Experiments on Tec eukaryotic expression vector construction and its effect on cell signaling
Experiments on Tec eukaryotic expression vector construction and its effect on cell signaling
Tec (Tyrosinekinaseexpressedinhepatocelluar carcinoma) is an important non-receptor tyrosine kinase, first cloned by Mano et al, from liver cancer tissue. The human Tec coding frame is an open reading frame consisting of 1893 bases and encodes a protein of 630 amino acids with a molecular weight of 73 ku, consisting of four parts: the PHdo main, THdomain, SH3domain, SH2domain and Kinase domain.
Operation method
Cell Culture Technology
Principle
It is believed that Tec has liver tissue and hematopoietic tissue distribution specificity and is mainly closely related to cytokine-mediated signaling pathways such as EPO, EGF, IL6, GM CSF, etc., which are involved in the regulation of hematopoietic cell, especially lymphocyte, proliferation and differentiation.
Materials and Instruments
Rat Move I. Preparation of materials For more product details, please visit Aladdin Scientific website.
Primers Mouse Recombinant Hepatocyte Growth Factor DMEM Trypsin Fetal Bovine Serum
PCR instrument PVDF
1. Cell line culture conditions: high sugar DMEM, 10% fetal bovine serum (FBS), 37℃, 5% CO2, incubator culture.
2. Culture conditions for human cervical cancer epithelial cells: high sugar DMEM, 10% fetal bovine serum, incubated at 37℃ with 5% CO2.II. Experimental steps
1. Acquisition of hybrid Tec ORFs
(1) Human Tec is highly homologous to rat and mouse Tec . Due to the lower annealing temperature of the upstream primers of the human Tec ORF coding frame, it is not suitable for direct PCR to retrieve it from the library, so a two-step method was adopted, firstly, the hybrid Tec reading frame was expanded in the rat library using the mouse Tec 5' primer and the human Tec 3 primer.
(2) Primers: upstream: 5 'ATG ATG GTG TCA TTC CCT GTC AAA-3', downstream: 5 -GC TCT AGA TCT TCC AAA AGT TTC TTC ACA-3', template: rat cDNA library.
(3) PCR system 25 ul, annealing temperature 42 ℃, 5 cycles, and then increase the annealing temperature to 48 ℃ , reaction 28 cycles.
(4) The PCR product was found to be a fragment of about 1.8 kb by 1% agarose electrophoresis, which was recovered, digested and ligated into PMD-T vector, and then 20 colonies were randomly picked after small-scale bacterial amplification, and then positive clones were screened by PCR and digest, and then sequenced to compare the sequences.2. The construction of human Tec eukaryotic expression vectors was confirmed by sequencing of heterologous
(1) The cross Tec ORF differs from the human Tec ORF by one base at the 5 end, which in turn synthesizes a new 5 primer: 5'ATG ATG GTG TCA TTCCCT GTC AAlA-3'; the downstream primer is still the downstream primer of the human Tee containing the Xba 1 cleavage site.
(2) Use hybrid T as template. The annealing temperature was 52 ℃ for 5 cycles, and then the annealing temperature was increased to 58 ℃ for 28 cycles.
(3) 1% agarose electrophoresis revealed a fragment around 1.8 kb. The fragment was ligated into pc-DNA 3.1 eukaryotic expression vector by T4 DNA ligase.
(4) The ligated product was used for small-scale bacterial transformation and confirmed by sequencing after PCR and digestion.3. pCDNA3.1 transient transfection of cells
(1) Hela cells were inoculated with 1×106 in 90 mm plates, and transfected with pCDNA3.1 plasmid 10 ug, liposome 12 ul and serum-free DMEM after growing to 70% density.
(2) After 10 hours of transfection, the cells were incubated in DMEM containing 10% FBS for 48 hours.
(3) Add RIPA (97.5% PBS, 1% NP 40, 0.5% sodium deoxycholate, 0.1% SDS) and lysed on ice for 30 min.
(4) The supernatant was centrifuged at 4°C and the total protein (10 g/group) was extracted and separated by 10% SDS-PAGE.
(5) Electrotransfer (100 mA, 120 min) to PVDF membrane.
(6) The membrane was closed in TBST for 3 h and incubated in TBST containing anti-Tec antibody (1:1,000) for 1 h at room temperature, and then secondary antibody of the corresponding station was added to bind to ECL luminescent detector to show the results.