Method for silencing tnfaip8 gene and its application in tumor therapy
A gene and silencing technology, applied in the field of biopharmaceuticals, can solve problems such as lack of research reports and achieve the effect of improving sensitivity
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Embodiment 1
[0046] Silence provided by the present invention TNFAIP8 Gene method, the technical principle is, by constructing a lentiviral vector carrying shRNA, then preparing lentiviral particles and infecting tumor cells, so that the tumor cells TNFAIP8 Gene silencing. Since this method depends on the construction of the shRNA lentiviral vector, the construction process of the shRNA lentiviral vector is described in detail in this example as follows.
[0047] In this example, LV10 lentiviral vector is used as lentiviral vector (LV10 lentiviral vector can express red fluorescent protein). The specific process of constructing LV10-shRNA viral vector is as follows: first design TNFAIP8 Gene expression siRNA target sequence, then artificially synthesize the corresponding shRNA sense strand and antisense strand sequence, after annealing treatment, connect the shRNA double strand and the LV10 linear vector formed after enzyme digestion, and construct the LV10-shRNA viral vector. Details Th...
Embodiment 2
[0093] The LV10-shRNA vector containing the shRNA sequence constructed in Example 1 (that is, the plasmid extracted after sequencing verification in Example 1) needs to be further transformed into 293T cells to prepare lentiviral particles before it can be further applied. This example focuses on LV10 - The shRNA vector transformation process, that is, the preparation process of lentiviral particles, is briefly introduced as follows.
[0094] The specific process of transforming 293T cells with LV10-shRNA vector to prepare lentiviral particles is as follows:
[0095] (1) Digest 293T cells one day in advance, inoculate 2×10 6 cells, add DMEM medium without penicillin and streptomycin, 37°C, 5% volume CO 2 Cultivate overnight under the condition;
[0096] (2) Add 100 μL of serum-free DMEM to a sterile 1.5mL centrifuge tube, add 2 μg of the shRNA-LV10 vector prepared in Example 1, 1.5 μg of the packaging plasmid psPAX2, and 0.5 µg shuttle plasmid pMD2.G, mix well;
[0097] Ta...
Embodiment 3
[0112] Infect tumor cells with the lentiviral particles prepared in Example 2 to produce siRNA in tumor cells and finally silence TNFAIP8 expression, the specific process is described below.
[0113] The specific process of lentiviral particles infecting tumor cells is as follows:
[0114] (1) First determine the optimal concentration of puromycin, that is, the lowest concentration of puromycin that can completely kill cells, specifically:
[0115] The tumor cells were digested and inoculated in 96-well culture plates with an inoculum size of 1.5×10 4 Each / well; in order to study the applicable tumor range of the lentiviral particles prepared by the present invention, human tumor cell lines HeLa, H1299, U251, and EC9706 were used respectively;
[0116] After the tumor cells were inoculated in the 96-well culture plate, 100 μL of medium containing puromycin was added to each well the next day, and the gradient setting of puromycin was set as: 0 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / m...
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