Bicistronic expression vector, expression system, preparation method and application

Abstract

The invention discloses a bicistronic expression carrier, an expression system, a preparation method and an application, and belongs to the technical field of genetic engineering. The expression vector contains the sequence of the nuclear matrix binding region, the sequence of the inner ribosome entry site and the screening marker. By inserting the target gene between the promoter and the IRES sequence, a bicis sequence of the target gene-IRES sequence-selection marker gene can be formed. anti subsequence. The structure of the expression vector is: MAR-promoter-target gene-IRES sequence-selection marker-PolyA-MAR. The vector uses a promoter to realize the simultaneous expression of the target gene and the screening marker, which can reduce the false positive cell clones caused by the existence of different expression cassettes, improve the screening rate of positive cell clones, and overcome the unbalanced expression of the target gene and the screening marker in traditional vectors Question: The inclusion of MAR sequences on the vector can overcome transgene silencing and achieve efficient and long-term expression of transgenes in host cells.

Description

technical field [0001] The invention relates to a bicistronic expression carrier, and also relates to a eukaryotic cell expression system containing the expression carrier, a preparation method and an application, belonging to the technical field of genetic engineering. Background technique [0002] Biopharmaceuticals is a high-tech industry in the 21st century, in which therapeutic recombinant proteins are an important part of biopharmaceuticals, and the current annual output value (including recombinant antibodies) in the world exceeds 100 billion US dollars. It is well known that prokaryotes and lower eukaryotes lack complex post-translational modification functions, so related therapeutic recombinant proteins need to be expressed in higher eukaryotic cells to be active, and nearly 70% of them use Chinese hamster ovary (Chinese hamster ovary , CHO) cell expression system. However, the CHO cell expression system also has defects such as low expression level of target prot...

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Problems solved by technology

In addition, in the CHO cell expression system, it is often difficult to obtain cell lines with high levels of sustained and stable expression. This is because the random integration of transgenes into cells makes some transgenes silent or the expression level is reduced. The clonal cell lines with stable and high expression were isolated from the above, which also brought great troubles to the industrial production of genetic engineering (A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol.Bioeng.2009)

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