
[0041] For this specific embodiment, the primers for PCR amplification of the CTSC gene are as follows:
[0042] The upstream primers were: (5′-3′): GGG ACA AGT TTG TAC AAA AAA GCA GGC TTC ACC ATGGGT CCC TGG ACC CAC TC;
[0043] The downstream primers were: (5′-3′): GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA TAATTT AGG AAT CGG TAT.
[0044] The PCR reaction systems of the CTSC genes are as shown in Table 1:
[0045] Table 1
[0046]
[0047]
[0048] The PCR reaction conditions were as follows: 94°C for 30 s, 98°C for 10 s, 55°C for 15 s, 72°C for 2 min for 30 cycles, 72°C for 5 min, and storage at 4°C.
[0049] Furthermore, after obtaining the CTSC gene, 0.6% agarose gel electrophoresis was used for identification, constant voltage 130V electrophoresis for 30 minutes, ultraviolet development was performed to identify the band of the CTSC gene, and after development, the gene fragment was purified using a gel recovery purification kit.
[0050] S103, digest the plenti-cmv-puro DEST vector with enzymes, and then connect the CTSC gene amplified in step 1 to the gap of the vector using seamless cloning to obtain a new circular plasmid DNA, which is the CTSC overexpression plasmid.
[0051] For this specific embodiment, it includes a ligation reaction via Gateway reaction BP and a ligation reaction via Gateway reaction LR.
[0052] Specifically, the specific method of the BP connection reaction through the Gateway reaction is:
[0053] 1. First, mix the amplified CTSC gene, pDONR, BP Clonase II Enzyme Mix (56481, invitrigen) and TE Buffer in proportion to prepare 5 μl of the first reaction solution, and let it stand at room temperature for 4 hours:
[0054] Prepare 5 μl of the first reaction solution and add the following components in the amounts shown in Table 2:
[0055] Table 2
[0056] Components Addition amount attB DNA 100ng pDON 100ng BP Clonase II Enzyme Mix (56481, invitrigen) 1μl TE Buffer Make up to 5 μl
[0057] Among them, attB DNA is the CTSC gene obtained by the amplification reaction.
[0058] 2. Then add 0.7 μl proteinase K to terminate the reaction to obtain BP reaction solution, at 37°C for 10 min;
[0059] 3. Then, the BP reaction solution was introduced into E. coli, 1 μl of BP reaction solution was mixed with 50 μl of Stabl3 (CD521, TRANS), placed on ice for 30 min, heat-shocked at 42°C for 45-60 s; placed on ice for 2 min; the first mixed solution was obtained; 700 μl of LB was added to the above mixed solution, and the mixture was vigorously shaken at 37°C for 1 h; the mixed solution was spread on a kanamycin agar culture plate and placed in a 37°C incubator overnight;
[0060] 4. Finally, pick 2-3 single colonies on the agar culture plate, transfer them to 20 ml LB culture medium respectively, culture them in a shaking incubator at 37°C for 12-16 hours, extract the plasmid, and obtain the entry plasmid pDONR-CTSC-Flag.
[0061] The specific method of LR ligation reaction through Gateway reaction is:
[0062] 1. First, pDONR-CTSC-Flag, plenti-cmv-puro-DEST, LR Clonase II Enzyme Mix (56484, Invitrigen) and TE Buffer were mixed in proportion to prepare 5 μl of the second reaction solution, and allowed to stand at room temperature for 2 h;
[0063] Prepare 5 μl of the second reaction solution and add the following components in the amounts shown in Table 3:
[0064] Table 3
[0065] Components Addition amount pDONR-CTSC-Flag 100ng plenti-cmv-puro-DEST 100ng LR Clonase II Enzyme Mix (56484, invitrigen) 1μl TE Buffer Make up to 5 μl
[0066] 2. Then add 0.7 μl proteinase K to terminate the reaction at 37°C for 10 min;
[0067] 3. Then, the LR reaction solution was introduced into E. coli, 1 μl of LR reaction solution was mixed with 50 μl of Stabl3, placed on ice for 30 min, and heat-shocked at 42°C for 45-60 s; placed on ice for 2 min to obtain a second mixed solution; 700 μl of LB (without antibiotics) was added to the second mixed solution, and vigorously shaken at 37°C for 1 h; the second mixed solution was spread on an agar culture plate containing Ampicilin (AC00339-0005, Diamond), and placed in a 37°C incubator overnight;
[0068] 4. Then pick 2-3 single colonies on the agar culture plates, transfer them to 20 ml LB culture medium (containing Ampicilin), and culture them in a shaking incubator at 37°C for 12-16 hours;
[0069] 5. Finally, gene sequencing and plasmid extraction (CW2105S, CWBIO) were performed to obtain the CTSC overexpression plasmid, and the plasmid was stored at -20°C.
[0070] After the CTSC overexpression plasmid was constructed, the in vitro expression experiment of CTSC exogenous protein was performed as follows:
[0071] BV2 cells were cultured in vitro. When the cells grew to the logarithmic growth phase and the number reached 60%, plasmid transfection was performed using lipo3000. The medium was changed 6 hours after transfection. CTSC expression was detected 48 hours after transfection: cell RNA was extracted for Real-time qPCR to detect the mRNA level of CTSC and protein lysate was used for Western blot to detect the CTSC protein level. The results are shown in the attached Figure 2 and attached Figure 3, transfected on mouse microglia (BV2), Real-time qPCR results showed that the CTSC RNA level in the overexpression group was more than 1000 times that in the normal group. In general, the CTSC mRNA level was at least 1000 times that of the normal group, and the CTSC overexpression group was more than four times that of the control group at the protein level.
[0072] A CTSC overexpression plasmid construction method of the present invention is used, firstly, total RNA is extracted from mouse tail tissue, reverse transcription is performed and then PCR amplification is performed to obtain the CTSC gene; the plenti-cmv-puro DEST vector is digested, and then the CTSC gene amplified in step one is connected to the gap of the vector by seamless cloning to obtain a new circular plasmid DNA, i.e., the CTSC overexpression plasmid. The CTSC overexpression plasmid obtained by the present invention improves the expression efficiency of exogenous proteins and increases the expression amount of CTSC protein by 4 times. The expression of CTSC protein is reliable and can provide tool support for subsequent upstream and downstream research of CTSC. In this way, the technical problem in the prior art that, when constructing a CTSC overexpression plasmid, the expression efficiency of CTSC protein is low and the expression amount is unstable, which makes it difficult to meet the needs of subsequent upstream and downstream research of CTSC is solved.
[0073] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.
Claims
1. A method for constructing a CTSC overexpression plasmid, characterized in that: The steps include: Step 1: Extract total RNA from mouse tail tissue; Step 2: Reverse transcription followed by PCR amplification to obtain the CTSC gene; Step 3: digest the plenti-cmv-puro DEST vector with enzymes, and then connect the CTSC gene amplified in step 1 to the gap of the vector using seamless cloning to obtain a new circular plasmid DNA, which is the CTSC overexpression plasmid.
2. The method for constructing a CTSC overexpression plasmid according to claim 1, characterized in that: In step 2, the primers for PCR amplification of the CTSC gene are as follows: The upstream primers were: (5′-3′): GGG ACAAGT TTG TAC AAAAAA GCA GGC TTCACCATG GGT CCCTGGACC CAC TC; The downstream primers were: (5′-3′): GGG GAC CAC TTT GTACAAGAAAGC TGG GTC CTA TAA TTT AGGAAT CGG TAT.
3. The method for constructing a CTSC overexpression plasmid according to claim 2, characterized in that: In step 2, after obtaining the CTSC gene, 0.6% agarose gel electrophoresis was used for identification, constant voltage 130V electrophoresis for 30 minutes, ultraviolet development was performed to identify the band of the CTSC gene, and after development, the gene fragment was purified using a gel recovery and purification kit.
4. The method for constructing a CTSC overexpression plasmid according to claim 3, characterized in that: In step three, the plenti-cmv-puro DEST vector is digested with enzymes, and then the CTSC gene amplified in step two is connected to the gap of the vector by seamless cloning to obtain a new circular plasmid DNA, i.e., the CTSC overexpression plasmid, including a Gateway reaction BP ligation reaction and a Gateway reaction LR ligation reaction.
5. The method for constructing a CTSC overexpression plasmid according to claim 4, characterized in that: The specific method of BP connection reaction through Gateway reaction is: First, the amplified CTSC gene, pDONR, BP Cl0nase II Enzyme Mix and TEBuffer were mixed in proportion to prepare 5 μl of the first reaction solution, and then allowed to stand at room temperature for 4 hours; Then, 0.7 μl of proteinase K was added to terminate the reaction, and the BP reaction solution was obtained at 37°C for 10 min; Then, the BP reaction solution was introduced into E. coli, 1 μl of BP reaction solution was mixed with 50 μl of Stabl3, placed on ice for 30 min, and heat-shocked at 42°C for 45-60 s; placed on ice for 2 min; the first mixed solution was obtained; 700 μl of LB was added to the above mixed solution, and the mixture was vigorously shaken at 37°C for 1 h; the mixed solution was spread on a kanamycin agar culture plate and placed in a 37°C incubator overnight; Finally, single colonies were selected from 2-3 agar culture plates, respectively transferred to 20 ml LB culture medium, cultured in a shaking incubator at 37° C. for 12-16 h, and plasmids were extracted to obtain the entry plasmid pDONR-CTSC-Flag.
6. The method for constructing a CTSC overexpression plasmid according to claim 5, characterized in that: The specific method of LR ligation reaction through Gateway reaction is: First, pDONR-CTSC-Flag, plenti-cmv-puro-DEST, LR Clonase II Enzyme Mix and TE Buffer were mixed in proportion to prepare 5 μl of the second reaction solution, and then allowed to stand at room temperature for 2 h; Then, 0.7 μl of proteinase K was added to terminate the reaction at 37°C for 10 min; Then the LR reaction solution was introduced into E. coli, 1 μl of 1LR reaction solution was mixed with 50 μl of Stabl3, placed on ice for 30 min, and heat-shocked at 42°C for 45-60 s; Place on ice for 2 minutes to obtain a second mixed solution; add 700 μl LB to the second mixed solution and shake vigorously at 37°C for 1 hour; spread the second mixed solution on an Ampicilin agar plate and place in a 37°C incubator overnight; Then pick 2-3 single colonies on the agar culture plates, transfer them to 20 ml LB culture medium, and culture them in a shaking incubator at 37°C for 12-16 hours; Finally, gene sequencing and plasmid extraction were performed to obtain the CTSC overexpression plasmid, and the plasmid was stored at -20°C.
7. A CTSC overexpression plasmid constructed by the method for constructing a CTSC overexpression plasmid according to claim 6, characterized in that: Application in the preparation of products for detecting or studying CTSC targets.
8. A CTSC overexpression plasmid constructed by the method for constructing a CTSC overexpression plasmid according to claim 6, characterized in that: Application in preparing products for treating, detecting or studying diseases associated with CTSC.