A kind of anti-tumor NK cell and its preparation method and application
A NK cell, anti-tumor technology, applied in the biological field, can solve problems such as life-threatening patients, and achieve the effects of wide treatment range, accurate identification and specific killing, and strong promoter effect.
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Embodiment 1
[0035] Example 1: Synthesis of chimeric co-stimulatory switch receptor encoding gene
[0036] Through the method of whole gene synthesis, a 450bp PD-1 extracellular segment (21-170aa), a 270bp NKG2D transmembrane and intracellular segment (44-132aa), and a 126bp co-stimulatory molecule 41BB intracellular segment ( 214-255aa), and connect them together to obtain the PD1-NKG2D-41BB gene fragment.
[0037] 1. Primer design
[0038] PD1-NKG2D-41BB is a DNA molecule containing 849bp. Every 60bp is a fragment, and there must be 10bp overlap between every two fragments, for example, 1-60bp is fragment 1, 50-110bp is fragment 2, 100-160bp is fragment 3, and so on. Design an upstream primer at the 5' end of Fragment 1, named as F 1 ; Design a downstream primer at the 3' end of fragment 2, named as R 1 ; Design an upstream primer at the 5' end of fragment 3, named as F 2 ; Design a downstream primer at the 3' end of fragment 4, named as R 2 , and so on. There are 9 pairs of upstr...
Embodiment 2
[0062] Example 2: Construction of recombinant lentiviral vector
[0063] Cloning the PD1-NKG2D-41BB gene fragment obtained in Example 1 into the pCDH-CMV-MCS-EF1-CopGFP lentiviral vector, such as figure 1 shown. The pCDH-CMV-MCS-EF1-CopGFP lentiviral vector and the PD1-NKG2D-41BB gene fragment were respectively digested with restriction endonucleases XbaI and EcoR I to obtain the linearized pCDH-CMV-MCS-EF1- The CopGFP lentiviral vector and the digested PD1-NKG2D-41BB gene fragment were incubated overnight at 16°C using the T4 DNA ligase system. Then transform the competent cells, screen the positive colonies, and extract the plasmids of the positive colonies to obtain the PD1-NKG2D-41BB-pCDH expression vector.
[0064] 1. Double digestion pCDH-CMV-MCS-EF1-CopGFP lentiviral vector
[0065] The enzyme digestion reaction system is as follows (100μl):
[0066]
[0067] Reaction conditions for enzyme cleavage: react at 37°C for 3 hours.
[0068] 2. Double digestion of PD1-...
Embodiment 3
[0076] Example 3: Packaging of lentivirus
[0077] 1. Extraction of lentiviral packaging plasmid and target plasmid
[0078] 1.1 Plasmid transformation
[0079] Take one 100 μL Stbl3 competent cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and two 100 μL Escherichia coli TOP10 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.); Plasmid PD1-NKG2D-41BB-pCDH was added to Stbl3 competent cells, and 1 μg of lentiviral packaging helper plasmids pSPAX2 and PMD2G were respectively added to 100 μL of E. coli TOP10 competent cells. Incubate on ice for 30 minutes, then immediately heat shock in a 42°C water bath for 90 seconds, and place in an ice bath for 2 minutes. Then, 800 μL of LB medium were added, mixed well, placed in a constant temperature shaking incubator at 37°C, and shaken at 100 rpm / min for 1 hour.
[0080] After the cultivation, 200 μL of the bacterial liquid was taken and spread on three LB solid medium (containing ampicillin...
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