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.

Pending Publication Date: 2021-05-11
XINXIANG MEDICAL UNIV
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Problems solved by technology

Publication number CN103642838B discloses a human and mammalian cell expression vector, which only inserts human β-globin MAR sequence (770bp) and EGFP (respectively 350,750bp), although it can mediate foreign genes in mammals Expressed in cells, but only one of the MARs is used, not the full length of the MAR sequence, and the foreign fragment comes from EGFP, the effect of its sequence itself on the expression of the transgene may cause some interference
At present, MAR is not very ideal in improving the expression level of transgenes. At the same time, studies on the location of expression vectors and the adaptability of distance effects have not been reported.

Method used

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  • Human and mammalian cell expression vector and system as well as construction method and application thereof
  • Human and mammalian cell expression vector and system as well as construction method and application thereof
  • Human and mammalian cell expression vector and system as well as construction method and application thereof

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Effect test

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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Abstract

The invention relates to a human and mammalian cell expression vector and system as well as a construction method and application thereof. The MAR1-68-Spacer-CMV-target gene-PolyA-MAR1-68 vector is constructed by inserting an MAR 1-68 sequence into the upstream of a promoter and the downstream of PolyA and inserting a segment of spacer DNA (Deoxyribonucleic Acid) sequence with the length of 500bp between the MAR sequence and the promoter at the same time. A Spacer segment in the vector is a neutral DNA sequence, and the transgenic expression level cannot be improved, so that the influence of a distance effect on transgenic expression can be really discussed. Experiments prove that the human and mammalian cell expression system containing the expression vector can significantly improve the expression level of carried target genes. Therefore, the mammalian cell expression vector and the expression system provided by the invention can overcome transgenic silencing, promote high-level stable expression of exogenous genes in host cells, and can be used for production of recombinant proteins.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a human and mammalian cell expression vector, an expression system, a construction method and application thereof. Background technique [0002] At present, more than 70% of global therapeutic recombinant proteins are produced by human and mammalian cell expression systems, and expression vectors are one of the important factors for recombinant protein production. In the current human and mammalian cell expression systems, a problem that needs to be solved urgently is the phenomenon of transgene silencing. The phenomenon that the expression of foreign genes in the host is suppressed is called transgene silencing. The occurrence of transgene silencing is related to the position where the transgene is integrated into the host cell gene, that is, a "position effect". In addition, the strength of transgene expression is affected by the sequence of the vector ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N5/10C12P21/00
CPCC12N15/85C12N15/113C12P21/00C12N2800/107
Inventor 张俊河王天云张继红樊振林杨雯雯王小引肖云喜
Owner XINXIANG MEDICAL UNIV
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