阅读说明：本技术 一种利用激活rna构建高效表达载体的方法 (Method for constructing high-efficiency expression vector by using activating RNA ) 是由 王斌 刘凌云 程霞 王定康 殷旭东 王德斌 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种利用激活RNA构建高效表达载体的方法,所述方法利用带近端增强子的OCT4基因启动子为靶启动子,通过体外重组技术,在所述靶启动子之前插入MAR序列,在所述靶启动子下游插入一个表达靶向所述靶启动子的特异小激活RNA(SaRNA)的shRNA表达框,构建第二表达载体pOCT4-M-SaRNA,第二表达载体在转染细胞32天以后,由OCT4启动子启动的EGFP表达量仍能持续稳定地高效表达,表达量显著高于商业载体pEGFP-C1；本发明所述方法构建的表达载体可以应用于抗体及蛋白药物的工业化生产领域或基因治疗,提高生产效率,降低生产成本,具有良好的社会效益和经济效益。(The invention discloses a method for constructing a high-efficiency expression vector by using an activating RNA, which comprises the steps of using an OCT4 gene promoter with a proximal enhancer as a target promoter, inserting a MAR sequence in front of the target promoter through an in vitro recombination technology, inserting a shRNA expression frame for expressing a specific small activating RNA (SaRNA) targeting the target promoter at the downstream of the target promoter, constructing a second expression vector pOCT4-M-SaRNA, wherein after the second expression vector transfects cells for 32 days, the EGFP expression amount started by the OCT4 promoter can be continuously and stably expressed with high efficiency, and the expression amount is obviously higher than that of a commercial vector pEGFP-C1; the expression vector constructed by the method can be applied to the field of industrial production of antibodies and protein drugs or gene therapy, improves the production efficiency, reduces the production cost and has good social benefit and economic benefit.)

1. A method for constructing a high-efficiency expression vector by using activating RNA, which is characterized by comprising the following steps:

(1) selecting a human OCT4 gene promoter with a proximal enhancer as a target promoter, wherein the sequence of the gene of the target promoter from a transcription initiation site to the enhancer is less than 1.5 kb; replacing a CMV promoter on a pEGFP-C1 vector by the target promoter to construct an intermediate vector I, namely a pOCT4 vector;

(2) inserting an shRNA gene expression frame aiming at the expression activation RNA gene of the target promoter into the MluI enzyme cutting site at the downstream of the exogenous gene expression frame of the intermediate vector I to construct a first expression vector, namely pOCT 4-SaRNA; the sequence of the shRNA gene expression cassette of the expression activation RNA gene aiming at the target promoter is shown in a table I;

table one:

the first expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

2. The method for constructing a high-efficiency expression vector by using activating RNA as claimed in claim 1, wherein the MAR sequence fragment is inserted into the Ase I enzyme cutting site before the exogenous gene expression frame of the intermediate vector I to construct an intermediate vector II; inserting an shRNA gene expression frame aiming at the expression activation RNA gene of the target promoter into an MluI enzyme cutting site at the downstream of the exogenous gene expression frame of the intermediate vector II to construct a second expression vector, namely pOCT 4-M-SaRNA; the second expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

3. The method for constructing a high-efficiency expression vector by using activating RNA as claimed in claim 1 or 2, wherein in the step of constructing the intermediate vector I, the target promoter sequence is amplified by using AgeI and AseI double-enzyme cutting site primers, wherein the primers are as follows:

OCT4 # forward primer: TAGTTATTAAT(Ase I) AGACCCAGGAGACTCAAAG

OCT4 # reverse primer: TGGCGACCGGT(Age I) AGCGCTAGTGATTACCCAAGA are provided.

4. The method for constructing a high expression vector using activating RNA as claimed in claim 2, wherein the MAR sequence is HUBB-LCR sequence.

Technical Field

The invention belongs to the technical field of biology, relates to an expression vector for recombinant antibody or protein drug production and the like and a construction method thereof, and particularly relates to a method for constructing a high-efficiency expression vector based on the interaction of a MAR sequence and an activating RNA.

Background

Recombinant antibody and protein drugs are special drugs which are frequently used clinically in recent years, but the wide application thereof is limited due to the expensive price thereof. In the cost constitution of recombinant antibodies and protein drugs, low production efficiency is an important factor. In the complicated production link of recombinant antibody and protein medicine, whether the vector can be expressed stably and efficiently is one of the key technologies affecting the production efficiency. There are many factors that affect high expression, the main ones are still position effect and silencing effect in transgene expression. The silencing effect often causes that the continuous and high-efficiency production of vectors which are transferred to animal cells for expression is difficult to realize, so that the production efficiency of recombinant antibodies and protein drugs is difficult to improve; the position effect is caused by abnormal transcription of the recombinant gene inserted near the heterochromosome, the position effect often causes a silencing effect, and the silencing effect of the transgene is related to factors such as chromatin remodeling and epigenetic modification.

There are many methods for overcoming the silencing effect of transgenes, but the effect is different, and an effective standard is not formed in the industry. Studies have shown that chromatin structure regulatory elements are significantly effective in overcoming position effects. Promoters are regulated by specific regulatory elements with the ability to overcome positional effects, among which Matrix Sequences (MARs) were the earliest DNA elements thought to topologically separate the genome into distinct domains (Phi-Van L, 1996). The organization of eukaryotic chromatin into chromatin domains requires the mediation of a nuclear Matrix (MAR), also known as a nuclear backbone, consisting of nuclear proteins, with DNA loops (loops) provided by protein fibers branching from the nuclear matrix to form a supporting structure. These independent DNA loops attached to the nuclear matrix or backbone isolate the transgene from the negative effects of the surrounding genome (insulating function) and thus overcome the transgene position effects (Seibler, et al, 2005; McKnight, et al, 1992), enhance gene transcription efficiency (Bode, et al, 2000); the nuclear matrix sequence also overcomes the transgene silencing effect by preventing DNA methylation from supporting histone acetylation to maintain long-term high transcription levels (Dang, et al, 2000; Kurre, et al, 2003).

On the other hand, RNA is also an effective means for regulating gene expression. RNA regulation mainly includes RNA interference (RNAi) and RNA activation (RNAa). RNA interference means that siRNA (double-stranded small RNA) can specifically silence the expression of a target gene by acting on mRNA or 3' UTR region of the gene; RNA activation targets the promoter of a gene, thereby increasing gene expression by affecting transcriptional or epigenetic regulatory mechanisms. Such small RNAs that activate gene promoters are called small activating RNAs (sarRNAs).

In the existing technical scheme, the small activating RNA is mainly used as a potential molecular drug for activating the expression up-regulation of in vivo special genes so as to change in vivo physiology or in vitro molecular effects. However, no solution has been reported for activating RNA to improve the expression ability of recombinant proteins or drug expression vectors. More importantly, although both the activating RNA and the MAR can be independently used for optimizing the promoter start efficiency of the foreign gene expression cassette in the recombinant protein expression vector, the technology or application of the activating RNA and the MAR which are jointly used for improving the promoter start efficiency and further improving the expression capacity of the expression vector by acting on a certain type of promoter is not reported.

Disclosure of Invention

The invention aims to solve the technical problem that the expression efficiency of the expression vector of the recombinant antibody and the protein drug is low at present, and provides a method for constructing a high-efficiency expression vector by using activating RNA.

In order to solve the technical problems, the method adopts the technical scheme that: the method for constructing the high-efficiency expression vector by using the activating RNA comprises the following steps:

(1) selecting a human OCT4 gene promoter with a proximal enhancer as a target promoter, wherein the sequence of the gene of the target promoter from a transcription initiation site to the enhancer is less than 1.5 kb; replacing a CMV promoter on a pEGFP-C1 vector by the target promoter to construct an intermediate vector I, namely a pOCT4 vector;

(2) inserting an shRNA gene expression frame aiming at the expression activation RNA gene of the target promoter into the MluI enzyme cutting site at the downstream of the exogenous gene expression frame of the intermediate vector I to construct a first expression vector pOCT 4-SaRNA; the sequence of the shRNA gene expression cassette of the expression activation RNA gene aiming at the target promoter is shown in a table I; the first expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

Furthermore, in the method, a MAR sequence fragment is inserted into an Ase I enzyme cutting site in front of an exogenous gene expression frame of the intermediate vector I to construct an intermediate vector II; inserting an shRNA gene expression frame aiming at the expression activation RNA gene of the target promoter into the MluI enzyme cutting site at the downstream of the exogenous gene expression frame of the intermediate vector II to construct a second expression vector, namely pOCT 4-M-SaRNA; the second expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

Further, in the method, in the step of constructing the intermediate vector I, a target promoter sequence is amplified by using a double enzyme cutting site primer with AgeI and AseI, wherein the primer is as follows:

OCT4 # forward primer: TAGTTATTAAT(Ase I) AGACCCAGGAGACTCAAAG

OCT4 # reverse primer: TGGCGACCGGT(Age I) AGCGCTAGTGATTACCCAAGA are provided.

Preferably, the MAR sequence of the invention is a HUBB-LCR sequence.

The invention selects human OCT4 gene promoter with proximal enhancer as target promoter, inserts MAR sequence (such as HUBB-LCR sequence) before the target promoter, and inserts a shRNA gene expression frame (specific small activation RNA expression frame) aiming at the expression activation RNA gene of OCT4 gene promoter after the target promoter (OCT 4) and its expression frame acted by MAR sequence, the shRNA expressed by the expression frame can generate activation small RNA aiming at the target promoter after being cut by in vivo enzyme. The expression vector constructed in the way is transfected into 293 cells or CHO cells, and can enable the expression level of the EGFP gene to be higher than that of the EGFP gene when the MAR alone regulates or only activates small RNA after 32 days of culture.

The second expression vector of the high-efficiency expression vector constructed by the method of the invention has the most obvious effect of continuous, stable and high-efficiency expression, and the main action mechanism is as follows: when the MAR interacts with the promoter with the proximal enhancer, the MAR-regulated promoter with the proximal enhancer changes histone modification on the promoter through a chromatin remodeling mechanism, so that the histone modification is more favorable for RNA activation, and high-efficiency expression is activated (the expression amount of the second expression vector is higher than the effect achieved by singly using the MAR or singly using the RNA activation); it can also be explained that when MAR and RNA are co-acting on the same promoter with a proximal enhancer, both co-acting on the same promoter with a proximal enhancer will achieve higher efficiency of expression, since both will have the effect of altering the appearance of the targeted region and activating gene expression. The theory is supposed that the two interfere the appearance modification of the target promoter from different mechanisms, so that the superposition appears on the function of overcoming the appearance silencing effect on the promoter with the near-end enhancer, finally the expression of the vector and the commercial vector (the expression can be continuously and stably carried out for more than 32 days) which is higher than that of the two when the two act independently is obtained, and the industrial production application is met to the maximum extent.

The invention has the beneficial effects that:

1. the invention screens the sequences with different lengths and sizes of the human OCT4 promoter to obtain the human OCT4 gene promoter with the characteristic of a proximal enhancer, namely the target promoter.

2. After the first expression vector and the second expression vector constructed by the method transfect mammalian cells, through a detection test of expression efficiency, the expression quantity of the expressed exogenous EGFP gene is better expressed, the expression quantity of the expressed exogenous EGFP gene is most obvious by using the second expression vector pOCT4-M-SaRNA, the expression quantity of the expressed exogenous EGFP gene is obviously higher than that of a commercial vector pEGFP-C1 on the EGFP gene, and is simultaneously higher than that when a MAR sequence acts on an OCT4 promoter alone or that when the SaRNA acts on an OCT4 promoter alone, and the expression quantity can still be stably expressed for more than 32 days after continuous culture, namely, the expression can be continuously, stably and efficiently expressed, the requirement of industrial production is met, the drug price is reduced, the popularization and the application are convenient, and good social benefits and economic benefits are achieved.

Drawings

FIG. 1 is a schematic diagram of the structure of the intermediate vector IpOCT 4;

FIG. 2 is a schematic diagram of the structure of an intermediate vector IIpOCT 4-LCR;

FIG. 3 is a schematic structural diagram of a first expression vector pOCT 4-SaRNA;

FIG. 4 is a schematic structural diagram of a second expression vector pOCT 4-M-SaRNA;

FIG. 5A is a fluorescent photograph of mammalian cells transfected with the second expression vector pOCT4-M-SaRNA for 14 days;

FIG. 5B is a fluorescent photograph of mammalian cells transfected with the second expression vector pOCT4-M-SaRNA for 32 days;

FIG. 6 is a graph comparing the results of fluorescence intensity tests of the recombinant vectors pOCT4-LCR, pOCT4-SaRNA and pOCT4-M-SaRNA with the commercial vector pEGFP-C1;

FIG. 7 is a graph showing the comparison of the expression levels of EGFP protein in the recombinant vectors pOCT4, pOCT4-LCR, pOCT4-SaRNA and pOCT4-M-SaRNA tested in ELISA assay with the commercial vector pEGFP-C1 (note: the difference in expression levels between the constructed vector and pEGFP-C1 is significant, p is less than 0.05; the difference in expression levels between the second expression vector and all other vectors is significant, p is less than 0.01);

FIG. 8 is a graph showing the comparison of EGFP gene expression levels when the recombinant vectors pOCT4, pOCT4-LCR, pOCT4-SaRNA and pOCT4-M-SaRNA were detected by qRT-PCR quantitation method with the commercial vector pEGFP-C1 (note: the difference in expression levels was significant for the constructed vectors compared with pEGFP-C1, p was < 0.05);

FIG. 9 is the HUBB-LCR sequence described in the examples;

FIG. 10 shows the promoter sequence of human OCT4 gene with proximal enhancer, the sequence from transcription initiation site to enhancer is less than 1.5 kb.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

The method for constructing the high-efficiency expression vector by using the activating RNA comprises the following steps:

(1) selecting a human OCT4 gene promoter with a proximal enhancer as a target promoter, wherein the sequence of the gene of the target promoter from a transcription initiation site to the enhancer is less than 1.5 kb; replacing a CMV promoter on a pEGFP-C1 vector by the target promoter to construct an intermediate vector I, namely a pOCT4 vector;

(2) inserting an shRNA gene expression frame aiming at the expression activation RNA gene of the target promoter into the MluI enzyme cutting site at the downstream of the exogenous gene expression frame of the intermediate vector I to construct a first expression vector pOCT 4-SaRNA; the sequence of the shRNA gene expression cassette of the expression activation RNA gene aiming at the target promoter is shown in a table I; the first expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

Furthermore, in the method, a MAR sequence fragment is inserted into an Ase I enzyme cutting site in front of an exogenous gene expression frame of the intermediate vector I to construct an intermediate vector II; inserting an shRNA gene expression frame aiming at the expression activation RNA gene of the target promoter into the MluI enzyme cutting site at the downstream of the exogenous gene expression frame of the intermediate vector II to construct a second expression vector, namely pOCT 4-M-SaRNA; the second expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

Further, in the method, in the step of constructing the intermediate vector I, a target promoter sequence is amplified by using a double enzyme cutting site primer with AgeI and AseI, wherein the primer is as follows:

OCT4 # forward primer: TAGTTATTAAT(Ase I) AGACCCAGGAGACTCAAAG

OCT4 # reverse primer: TGGCGACCGGT(Age I) AGCGCTAGTGATTACCCAAGA are provided.

Preferably, the MAR sequence of the invention is a HUBB-LCR sequence.