ShRNA of CXCR4 gene and application of shRNA
A gene and expression vector technology, applied to the shRNA of the CXCR4 gene and its application fields, can solve problems such as the difficulty of human T cells
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Embodiment 1
[0078] siRNA and shRNA sequence design of embodiment 1 CXCR4 gene
[0079] The full-length mRNA sequence of the CXCR4 gene published by GenBank (NM_003467.2) was analyzed, the specific mRNA was obtained by Blast comparison, and the siRNA of CXCR4 mRNA was designed. The target position of each siRNA was as follows: figure 1 The specific sequence information is shown in Table 1.
[0080] Table 1 6 specific siRNAs of CXCR4 gene
[0081] serial number target nucleotide sequence target position SEQ ID NO:1 gcaaggcagtccatgtcatct 421～441 SEQ ID NO:2 ggatcagcatcgactccttca 868～888 SEQ ID NO:3 gcacatcatggttggccttat 701～721 SEQ ID NO:4 agataactacaccgaggaaat 122～142 SEQ ID NO:5 cctgttcttaagacgtgattt 1512～1532 SEQ ID NO:6 tcctgtcctgctattgcatta 739～759
[0082] According to the siRNA sequence, design the corresponding shRNA, the shRNA includes the sense strand (S) and the antisense strand (A), the shRNA sequence is composed...
Embodiment 2
[0086] The construction of the shRNA lentiviral expression vector of embodiment 2 CXCR4 gene
[0087] The pCHD vector was double-enzymatically digested with XhoI / EcoRV, and recovered by gel cutting; primer annealing was completed by designing specific primers based on shRNA; the shRNA and the recovered vector were recombined to form circular fragments, and transformed into the prepared bacterial competent cells , select a single clone colony for sequencing identification, and the correct clone is the successful construction of the shRNA silencing vector.
[0088] Specific steps are as follows:
[0089] (1) Double enzyme digestion of the vector
[0090] ① Cultivate the cloning strain containing pCDH empty vector overnight, take 5mL of fresh bacterial liquid and use QIAGEN plasmid extraction kit to extract the plasmid;
[0091] ②Take 2 μg of the extracted pCDH, and digest it with the corresponding restriction endonuclease at 37°C for about 1 hour. The enzyme digestion system i...
Embodiment 3
[0112] Example 3 lentiviral packaging
[0113] The lentiviral vector constructed in Example 2 was packaged with lentivirus, using a four-plasmid system, and the specific steps were as follows:
[0114] (1) The four-plasmid system includes gag / pol, Rev, VSV-G and lentiviral vector pCDH-shCXCR4-1-6 or negative control plasmids required for packaging lentiviral vectors. Add the above plasmids to a certain volume of serum-free DMEM In the culture medium, place it for 15 minutes after mixing;
[0115] (2) Add the above mixture into a T75 culture flask lined with 293T cells, mix gently, and transiently transfect 293T cells, at 37°C, 5% CO 2 Cultivate in a cell incubator for 6 hours;
[0116] (3) Replace the fresh medium after 6 hours, continue the culture, and add 10 mM sodium butyrate solution, and collect the culture supernatant after 72 hours for lentivirus purification and detection.
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