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

Inactive Publication Date: 2012-10-31
西藏自治区人民医院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Christoph L. speculated that the level of CD14 may affect the TLR auxiliary regulation effect in vivo, thus affecting some anticancer therapy or antiviral response

Method used

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  • Construction of CD14 eukaryotic expression vector and method for preparing high-expression cell strain by using CD14 eukaryotic expression vector
  • Construction of CD14 eukaryotic expression vector and method for preparing high-expression cell strain by using CD14 eukaryotic expression vector
  • Construction of CD14 eukaryotic expression vector and method for preparing high-expression cell strain by using CD14 eukaryotic expression vector

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Experimental program
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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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Abstract

The invention discloses a construction of a eukaryotic expression vector and a method for preparing a high-expression cell strain by using the eukaryotic expression vector. According to the invention, a CD14 overall-length gene is cloned to a pcDNA3.1-EGFP eukaryotic expression vector labeled by a fluorescent protein, gastric cancer SGC-7901 cells are transfected by using the constructed eukaryotic expression vector, and a CD14 stable-overexpressed gastric cancer cell line is established.

Description

technical field [0001] The invention relates to the construction of a eukaryotic expression vector and the preparation of a stable expression cell line using the vector. Specifically, the present invention clones the full-length CD14 gene into a pcDNA3.1-EGFP eukaryotic expression vector with a fluorescent protein tag, and transfects gastric cancer SGC-7901 cells with the constructed eukaryotic expression vector to establish stable overexpression of CD14 gastric cancer cell lines. Background technique [0002] Gastric cancer poses a great threat to human health. Gastric cancer ranks second among the deaths caused by various tumors, and it has attracted widespread attention at home and abroad because of its high incidence and universality. The occurrence of gastric cancer is a complex process, including the interaction of multiple factors and multiple genes. The activation of some proto-oncogenes and the inactivation mutation of tumor suppressor genes are the main reasons fo...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/79C12N15/66C12N5/10C12Q1/68C07K14/705C12R1/91
Inventor 李康旦增
Owner 西藏自治区人民医院
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