Apolipoprotein and preparation method thereof

Abstract

The invention relates to the field of protein recombination, in particular to apolipoprotein and a preparation method thereof, the apolipoprotein is obtained by constructing plasmids through sequence synthesis, transforming, transfecting with sf9 insect cells and amplifying to obtain P4-generation viruses, the P4-generation viruses are expressed in Hi cells and purified to obtain the apolipoprotein, and the constructed apolipoprotein is accurate and stable.

Description

Technical Field

[0001] The present invention relates to the field of protein recombination, and in particular to an apolipoprotein and a preparation method thereof. Background Art

[0002] Atherosclerotic cardiovascular disease. Since the 1990s, the medical community has discovered that lowering serum total cholesterol levels can prevent the formation of atherosclerosis and reduce the incidence, development and mortality of atherosclerotic cardiovascular disease. At present, the only drugs in the world that can lower serum total cholesterol are HMG-CoA reductase inhibitors that inhibit the synthesis of endogenous cholesterol, namely statins. Statins have the effect of preventing the expansion of atherosclerotic plaques, but statins cannot remove cholesterol accumulated in the inner wall of blood vessels and atherosclerotic plaques, and cannot make atherosclerotic plaques on the inner wall of blood vessels disappear. It is even more impossible to treat acute cardiovascular diseases caused by the formation of atherosclerotic plaques, such as myocardial infarction, angina pectoris and other high-mortality diseases. At present, the only means of treating acute cardiovascular diseases caused by the formation of atherosclerotic plaques is cardiac surgery, such as coronary artery bypass grafting, catheterization and stent dilatation. Due to the limitations of these surgical treatments that only have short-term relief of symptoms, high surgical risks and high recurrence rates, the medical community has been seeking a "high-efficiency drug" solution in the past few decades.

[0003] Apolipoprotein CI has multiple physiological functions: ① Inhibits the uptake of TG-rich lipoproteins by liver lipoprotein receptors: ApoC-I inhibits the binding of ApoE-mediated β-VLDL to LDL receptors and LDL receptor-related proteins by replacing ApoE in β-VLDL or masking and changing the structure of ApoE, thereby delaying the clearance of TG-rich lipoproteins; ② Activates phosphatidylcholine cholesterol acyltransferase (LCAT) and promotes cholesterol esterification. ApoA-I is the strongest activator of LCAT, and ApoC-I can also activate LCAT, with an activation capacity of about 78% of ApoA-I; ③ Inhibits cholesterol ester transfer protein (CETP). In vitro experiments have shown that the peptide synthesized according to the 38 amino acid sequence at the N-terminus of ApoC-I can inhibit the activity of CETP. Therefore, ApoC-I plays a very important role in the regulation of lipid metabolism. At present, ApoC-I is mostly isolated and purified from plasma, with high cost and low yield. Therefore, it is necessary to establish and develop methods for recombinant expression and purification of ApoC-I to meet the needs of laboratory research. Summary of the invention

[0004] In view of the existing problems, the present invention provides an apolipoprotein and a preparation method thereof. The invention constructs a plasmid by sequence synthesis, transforms it, and then transfects and amplifies it with sf9 insect cells to obtain a P4 generation virus, and then expresses it in Hi cells and purifies it to obtain apolipoprotein.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In one aspect, the invention provides a method for constructing an apolipoprotein, comprising the following steps:

[0007] (1) Construction of expression plasmid;

[0008] (2) transforming the expression plasmid described in step (1) and extracting the recombinant plasmid again;

[0009] (3) transfecting the recombinant plasmid described in step (2) with sf9 insect cells to obtain P1 virus;

[0010] (4) Amplifying the P1 virus to obtain the P4 virus;

[0011] (5) expressing the P4 virus in Hi cells;

[0012] (6) Purify the protein and verify it.

[0013] In a specific embodiment, the transformation culture medium in step (2) contains three antibiotics, including kanamycin, gentamicin, and tetracycline.

[0014] In a specific embodiment, the amplification described in step (4) includes amplifying the P1 generation virus for 2-4 days to obtain the P2 generation virus, preferably amplifying for 3 days.

[0015] In a specific embodiment, the P2 generation virus is obtained by amplifying the P3 generation virus in 2-4 days, and preferably for 3 days.

[0016] In a specific embodiment, the P3 generation virus is obtained by amplifying the P4 generation virus in 2-4 days, and preferably for 3 days.

[0017] In a specific embodiment, the ratio of the P4 virus to the Hi cells in step (5) is 1:30-1:50.

[0018] In another aspect, the present invention provides an apolipoprotein constructed by the construction method described in the first aspect.

[0019] In a specific embodiment, the amino acid sequence of the apolipoprotein is shown in SEQ ID NO.1. According to the present invention, the amino acid sequence of PVY-CI-C-STREP II (SEQ ID NO.1) is as follows:

[0020] In a specific embodiment, the gene size of the apolipoprotein is 4000-4200 bp, preferably 4100 bp.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The invention constructs a plasmid by sequence synthesis, performs transformation, and then uses sf9 insect cells for transfection and amplification to obtain a P4 generation virus, and purifies the apolipoprotein after expression in Hi cells. The apolipoprotein constructed by the invention is accurate and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 To verify the expression of CI protein;

[0024] Figure 2 To construct the recombination status of Bacmid vector;

[0025] FIG3 shows the toxicity of the constructed vectors, wherein FIG3(A) is a control group and FIG3(B) is an experimental group;

[0026] Figure 4 This is the mortality rate after the constructed vector is expressed;

[0027] Figure 5 This is the affinity chromatography result of CI protein;

[0028] Figure 6 This is the gel chromatography purification result of CI protein;

[0029] Figure 7 This is the CI protein spectrum comparison result. DETAILED DESCRIPTION

[0030] To further explain the technical means and effects of the present invention, the technical solution of the present invention is further described below with reference to the accompanying drawings and through specific implementation methods, but the present invention is not limited to the scope of the embodiments.

[0031] Example 1 Construction of expression plasmid

[0032] The protein sequence of the target protein was obtained from the National Center for Biotechnology Information (NCBI) of the United States and sent to Suzhou Genewise Biotechnology Co., Ltd. for plasmid construction. By means of molecular cloning, the CI gene fragment was cloned into the pFastBac1 expression vector to obtain the recombinant plasmid of the insect-baculovirus expression system of the CI protein, as follows:

[0033] PVY-CI-C-STREPII:MSLDDVIKNFDERNEIIDFELSEDTIRTSSVLDTKFSDWWDRQIQMGHTLPHYRTEGHFMEFTTRATAVQVANDIAHSEHLDFLVRGAVGSGKSTGLPVHLSVAGSVLLIEPTRPLAENVFKQLSSEPFFKKPTLRMRGNSIFFGSSPISVMTSGFALHYFAN NRSQLAQFNFVIFDECHVLDPSAMAFRSLLSVYHQACKVLKVSATPVGREVEFTTQQPVKLIVEDTLSFQSFVDAQGSKTNADVVQFGSNVLVYVSSYNEVDTLAKLLTDKNMMVTKVDGRTMKHG CLEIVTKGTSARPHFVVATNIIENGVTLDIDVVVDFGLKVSPFLDIDNRSIAYNKVSVSYGERIQRLGRVGRFKKGVALRIGHTEKGIIEIPSMIATEAALACFAYNLPVMTGGVSTSLIGNCTVRQ VKTMQQFELSPFFIQNFVAHDGSMHPVIHDILKKYKLRDCMTPLCDQSIPYRASSTWLSVSEYERLGVALEIPKQAKIAFHIKEIPPKLHEMLWETVVKYKDVCLFPSIRASSISKIAYTLRTDLFA IPRTLILVERLLEEERVKQSQFRSLIDEGCSSMFSIVNLTNTLRARYAKDYTAENIQKLEKVRSQLKEFSNLDGSACEENLIKRYESLQFVHHQGSGGWSHPQFEKGGGSGGGSGGGSWSHPQFEK.

[0034] Example 2 Recombinant pFastBac1 plasmid transforms DH10Bac TM E. coli

[0035] a. Take 100 μL of MAX DH10Bac TM Keep competent cells on ice.

[0036] b. Add 50-100 ng of recombinant pFastBac1 plasmid to 100 μL DH10Bac TM In competent cells, gently tap the tube wall with your finger and place on ice for 30 minutes.

[0037] c. Heat shock at 42℃ for 45 seconds, put the EP tube back on ice and let it stand for 2 minutes.

[0038] d. Add 900 μL LB medium to each EP tube in a clean bench.

[0039] e. Incubate at 37°C and 225 rpm for 4 hours.

[0040] f. After 4 hours of culture, take 100 μL of bacterial solution and apply it to a triple-antibody plate (LB plate, containing: 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml Bluo-gal, and 40 μg / ml IPTG). The remaining bacterial solution is temporarily stored at 4°C. Depending on the number of colonies growing on the plate, determine whether the amount of bacterial solution to be applied needs to be increased or decreased.

[0041] g. Incubate the plate upside down in a 37°C oven for 48 h.

[0042] h. Select uniform white spots and add each single clone to 50 μL-3 ml LB liquid culture medium containing three antibiotics (50 μg / ml kanamycin, 7 μg / ml gentamicin and 10 μg / ml tetracycline) and culture at 37°C and 225 rpm. 50 μL of bacterial liquid can be cultured for half an hour for identification.

[0043] i. Use M13 primer to PCR to identify whether the white spot is a positive clone.

[0044] M13 Forward(-40)5′d[GTTTTCCCAGTCACGAC]3′

[0045] M13 Reverse5′d[CAGGAAACAGCTATGAC]3′

[0046] 50μL PCR system:

[0047]

[0048] PCR program setting: (steps 2-4 cycle 30 times)

[0049]

[0050] 1% agarose gel was used to identify the bands. If the Bacmid recombination was successful, the band size would be 2300 bp plus the bp of the target gene. A large and uniform white spot clone was selected for transfer to the bacterial solution.

[0051] j. The white spot bacterial solution identified as positive by PCR was transferred to 3 mL of LB medium containing kanamycin, gentamicin and tetracycline and continued to be cultured at 37°C and 225 rpm for about 24 hours. When the bacterial solution concentration OD600 was about 0.6, the recombinant Bacmid was extracted using the mini-plasmid extraction method (Tiangen mini-extraction kit).

[0052] The expression of plasmid was verified by western blotting. Figure 1 As shown, it indicates that the construct can express the target protein.

[0053] Example 3 Transfection of Bacmid with sf9 Insect Cells

[0054] a. Prepare the sf9 cells in suspension culture in advance. The cells are in the logarithmic growth phase and the density is about 1.5-2.5×10 6 cell / mL.

[0055] b. Dilute the cell solution 4 times (50 μL cell solution + 150 μL culture medium), and then count the cells using a cell counting plate. Count the number of cells in four large grids (16 small grids), and multiply by 104 to get the cell density. Spread a six-well plate (hole diameter 35 mm), 8-9×10 cells per well. 5 Place the six-well plate in a 27°C incubator for about 1 hour to allow the cells to fully adhere to the wall.

[0056] c. In a clean bench, take 6-8μL of transfection reagent (FuGene6) + 100μL of antibody-free culture medium, mix gently, label tube 1, and let stand for 5 minutes. Bacmid 2μg + 100μL of antibody-free culture medium, mix gently, label tube 2, and let stand for 5 minutes. Transfer the solution in tube 1 to tube 2 (total volume is about 210μL), mix gently, and incubate for 15-30 minutes.

[0057] d. Aspirate the culture medium in the six-well plate with a pipette, add 2 ml of antibiotic-free culture medium to each well and wash twice. Do not discard the culture medium after the second wash.

[0058] e. Add 0.8 ml of antibody-free culture medium to each tube of Bacmid and FuGene6 mixed in step c, and transfer the culture medium in the six-well plate to the

[0059] The base was discarded, the mixture was gently mixed and then slowly added along the wall of the six-well plate.

[0060] f. Place in a 27°C incubator and incubate for 5 hours.

[0061] g. Discard the mixed solution in the six-well plate and add 2 mL of triple-antibody culture medium (containing three antibiotics: 50 units / ml penicillin, 50 μg / ml streptomycin and 0.125 μg / ml Antimycotic).

[0062] h. Incubate in a 27℃ incubator. Observe the transfection phenomenon.

[0063] In the early stage (within 24 hours), the cell diameter increases by about 25-50%.

[0064] In the late stage (24-72h), compared with the control group, the cells transfected with Bacmid stopped growing and granular substances appeared, which were budding virus particles, and the cells vesiculated.

[0065] At a later stage (after 72 h), cells lyse and the monolayer of cells floats.

[0066] i. If the transfection phenomenon is consistent with the above, the virus can be collected within 72 hours: 2 mL of supernatant is collected, centrifuged at 500 g for 5 min, and then transferred to a clean EP tube. Wrap it in tin foil and store it at 4°C away from light to obtain the P1 virus.

[0067] Transform the recombinant plasmid into DH10 Bac competent medium and culture it in triple antibody plate to obtain blue white spots. Pick the white spots for culture and extract Bacmid. PCR is used to identify whether the bacmid is successfully recombined. The results are as follows: Figure 2 As shown, the size of the successfully recombined Bacmid is 2300 bp plus the bp number of the target fragment. The bp number of the CI gene is 1800, so the PCR product band size of the successfully recombined Bacmid is 4100 bp. The results show that the construction is successfully recombined.

[0068] Example 4: Poisoning

[0069] P1~P2:

[0070] 1) 10 cm dish: 5 mL culture medium + 5 mL sf9 cells (total cell number is 1×10 7 ).

[0071] 2) After the cells adhered, add 1 mL of P1 to each dish and culture for 3 days.

[0072] 3) Centrifuge at 500 g for 5 min and transfer the supernatant to obtain the P2 virus.

[0073] P2~P3:

[0074] 1) Spread a 10cm plate.

[0075] 2) Add 150 μL of P2 to each plate and culture for 3 days (200-500 μL, depending on virulence).

[0076] 3) Centrifuge at 500g for 5 min, transfer the supernatant after centrifugation to obtain P2 virus.

[0077] 4) P3~P4:

[0078] 1) Medium bottle 400mL, density 2×10 6 .

[0079] 2) Add 300-400 μL P3 per 100 mL of cells and culture for 3 days (200 μL).

[0080] 3) Centrifuge at 300g for 5 min and transfer the supernatant, which is the P4 poison.

[0081] One Bacmid was selected and transfected into sf9 cells, and four rounds of virus expansion were performed. The results of P3 virus expansion showed that compared with the control group cells, the virus-expanded cells were obviously swollen and vacuolated, and the Bacmid successfully infected the cells, as shown in Figure 3.

[0082] Example 5 Protein Expression

[0083] P4 virus was added to 350ml Hi5 cells at a ratio of 1:30-1:50 for expression. The best harvest time is between 24 and 48 hours, and 50% cytopathic effect is usually considered appropriate under microscopic examination.

[0084] P4 virus was used to infect Hi5 cells for protein expression. After 48 hours, the cell death rate reached 50%. Figure 4 Subsequent purification was performed immediately as indicated.

[0085] Example 6 Purification of protein

[0086] Collect cells by centrifugation at 3000rpm for 10min in a floor centrifuge. Resuspend 700mL of cells with 50mL of buffer 1 (20mM Tris, 500mMNaCI, pH 8.0), and place the resuspended liquid in a 100mL plastic container. Use an ultrasonic disruptor to break for 30min (10% power, supersonic 4s stop 6s), and then use a vertical low-temperature floor centrifuge for high-speed centrifugation for 30min (12000rpm). Discard the precipitate and take the supernatant, filter it with a 0.22μm needle filter, and collect the filtrate in a clean 100mL plastic bottle. Use a peristaltic pump to remove the storage solution in the HisTrap 5ml pre-packed column (GE Healthcare) and Strep HP 5mL (GEHealthcare) with deionized water filtered with a 0.22μm filter membrane, and then equilibrate the affinity column with 25mL buffer 1. Use a peristaltic pump to hang the prepared supernatant on the equilibrated affinity column. The affinity column bound to the target protein is eluted and purified using AKTA.

[0087] 1) Strep HP column

[0088] First, wash pumps A and B with deionized water filtered through a 0.22μm filter membrane. Balance pump A with buffer 1 (20mMTris, 500mMNaCl, pH 8.0). After balancing, connect the column to the machine, rinse the column with buffer 1 at a flow rate of 2mL / min, and collect the flow-through peak. After the UV is smooth, pause the machine, put pump A into the desthiobiotin solution filtered through a 0.22μm filter membrane, and elute the target protein. Collect the protein corresponding to the peak, identify the collected samples by SDS-PAGE, and determine the component where the target protein is located.

[0089] The target protein containing the Strep tag was captured using the Strep HP affinity column and eluted using AKTA. The eluate was verified by SDS-PAGE. The results showed that the target protein with the Strep tag as the purification tag was captured. Figure 5 shown.

[0090] 2) Gel filtration chromatography

[0091] The protein was further purified using a Superose 6 column with a buffer of 20 mM Tris, 150 mM NaCl, pH 8.0. The protein corresponding to the peak was collected, and the collected samples were identified by SDS-PAGE to determine the component where the target protein was located.

[0092] After the eluted protein was concentrated, gel filtration chromatography was performed using a Superose 6 Increase column to obtain a high-purity target protein. Figure 6 shown.

[0093] At the same time, the purified target protein was sent to the mass spectrometry platform of the Institute of Microbiology, Chinese Academy of Sciences for mass spectrometry verification. Figure 7 Show: The protein is indeed PVY-CI protein.

[0094] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing an apolipoprotein, characterized in that: The following steps are involved: (1) Construction of expression plasmid; (2) transforming the expression plasmid described in step (1) and extracting the recombinant plasmid again; (3) transfecting the recombinant plasmid described in step (2) with sf9 insect cells to obtain P1 virus; (4) Amplifying the P1 virus to obtain the P4 virus; (5) expressing the P4 virus in Hi cells; (6) Purify the protein and verify it.

2. The construction method according to claim 1, characterized in that: The culture medium for transformation in step (2) contains three antibiotics, including kanamycin, gentamicin and tetracycline.

3. The construction method according to claim 1, characterized in that: The amplification described in step (4) includes amplifying the P1 generation virus for 2-4 days to obtain the P2 generation virus, preferably amplifying for 3 days.

4. The construction method according to claim 3, characterized in that: The P2 generation virus is used to obtain the P3 generation virus in 2-4 days, and preferably the amplification takes 3 days.

5. The construction method according to claim 4, characterized in that: The P3 generation virus is used to obtain the P4 generation virus in 2-4 days, and preferably the amplification takes 3 days.

6. The construction method according to claim 1, characterized in that: In step (5), the ratio of P4 virus to Hi cells is 1:30-1:

50.

7. An apolipoprotein constructed by any one of the construction methods of claims 1-6.

8. The apolipoprotein according to claim 7, characterized in that The amino acid sequence of the apolipoprotein is shown in SEQ ID NO.

1.

9. The apolipoprotein according to claim 7 or 8, characterized in that The gene size of the apolipoprotein is 4000-4200 bp.

10. The apolipoprotein according to claim 9, characterized in that The gene size of the apolipoprotein is 4100 bp.

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