Construction of CD14 eukaryotic expression vector and method for preparing high-expression cell strain by using CD14 eukaryotic expression vector
A technology for eukaryotic expression vectors and cells, applied to cells modified by the introduction of foreign genetic material, receptors/cell surface antigens/cell surface determinants, using vectors to introduce foreign genetic materials, etc., can solve the effect of TLR auxiliary regulation issues affecting anticancer therapy or antiviral responses
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Embodiment 1
[0039] The design of the specific primer of a kind of CD14 gene of embodiment 1
[0040] According to the mRNA sequence of CD14 in GenBank (NM_000591), design the upstream and downstream primers including the entire CD14 coding region. The sequence of the upstream primer F1 is shown in the sequence table SEQ NO.1. The 5' end of F1 introduces a HindIII restriction site, and the downstream primer The sequence of R1 is shown in SEQ NO.2, the 5' of R1 is introduced with a BamHI restriction site, and the amplified length is 1153bp. At the same time, the sequence of the CD14 fragment amplification primer F2 designed for RT-PCR identification after cell transfection is shown in the sequence table SEQ NO.3, the sequence of R2 is shown in the sequence table SEQ NO.4, and the sequence of the internal reference primer β-actin upstream primer F3 is shown in The sequence listing is SEQ NO.5, and the sequence of the downstream primer R3 is shown in the sequence listing SEQ NO.6. The amplif...
Embodiment 2
[0041] Example 2 Construction of CD14 eukaryotic expression vector
[0042] 1 Materials and methods
[0043] 1.1 Materials
[0044] Escherichia coli DH5α, gastric cancer cell line SGC-7901, pcDNA3.1-EGFP eukaryotic expression vector; total RNA extraction kit; TIAN Script cDNA first-strand synthesis kit; 2×Taq PCR Master Mix were purchased from Tiangen Biochemical Technology Co., Ltd. Company; pMD19-T vector was purchased from Dalian Bao Biological Engineering Co., Ltd.; HindIII and BamHI endonuclease were purchased from NEB Company; Lipofectamine2000, G418 and Lipofectamine were purchased from Invitrogen Company; RPMI1640 and fetal bovine serum (FBs) were purchased from GibcoBRL Company . Other reagents were of domestic analytical grade.
[0045] 1.2 Method
[0046] 1.2.1 Extraction of total RNA from gastric cancer cell line SGC-7901
[0047] Collect the cultured SGC-7901 cells, add 1ml RZ lysate, and let stand at room temperature for 5min. Add 200 μl of chloroform, mix ...
Embodiment 3
[0058] 1 Materials and methods
[0059] 1.1 Materials
[0060] Gastric cancer cell line SGC-7901, pcDNA3.1-EGFP eukaryotic expression vector, recombinant vector pcDNA3.1-EGFP-CD14; total RNA extraction kit TIAN Script cDNA first-strand synthesis kit; 2×Taq PCR Master Mix was purchased from Tiangen Biochemical Technology Co., Ltd.; liposome Lipofectamine2000 and G418 were purchased from Invitrogen; RPMI1640 and fetal bovine serum (FBs) were purchased from GibcoBRL. Other reagents were of domestic analytical grade, and primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
[0061] 1.2 Method
[0062] 1.2.1 Cell transfection and screening of stably transfected cell lines
[0063] 1) Remove the antibiotics in the medium before transfection, inoculate SGC-7901 cells in the logarithmic growth phase into 6-well plates and culture them for 24 hours, and perform transfection when the cells grow to 80%-90% confluent.
[0064] 2) Transfection using liposome method, g...
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