阅读说明：本技术 非强启动式的外源基因表达法及其在具有毒性的目标蛋白表达中的应用 (Non-strong start type exogenous gene expression method and application thereof in expression of target protein with toxicity ) 是由 冯磊 陈丽 侯继波 于 2018-06-15 设计创作，主要内容包括：本发明公开一种非强启动式的外源基因表达法及其在CHO细胞中的应用该方法,通过定点整合的方式,将外源蛋白编码序列定点整合插入至宿主细胞自身基因的启动子下游,并利用宿主自身基因的启动子以及相关转录起始或翻译起始调控序列实现外源基因的转录、表达。利用宿主细胞自身的启动子以及调控机制,实现外源基因非强启动式的表达,从而避免由于外源毒性蛋白在宿主细胞强启动式表达造成的细胞死亡,和加速死亡,实现先增殖后表达,从而在宿主细胞生长后期获得大量毒性目标蛋白。填补了目前现有技术中的空白,为有关毒性蛋白基因工程技术的研究发展开拓了新的方向。(The invention discloses a non-strong start type exogenous gene expression method and application thereof in CHO cells. The expression of the exogenous gene in a non-strong start mode is realized by utilizing a promoter and a regulation mechanism of a host cell, so that the cell death caused by the strong start expression of the exogenous toxic protein in the host cell is avoided, the cell death is accelerated, the expression after proliferation is realized, and a large amount of toxic target protein is obtained in the later growth stage of the host cell. Fills the blank in the prior art and develops a new direction for the research and development of the related toxic protein gene engineering technology.)

1. The non-strong start type exogenous gene expression method is characterized in that: the method is characterized in that a foreign protein coding sequence is inserted into the downstream of a promoter of a host cell self gene in a site-specific integration manner, and transcription and expression of the foreign gene are realized by utilizing the promoter of the host self gene and a related transcription initiation or translation initiation regulatory sequence.

2. The method for expressing a foreign gene according to claim 1, wherein: the foreign protein coding sequence does not contain a promoter.

3. The method for expressing a foreign gene according to claim 1, wherein: the method specifically comprises the following steps:

s1: selecting host cells, selecting endogenous proteins meeting the expression time requirements of target exogenous proteins according to the expression time of different endogenous proteins in the host cells, and determining the expression cassette sequences of the endogenous protein genes according to GenBank;

s2: selecting a gene editing system for site-directed insertion;

s3: selecting sites which meet the conditions in an expression cassette sequence of an endogenous protein gene as fixed-point insertion points according to the following rules, wherein the sites are a. located at the upstream of a transcription initiation site of the endogenous protein gene, b. located at the downstream of a promoter of the endogenous protein gene, and c. meet the identification operation requirements of a fixed-point insertion gene editing system;

s4: designing and synthesizing a fluorescent marker exogenous gene sequence comprising an upstream homologous sequence of the insertion site, a loxP511 sequence, an eGFP coding sequence, a loxP sequence, an IRES2 sequence and a downstream homologous sequence of the insertion site according to the insertion site;

s5: inserting the foreign gene sequence obtained in S4 into the insertion point obtained by the S3 screening using the gene editing system selected in S2;

s6: obtaining recombinant cell clones which are integrated and inserted with exogenous gene sequences at different insertion sites through PCR specific amplification fragments and sequencing verification;

s7: observing the transcription levels of eGFP in the recombinant cells subjected to point-fixed integral insertion of different insertion sites, selecting insertion sites with eGFP transcription levels basically consistent with the transcription levels of the selected endogenous genes in S1, and respectively marking the insertion sites as a targeting site 1, a targeting site 2 and a … … targeting site n;

s8: recording and forming an insertable site form by taking host cell-endogenous protein-targeted site n as a format;

s9: replacing a target protein coding sequence and an eGFP coding sequence between loxP511 and loxP of an exogenous gene sequence by adopting a Cre/loxP system to obtain a target exogenous gene sequence;

s10: and inserting the target foreign gene sequence in the S9 into one or more host cell-endogenous protein-targeting site n insertion sites in the S8 according to the mode of S5 to obtain the target protein.

4. The method for expressing a foreign gene according to claim 1, wherein: the host cell is a CHO cell.

5. The method for expressing a foreign gene with non-strong priming according to claim 4, wherein: the endogenous protein is a thioredoxin interacting protein.

6. The method for expressing a foreign gene with non-strong start according to claim 5, wherein: the CRISPR/Cas9 system was used as a site-directed insertion site system.

7. The method for expressing a foreign gene with non-strong start according to claim 6, wherein: the step of inserting exogenous gene into CRISPR/Cas9 system at fixed point includes 3 transfection operation vectors: 1) a homologous recombination vector containing upstream and downstream homologous sequences and an insertion sequence constructed according to the upstream and downstream sequences of the insertion site, 2) a sgRNA vector capable of identifying the insertion site is constructed, and 3) a vector containing the complete Cas9 expression cassette is constructed.

8. The method for expressing a foreign gene according to claim 1, wherein: the transcriptional level of eGFP was investigated by the change in transcriptional level of the fluorescent reporter gene.

9. Use of the non-strong start foreign gene expression method of claims 1-8 for the expression of target proteins with toxicity.

10. An exogenous gene expression cassette without a promoter.

Technical Field

The invention discloses a non-strong start type exogenous gene expression method and application thereof in CHO cells, belonging to the field of biotechnology, in particular to the aspect of gene recombination modification in the field of biotechnology.

Background

Exogenous genes are named relative to endogenous genes, which for a cell are sequences of their genome, i.e., the gene (DNA) sequence that the organism itself has, and exogenous genes are segments of genes from other species, or artificially synthesized. We introduce the exogenous gene into the host cell and carry out high-efficiency expression through the host cell, thereby harvesting the target protein compiled by the exogenous gene.

In the prior art, exogenous genes are introduced into gene segments of host target cells by genetic engineering techniques or viral infection and the like.

Viral infection is a random route of introduction by infecting host cells with recombinant viruses and naturally selecting the insertion site to randomly integrate the foreign gene into the endogenous gene of the host cell. This is a random integration approach.

Scientists have found regular sites through studies on the infection process and sequencing of the recombinant gene after infection. Thereby gradually forming a stage of site-directed genomic integration and recombinant expression.

The site-specific integration refers to the integration of a complete gene expression cassette into a certain site of chromosome by using genetic engineering technology.

The gene fragment for recombination is a complete gene expression cassette, whether by genetic engineering techniques or by viral infection. After the complete gene expression cassette is integrated into a certain site of chromosome, the expression of recombinant protein is realized by means of exogenous promoter in the expression cassette. In order to ensure the expression of the exogenous gene, a strong promoter or a mode of weakening the endogenous gene promoter is generally adopted in the prior art to realize the strong start of the exogenous gene.

In the process, whether the expression of the endogenous gene is influenced, whether the endogenous gene can be normally transcribed and whether the endogenous gene can be normally expressed do not need to be considered. Therefore, the integration site of the foreign gene is selected so as to be located in a region where the host cell chromosome is relatively stable and transcription is active, and it is sufficient that the insertion sequence is not lost with the replication of the chromosome and transcription is efficiently performed. As for this insertion site, there is no need to consider the transcription, expression, etc. of the gene associated with the host cell itself.

With the continuous development of recombinant protein expression technology, more and more target proteins are designed by means of gene recombination and obtained by means of host cell expression.

This includes cytotoxic proteins whose expression can affect the host cell, cause it to die, or accelerate its death. Host cells are vectors for recombinant protein expression, and if the host cells die, no more protein is available. In the host cell gene, increasing the insertion site makes it possible to obtain more target proteins in the early stage, but the toxicity of these proteins makes the host cell die more quickly. Therefore, the prior art has not solved how to obtain cytotoxic proteins in host cells in high yields by using gene recombination techniques.

Disclosure of Invention

The invention aims to solve the problem that the target protein with toxicity in the prior art is easy to cause the death of host cells, so that the expected recombinant transcription expression effect cannot be obtained.

In order to solve the problem, the invention discloses a non-strong start type exogenous gene expression method, which inserts an exogenous protein coding sequence into the downstream of a promoter of a host cell self gene in a site-specific integration way, and realizes the transcription and expression of an exogenous gene by utilizing the promoter of the host self gene and a related transcription initiation or translation initiation regulatory sequence.

The method specifically comprises the following steps:

s1: selecting host cells, selecting endogenous proteins meeting the expression time requirements of target exogenous proteins according to the expression rules of different endogenous proteins in the host cells, and determining the expression cassette sequences of the endogenous protein genes according to GenBank;

s2: selecting a gene editing system for site-directed insertion;

s3: selecting sites which meet the conditions in an expression cassette sequence of an endogenous protein gene as fixed-point insertion points according to the following rules, wherein the sites are a. located at the upstream of a transcription initiation site of the endogenous protein gene, b. located at the downstream of a promoter of an endogenous protein coding gene, and c. meet the identification operation requirements of a fixed-point insertion gene editing system;

s4: designing and synthesizing a fluorescent marker exogenous gene sequence comprising an upstream homologous sequence of the insertion site, a loxP511 sequence, an eGFP coding sequence, a loxP sequence, an IRES2 sequence and a downstream homologous sequence of the insertion site according to the insertion site;

s5: inserting the foreign gene sequence obtained in S4 into the insertion point obtained by the S3 screening using the gene editing system selected in S2;

s6: obtaining recombinant cell clones which are integrated and inserted with exogenous gene sequences at different insertion sites through PCR specific amplification fragments and sequencing verification;

s7: observing the transcription levels of eGFP in the recombinant cells subjected to point-fixed integral insertion of different insertion sites, selecting insertion sites with eGFP transcription levels basically consistent with the transcription levels of the selected endogenous genes in S1, and respectively marking the insertion sites as a targeting site 1, a targeting site 2 and a … … targeting site n;

s8: recording and forming an insertable site form by taking host cell-endogenous protein-targeted site n as a format;

s9: replacing a target protein coding sequence and an eGFP coding sequence between loxP511 and loxP of an exogenous gene sequence by adopting a Cre/loxP system to obtain a target exogenous gene sequence;

s10: and inserting the target foreign gene sequence in the S9 into one or more host cell-endogenous protein-targeting site n insertion sites in the S8 according to the mode of S5 to obtain the target protein.

Since the target protein does not contain a promoter when inserted, and transcription and translation are initiated by the promoter of the host cell itself, the expression of the target protein is initiated by the expression of the endogenous protein at the site of insertion. The initiation, transcription initiation, and translation initiation of the endogenous protein in the host cell are controlled by the regulatory sequence, and thus the expression of the target protein is controlled by the regulatory sequence. Thereby realizing a non-strong start type exogenous gene expression method. By the non-strong start type exogenous gene expression method, the exogenous gene can select different endogenous proteins according to the requirement of expression time, so that the exogenous gene can be expressed in different growth stages of host cells.

In addition, by using the non-strong start type foreign gene expression method, the toxic protein can be expressed in the later stage of the growth of the host cell by inserting the target protein coding sequence with toxicity into the protein expressed in the later stage of the growth of the host cell. Since the host cell has already a certain scale through a large amount of growth and proliferation in the early and middle stages, the toxic protein is expressed at this time, the amount of the target protein obtained is larger, and the expression has a small influence on the growth-death course of the host cell itself.

Further preferably, the host cell is a CHO cell.

CHO cell is the recombinant protein expression host cell which is most widely applied in the biological medicine industry at present, and a plurality of fixed-point integration methods which can be applied to the CHO cell are provided, and most of the methods are homologous recombination technologies of specific integration sites, and genetic engineering operations such as gene fragment replacement, gene knockout, gene knock-in and the like are realized between recombinase specific recognition sites on a genome and an exogenous DNA carrier by means of site-specific recombinase. Therefore, the CHO cell is used as a host cell which is more mature and stable.

More preferably, the endogenous protein is thioredoxin interacting protein (Txnip).

Further, preferably, the CRISPR/Cas9 system is used as a site-directed insertion site system.

The sequence satisfying the requirement for the identification operation of the site-specific insertion gene editing system is (5' -N (N … N)₁₉NGG-3’）。

Further, the invention also discloses a step of site-specific insertion of a foreign gene by using a CRISPR/Cas9 system, which comprises 3 transfection operation vectors: 1) a homologous recombination vector containing upstream and downstream homologous sequences and an insertion sequence constructed according to the upstream and downstream sequences of the insertion site, 2) a sgRNA vector capable of identifying the insertion site is constructed, and 3) a vector containing the complete Cas9 expression cassette is constructed.

Still further preferably, the method further comprises the steps of: 3 vectors were transfected into CHO cells, subcultured to day 5 after transfection, and CHO cells with green fluorescence were sorted into 96-well plates using a flow cytometer and allowed to contain only one cell clone per well.

Meanwhile, the invention further discloses a mode for inspecting the transcription and expression level of eGFP in the recombinant cells inserted with different insertion sites by site-specific integration, which comprises the following steps: within one culture cycle, for CHO cells for 6 days, the transcription level of the green fluorescent reporter gene and the change in the green fluorescence intensity level were examined.

The invention also discloses an exogenous gene expression sequence without a promoter, which is different from the prior exogenous gene expression cassette, does not contain the promoter and does not contain a strong promoter, and the exogenous gene expression sequence is applied to the non-strong start type exogenous gene expression method disclosed by the invention and is started by the promoter in the endogenous protein gene expression cassette.

The invention is an innovative invention and solves the dilemma that toxic protein in the industry cannot be expressed in high yield at present. By utilizing the promoter and the regulation mechanism of the host cell, the non-strong start expression of the exogenous gene is realized, so that the cell death and accelerated death caused by the strong start expression of the exogenous toxic protein in the host cell are avoided, the expression after proliferation is realized, and a large amount of toxic target protein is obtained in the later growth stage of the host cell. Fills the blank in the prior art and develops a new direction for the research and development of the related toxic protein gene engineering technology.

Drawings

FIG. 1 is a schematic diagram of the position characteristics of a target recognition site in a Txnip gene sequence of CHO-K1 cells.

FIG. 2 is a schematic diagram of a site-directed integration vector.

FIG. 3 is the electrophoresis chart of the PCR product of CHO cell clone.

FIG. 4 is a graph showing the results of eGFP transcription level determination of CHO site-directed integration recombinant clones.

FIG. 5 is a graph showing the eGFP expression level of the recombinant CHO cell clone 10-6-G inserted by site-directed integration.

FIG. 6 is a schematic diagram showing the results of endogenous non-strong start expression of CSFV E2 protein truncated at the C-terminus.

Detailed Description

In order that the invention may be better understood, we now provide further explanation of the invention with reference to specific examples.