SiRNA silencing human RAN gene, recombinant vector and application
A recombinant vector and silencing technology, applied in the field of molecular biology and biopharmaceuticals, can solve the problem that mRNA cannot be translated into protein
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Embodiment 1
[0018] This example is used to illustrate the construction method of siRNA for silencing human RAN gene provided by the present invention.
[0019] According to the nucleotide sequence of the RAN gene reported in Genbank, five siRNA target sequences that inhibit the expression of the RAN gene were designed with reference to the siRNA design principles, respectively SEQ ID NO: 1-5, and a random control sequence SEQ ID NO was designed. 6, as follows:
[0020] SEQ ID NO. 1: TAAGTCGTGCTCATACTGT;
[0021] SEQ ID NO. 2: GTCATTTGACTGGTGAATT;
[0022] SEQ ID NO. 3: GTTCAAACTTGTATTGGTT;
[0023] SEQ ID NO. 4: CCCTAACTTGGAATTTGTT;
[0024] SEQ ID NO.5: GGATATTAAGGACAGGAAA;
[0025] SEQ ID NO. 6: TTCTCCGAACGTGTCACGT.
[0026] According to Blast gene homology analysis, the sequence SEQ ID NO.1-5 is not homologous to any human non-RAN gene sequence, so as to ensure the specificity of gene suppression.
[0027] For the above target sequences, referring to the design strategy of siRNA,...
Embodiment 2
[0059] This example is used to illustrate the construction method of the siRNA recombinant vector for silencing human RAN gene provided by the present invention.
[0060] The vector used was the lentiviral vector pGC-LV (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.). Its frame structure is:
[0061] hU6-MCS-Ubiquitin-EGFP-IRES-puromycin.
[0062] The construction method of the recombinant vector is as follows:
[0063] Put the sense strand and antisense strand of the 5 pairs of shRNA expression framework DNA sequence SEQ ID NO.1'-5' of the siRNA in Example 1 in an annealing buffer (10mM Tris-HCl, 1mM EDTA, 0.1mMNaCl) in a 90°C water bath 15min, 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 a double-stranded DNA sequence Using T4 DNA ligase to ligate the ...
Embodiment 3
[0066] This example is used to illustrate the inhibitory effect of the siRNA recombinant vector for silencing human RAN gene provided by the present invention on the expression of RAN gene in human hepatocyte HL-7702.
[0067] 1. Cell culture
[0068] 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%. At the same time, the cultured cells were divided into 6 groups, namely 5 experimental groups and 1 control group.
[0069] 2. Gene transfection
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