Preparation method and use of medicine over-expressing PCNP gene
A GV230-PCNP, overexpression technology, applied in gene therapy, drug combination, anti-tumor drugs, etc., can solve problems such as lack of research reports
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Embodiment 1
[0051] The present embodiment provides a kind of construction method of the plasmid of overexpression PCNP gene, it comprises the following steps:
[0052] 1. First, according to the PCNP gene sequence (NM_020357), design primers for PCR amplification of the target gene. The specific sequence is as follows:
[0053] Forward primer:
[0054] 5'-TACCGGACTCAGATCTCGAGCGCCACCATGGCGGACGGGAAGGCGGGAGAC-3' (as shown in SEQ ID NO: 1);
[0055] Reverse primer:
[0056] 5'-GATCCCGGGCCCGCGGTACCGTaTTGTCTTGGTCATGGACATTTC-3' (shown in SEQ ID NO: 2).
[0057] Digest the restriction enzyme sites XhoI and KpnI in the GV230 vector (carrier sequence: CMV-MCS-EGFP-SV40-Neomycin) to form a linearized GV230 vector;
[0058] 2. Acquisition of target gene fragments
[0059] The primers synthesized by Jinweizhi Biotechnology (Beijing) Co., Ltd. were dissolved in double distilled water to prepare a solution with a concentration of 100 μM. The 50 μL reaction system was designed as follows:
[0060] 5...
Embodiment 2
[0097] This example provides the application of the plasmid overexpressing the PCNP gene prepared in the above example in the preparation of a drug for treating human neuroblastoma. The plasmid overexpressing the PCNP gene was transfected into human neuroblastoma cells as a drug for the treatment of human neuroblastoma, a stable strain was obtained by G418 screening, and finally the PCNP gene was overexpressed. The specific operation process was introduced as follows:
[0098] 1. First determine the optimal concentration of geneticin G418, that is, the lowest concentration of G418 that can completely kill cells, specifically:
[0099] Human neuroblastoma cells SH-SY5Y and SK-N-SH were digested and inoculated in 96-well culture plates with an inoculation amount of 1×104 cells / well;
[0100] After the tumor cells were inoculated in the 96-well culture plate, 100 μL of medium containing G418 was added to each well the next day, and the G418 gradient setting was as follows: 100 μg...
Embodiment 3
[0132] In order to detect the influence of the overexpressed PCNP gene on tumor cell proliferation in the above-mentioned embodiment 2, the inventor has done further detection experiments, and the relevant process is introduced as follows:
[0133] 1. First, the MTS method was used to determine the effect of PCNP overexpression on tumor cell proliferation. The specific process was as follows:
[0134] The tumor cells of the negative control and PCNP gene overexpression in Example 2 were digested and counted respectively, spread to a 96-well culture plate, and the inoculation amount was 8×10 3 Each well, 6 auxiliary wells for each group, add 100 μL medium to each well, and incubate in an incubator at 37°C;
[0135] After 24 hours, add 20 μL and 5 mg / mL MTS solution to each well, and continue to culture in the incubator for 2 hours;
[0136] Then the cells were taken out, and the absorbance value of each well was measured at 490 nm using a microplate reader, and the inhibition ...
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