Application of 2s-4b protein of peanut to induction of cell apoptosis
A 2s-4b, inducing cell technology, applied in the application of peanut 2s-4b fusion protein in inducing apoptosis, can solve the problem of less than 10% cure rate, limited early diagnosis of lung cancer, and research on the medicinal value of 2s albumin Fewer problems, etc., to achieve the effect of facilitating industrial production and efficient expression
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[0038] Example 1 2s-4b cDNA cloning
[0039] 1. Using the conventional Trizol method in the art to extract peanut total RNA.
[0040] 2. RT-PCR detection
[0041] 3. Synthesis of First-Strand cDNA and 3'-cDNA from Peanut Total RNA
[0042] Use TaKaRa PrimeScriptTM 1st Strand cDNA Synthesis Kit for reverse transcription to synthesize the first strand of cDNA. For the reaction system and reaction conditions, please refer to the relevant kit instructions ( figure 1 ).
[0043] 4. Obtaining the 2s-4b Gene Degenerate Primer Fragment
[0044] According to the 2s protein MS-MS results obtained in our laboratory, the design 2s-4b The degenerate upstream and downstream primers are shown in SEQ ID NO:6~7. Where R=A / G, Y=C / T, M=A / C, K=G / T, S=C / G, W=A / T, H=A / C / T, B=C / G / T,=A / C / G, D=A / G / T, N=A / C / G / T. Prepare a 60 μL PCR reaction in a 200 μL Eppendorf thin-walled tube.
[0045] 5. Obtaining the 3' end of 2s-4b cDNA
[0046] According to the sequencing results obtained by deg...
Example Embodiment
[0058] Example 2 pET - 32a - 2s-4b Construction and identification of prokaryotic expression vector
[0059] 1. 2s-4b Purification of Gene PCR Products
[0060] Using the TaKaRa DNA Fragment Purification Kit for 2s-4b PCR products were purified.
[0061] 2. 2s-4b Double digestion of gene purification product and pET-32a plasmid
[0062] 3. Gel recovery and purification of enzyme digestion products
[0063] 2s-4b Purification of the digested product was carried out using TaKaRa DNA Fragment Purification Kit.
[0064] 4. 2s-4b Ligation of gene to pET-32a plasmid
[0065] Ligation was performed using T4 DNA Ligase from TaKaRa Corporation.
[0066] 5. Preparation of E. coli Competent Cells
[0067] 6. Recombinant plasmid pET-32a- 2s-4b transformation
[0068] 7. Detection of recombinant plasmids
[0069] Identification of pET-32a plasmid ligation in E. coli on resistant plates by PCR 2s-4b Full-length cDNA fragments. pET-32a- 2s-4b Double enzyme...
Example Embodiment
[0070] Example 3 AtACDO1 homologous gene OsACDO1 Antisense Subtractive Vectors and Forward Overexpression
[0071] 1. Inducible expression of 2s-4b fusion protein
[0072] (1) Transform the pET-32a-2s-4b recombinant plasmid into Escherichia coli Rosetta-gami respectively.
[0073] (2) Pick a single colony and inoculate it in 2 ml LB liquid medium containing four antibodies, and incubate at 37 °C, 200 rpm for 12 h.
[0074] (3) Take 200 μl of the initial shaking bacteria solution in 20 ml of LB liquid medium containing four antibodies, and incubate at 37 °C, 200 rpm for 4-5 h (determination of OD590 of the bacteria solution = 0.5-1.9).
[0075] (4) When the OD590 of the bacteria solution reaches 0.5~1.9, take out 1 ml of the bacteria solution, centrifuge at 10,000 × g for 1 min at room temperature, discard the culture solution, add 100 μl of sterilized PBS to resuspend the bacteria, and store at -20 °C.
[0076] (5) Add IPTG to the remaining culture medium to make the...
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