Recombinant adenovirus, preparation method and application thereof
A technology of recombinant adenovirus and toxin, which is applied in the field of recombinant adenovirus, can solve the problem of undiscovered glioma and achieve the effect of efficient infection time
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Embodiment 1
[0030] Example 1 Construction of recombinant adenovirus
[0031] (1) Optimized synthesis and identification of BmK CT gene and construction and identification of intermediate plasmid pShuttle-IRES-hrGFP-2-BmK CT
[0032] The known BmK CT gene sequence was optimized according to the preferred codon table of mammals, handed over to Shanghai Sangon Bioengineering Technology Co., Ltd. for artificial synthesis and cloned into the vector pUC57, and Xho I and HindIII restriction sites were added to the two ends of the sequence respectively , for enzyme digestion identification ( figure 1 ).
[0033] The cloning vector pUC57 carrying the optimized BmK CT gene was transformed into Escherichia coli DH5a for massive amplification, and the plasmid was extracted and identified by PCR and enzyme digestion.
[0034] Primers were designed, and EcoRV and Xho I restriction sites were added to both ends of the BmK CT gene by PCR method ( figure 2 ), the PCR product was recovered after diges...
Embodiment 2
[0039] Example 2 Experiment of recombinant adenovirus inhibiting the proliferation and spread of glioma cells
[0040] (1) Concentration determination and purity identification of recombinant adenovirus
[0041] Densitometry was used to determine the concentration of adenovirus. The blank tube was 960 μl of PBS, 40 μl of 10% SDS; the measuring tube was 860 μl of PBS, 100 μl of recombinant virus solution, and 40 μl of 10% SDS. After incubation at 56°C for 1 hour, the supernatant was collected by centrifugation. UV spectrophotometer measures OD260 value and OD280 value, virus titer=A260×dilution factor×10 12 The ratio of vp / ml; A260 / A280 reflects the purity of the adenovirus, and >1.3 indicates that the purity of the adenovirus is high. The virus assay A260=0.246 of this experiment package, A280=0.170; Virus titer is 2.46×10 12 , A260 / A280=1.45, the purity is higher.
[0042] The identification of the recombinant adenovirus adopts the extracted and purified Ad-BmK CT virus ...
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