Recombinant human esoderma colyone adenovirus, and its preparing method and use
An endostatin and adenovirus technology, applied in the field of genetic engineering, can solve the problems of complex and time-consuming protein purification process, large clinical application limitations and high cost, and achieve growth and migration inhibition, growth and migration inhibition, and increase administration time. The effect of the interval
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[0050] Example 1 Preparation of human endostatin gene
[0051] In this example, the RT-PCR method was used to amplify human endostatin cDNA fragments from the total RNA of human liver.
[0052] Design and synthesize RT-PCR primers based on the human endostatin gene sequence. In order to insert the PCR product directly into the cloning vector-pUC18 (purchased from Invitrogen), the 5'and 3'primers are designed with BaMH I restriction sites Point, the primer sequence is as follows:
[0053] 5’ Primer: 5’CGG GAT CCA CAG CCA CCG CGA CTT CCA GCC 3’
[0054] 3’ Primer: 5’GCG GAT CCT ACT TGG AGG CAG TCA TGA AGC 3’
[0055] The RT-PCR conditions are as follows:
[0056] After the RT-PCR reaction mixture was denatured at 50°C for 30 minutes, the reaction was carried out under the following conditions:
[0057] Reverse transcription reaction: denaturation at 94°C for 30 seconds; annealing at 50°C for 30 seconds; extension at 68°C for 1 minute. Reaction for 10 cycles.
[0058] PCR reaction: d...
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[0060] Example 2 Preparation of human endostatin gene encoding human IL-2 gene signal peptide
[0061] With reference to the cDNA sequence of human IL-2 (GI: 28178860), the PCR primers were designed and synthesized as follows:
[0062] 5’ Primer: 5’CGG ATC CAT GTA CAG GAT GCA ACT CCT GTC TTG CAT TGCACT AAG TCT TGC ACT TGT CAC GAA TTC GGC CCA CAG CCA CCG CGA CTTCCA GCC 3’
[0063] 3’ Primer: 5’GCG GAT CCT ACT TGG AGG CAG TCA TGA AGC 3’
[0064] Both the 5'primer and the 3'primer are designed with BamH I restriction sites, which are directly inserted into the shuttle vector pAdenovator-CMV5; the italic part of the 5'primer is the signal peptide sequence of human IL-2.
[0065] Then use pUC18-endo as a template to amplify the human endostatin DNA fragment containing the IL-2 gene signal peptide. The PCR conditions are as follows:
[0066] Denaturation at 94°C for 30 seconds; annealing at 55°C for 30 seconds; extension at 72°C for 30 seconds. Reaction for 30 cycles. Then extend for an...
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[0068] Example 3 Construction and screening of recombinant adenovirus
[0069] 1. The cloned human endostatin gene containing human IL-2 signal peptide was constructed into pAdenoVator-CMV5 shuttle vector, and then combined with the circular plasmid pAdenoVatorΔE1E3 (purchased from Qbiogene) containing adenovirus genome (E1, E3 deletion) ) Co-transform Escherichia coli BJ5183 (purchased from Qbiogene) with conventional electroporation methods, and screen recombinant pAd-endo for identification. The results showed that the recombinant pAd-endo(1) and pAd-endo(3) with two recombination sites were screened separately (see Figure 3). The homologous recombination site is located in the left arm to cut out a fragment of 3.0 kb, and the recombination site is located in the replicon to cut out a fragment of 4.5 kb in size.
[0070] 2. The linearized recombinant plasmid pAd-endo and lipofectamine (purchased from Invitrogen) were co-transfected into 293 cells (purchased from ATCC) using con...
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