Interference fragments and their applications
A technology of fragments and uses, applied in the field of interference fragments and its applications, can solve problems such as adverse drug events
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Embodiment 1
[0037] 1. Plasmid construction:
[0038] Set the control NC (negative control), and construct three shRNA lentiviral vectors of PD-1 according to the following methods:
[0039] (1) Experimental materials
[0040] 1. DNA oligo was designed using Designer3.0 (Genepharma) software, and the primers were synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd.
[0041] 2. List of instruments used in the experiment:
[0042]
[0043] 3. List of reagents used in the experiment:
[0044]
[0045]
[0046] (2) Construction process
[0047] 1. Oligo Designer 3.0
[0048] When the inventor designed the template, the loop structure in the LV3-shRNA template selected TTCAAGAGA to avoid the formation of a termination signal; GATCC was added to the 5' end of the sense strand template, which was complementary to the sticky end formed after digestion with BamHI; the antisense strand template AATTC is added to the 5' end, which is complementary to the sticky end formed a...
Embodiment 2
[0215] Taking the PD791 lentiviral vector obtained in Example 1 as an example, using the PD791 lentiviral vector, the interference fragment carried by it was transferred into immune cells to prepare immune cells with inhibited expression of PD-1. The specific steps are as follows:
[0216] 1 Isolation of peripheral blood mononuclear cells
[0217] Cells isolated from 100ml of peripheral blood collected from an AFP-positive liver cancer patient were transferred to a 50ml centrifuge tube and centrifuged (1800rpm, 10min), the supernatant was sucked off, and the supernatant was slowly added to the lymphocyte separation Liquid Ficoll solution (GE), the volume ratio is 1:1, centrifuged (2000rpm, 20min);
[0218] Next, collect the white flocculent cells at the interface, add PBS, gently blow and mix, and centrifuge (1500rpm, 10min);
[0219] Next, repeat centrifugation and washing for a total of 3 times;
[0220] Then, collect the cells and blow and mix gently, add 20ml of basal me...
Embodiment 3
[0237] Embodiment 3: clinical experiment
[0238] The PD791-CTL cells prepared in Example 1 were reinfused into the patient, and the effect of reinfusion was observed, as follows:
[0239] 20 AFP-positive liver cancer patients were divided into 2 groups (10 patients in group A and 10 patients in group B), wherein, for group A, after obtaining sufficient CTL cells on the 14th and 15th days according to the method in Example 1 , to reinfuse the patient, the amount of cells reinfused each time is 1~2*10 9 ; Group B did not reinfuse cells. One blood collection and two consecutive reinfusions constituted a course of treatment. After 3 courses of treatment, observation was carried out.
[0240] It was found that after three courses of treatment, there was a significant difference between group A and group B. After reinfusion, all the patients in group A felt less tired, more appetite, less pain, and more weight gain. Among them, the AFP index of 8 patients decreased; one patient...
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