siRNA (small interfering RNA) of CDT1 gene of silent person, recombinant vector and application or siRNA and recombinant vector
A recombinant vector, silencing technology for molecular biology and biopharmaceutical applications
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Embodiment 1
[0017] This example is used to illustrate the construction method of siRNA for silencing human CDT1 gene provided by the present invention.
[0018] According to the nucleotide sequence of the CDT1 gene reported in Genbank, three siRNA target sequences that inhibit the expression of the CDT1 gene were designed with reference to the siRNA design principles, respectively SEQ ID NO: 1-3, and a random control sequence SEQ ID NO.4 was designed at the same time ,As follows:
[0019] SEQ ID NO. 1: GGACACCATCTCTGAGCTT;
[0020] SEQ ID NO.2: GGAGGTCAGATTACCAGCT;
[0021] SEQ ID NO. 3: CCTACGTCAAGCTGGACAA;
[0022] SEQ ID NO. 4: TTCTCCGAACGTGTCACGT.
[0023] According to Blast gene homology analysis, the sequence of SEQ ID NO: 1-3 is not homologous to any human non-CDT1 gene sequence, so as to ensure the specificity of gene suppression.
[0024] For the above target sequences, refer to the siRNA design strategy, and design four pairs of shRNA (short hairpin RNA, shRNA) expression fr...
Embodiment 2
[0048]This example is used to illustrate the construction method of the siRNA recombinant vector for silencing human CDT1 gene provided by the present invention.
[0049] The vector used was the lentiviral vector pGC-LV (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.). Its frame structure is:
[0050] hU6-MCS-Ubiquitin-EGFP-IRES-puromycin.
[0051] The construction method of the recombinant vector is as follows:
[0052] The sense and antisense strands of the 4 pairs of shRNA expression framework DNA sequences SEQ ID NO.1'-4' of the siRNA in Example 1 were respectively placed in annealing buffer (10mM Tris-HCl, 1mM EDTA, 0.1mM NaCl) at 90°C Water bath for 15 minutes, then naturally cool to room temperature, react to synthesize double-stranded DNA (dsDNA) sequence, digest the double-stranded DNA sequence with EcoRI and AgeⅠ, and use EcoRI and AgeⅠ to digest the lentiviral vector pGC-LV at the same time to produce dsDNA. Using T4 DNA ligase to ligate the dou...
Embodiment 3
[0055] This example is used to illustrate the inhibitory effect of the siRNA recombinant vector for silencing human CDT1 gene provided by the present invention on the expression of CDT1 gene in human hepatocyte HL-7702.
[0056] 1. Cell culture
[0057] The normal human liver cells HL-7702 in good condition were inoculated in 6-well cell culture plates in RPMI1640 medium containing 20% (v / v) calf serum, 100 U / ml penicillin and 100 mg / L streptomycin ( USA, Invitrogen Company), at 37°C, 5% (v / v) CO 2 cultured under the same conditions until the cell confluency reached about 80%.
[0058] At the same time, the cultured cells were divided into 4 groups, namely 3 experimental groups and 1 control group.
[0059] 2. Gene transfection
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