Human and mammalian cell expression vector and system as well as construction method and application thereof
An expression vector, mammalian technology, applied in the field of genetic engineering, can solve the problems of influence interference, suboptimal transgene expression, the position of the expression vector, and the research on the adaptability of distance effect has not been reported.
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Embodiment 1
[0040] Example 1 Construction of expression vectors containing MAR in different positions of the expression cassette
[0041] 1.1 PCR amplification of EGFP
[0042] Design the following primers with reference to the sequence of the Enhanced green fluorescent protein (EGFP) gene fragment (GenBank accession number: U55763.1, bases 613-1329): P1: 5′-CCG GATATC ATGGTGAGCAAGGGC-3';P2:5'-CGC GCTAGC TCACTTGTACAGCTC-3', the 5' ends of the primers were respectively introduced into EcoR V and Nhe I restriction sites. Using the extracted pEGFP-C1 plasmid as a template, conventional PCR method was used for amplification. The PCR system is as follows:
[0043]
[0044] The PCR program was as follows: 30 cycles of 95°C for 3min, 94°C for 40s, 60°C for 30s, 72°C for 40s, and 72°C for 3min. The amplified product was recovered by agarose gel electrophoresis and sent to the biological company for sequencing verification. The results showed that the amplified DNA fragment was completely...
Embodiment 2
[0059] Embodiment 2 Contains the expression vector construction of MAR and spacer DNA sequence
[0060] 2.1 Synthesis of Spacer DNA fragments
[0061] Referring to the Spacer DNA fragment sequence (NCBI Ref Seq: NM_011682.4), a 500bp Spacer DNA was synthesized first, and the gene synthesis was completed by Beijing Zhongmeitech Biological Company.
[0062] 2.2 Construction of expression vectors containing distance fragments
[0063] Using the seamless cloning method, construct the 500bp Spacer DNA fragment into the pIRES-MAR-CMV-EGFP-PolyA, pIRES-CMV-EGFP-PolyA-MAR and pIRES-MAR-CMV-EGFP-PolyA-MAR vectors respectively Upstream of the CMV expression cassette, select the correct plasmids verified by restriction enzyme digestion, perform sequencing verification, and construct vectors containing MAR 1-68 and distance fragments at different positions, and the vectors are respectively named pIRES-MAR-Spacer-CMV-EGFP-PolyA, pIRES -Spacer-CMV-EGFP-PolyA-MAR and pIRES-MAR-Spacer-CMV-E...
Embodiment 3
[0064] Embodiment 3 contains EPO, the vector construction of MAR and spacer DNA sequence
[0065] (1) Construction of pIRES-CMV-EPO vector containing erythropoietin (EPO)
[0066] PCR primers P7 and P8 were designed according to the cDNA sequence of human EPO gene (NCBI Ref Seq: NM_000799.4). In order to realize directional cloning, the 5′ ends of the primers were respectively introduced with EcoR V / NheI restriction sites. The primer sequences are as follows: P7: 5′-CCG GATATC ATGGGGGTGCACGAA-3';P8:5'-CGC GCTAGC TCATCTGTCCCCTGT-3'. Using the extracted human peripheral blood genomic DNA as a template, human EPO was amplified by PCR. The PCR system is the same as before, and the PCR conditions are as follows: 95°C for 3min, 94°C for 40s, 60-56°C for 30s, 72°C for 40s, 4 cycles for each annealing temperature, and finally 55°C for 30 cycles, 72°C for 3min. The amplified product was recovered by agarose gel electrophoresis and sent to the biological company for sequencing ver...
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