Use of human nob1 gene and related medicines
A gene and drug technology, applied in the field of the use of human NOB1 gene and related drugs, can solve the problem of tumor cell proliferation that has not yet occurred
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Embodiment 1
[0067] Example 1: Preparation of RNAi lentivirus against human NOB1 gene
[0068] 1. Screening for effective siRNA targets against human NOB1 gene
[0069] Select the coding region sequence of the human NOB1 (NM_014062) gene from Genbank, and obtain a sequence of 20 bases starting from every other base; using the design software of Shanghai Jikai Gene Chemical Technology Co., Ltd., using the human NOB1 mRNA sequence as a template, Determine 20 effective siRNA target sequences (SEQ ID NO: 1-20), as shown in Table 1:
[0070] Table 1 is targeted at the siRNA target sequence of human NOB1 gene
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[0073] For the siRNA target (taking SEQIDNO: 20 as an example) synthetic double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI restriction endonuclease at both ends; Act on pGCSIL-GFP carrier ( Provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to make it linearized (the reaction system is shown in Table 4, 37° C., reacti...
Embodiment 2
[0092] Example 2: Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of NOB1 gene
[0093] Human gastric cancer SGC-7901 cells, liver cancer SMMC-7721 cells, colon cancer RKO cells, lung cancer H1299 cells and pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5× 10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (the MOI value of SGC-7901 and SMMC-7721 is 20, the MOI value of H1299 cell, Panc-1 and RKO cell is 10) value, add the virus that suitable amount is prepared in embodiment 1, cultivate 24h and replace culture After 5 days of infection, the cells were collected. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV instruction manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see ...
Embodiment 3
[0100] Example 3: Detection of proliferation ability of tumor cells infected with siNOB1-Lentivirus lentivirus
[0101] Human gastric cancer SGC-7901 cells, liver cancer SMMC-7721 cells, colon cancer RKO cells, lung cancer H1299 cells, and pancreatic cancer Panc-1 cells in logarithmic growth phase were digested with trypsin to make cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (the MOI value of SGC-7901 and SMMC-7721 was 20, and the MOI value of H1299 cells, Panc-1 and RKO cells was 10), an appropriate amount of siNOB1-Lentivirus virus was added, and the culture medium was replaced after 24 h. After the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell densi...
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