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Leader sequence for improving non-cap-dependent translation efficiency and application thereof

A leading sequence and high-efficiency technology, applied in the field of genetic engineering, can solve problems such as low efficiency of downstream gene expression, protein heterogeneity, and incomplete cutting

Pending Publication Date: 2022-07-29
XINXIANG MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current bicistronic and polycistronic vectors constructed by IRES, 2A peptide vectors have incomplete cleavage characteristics when expressing antibodies, which will cause protein heterogeneity, and the downstream gene expression efficiency mediated by IRES vectors is low

Method used

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  • Leader sequence for improving non-cap-dependent translation efficiency and application thereof
  • Leader sequence for improving non-cap-dependent translation efficiency and application thereof
  • Leader sequence for improving non-cap-dependent translation efficiency and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Construction of monocistronic vector containing TurboRFP gene

[0028] (1) Synthesis of TurboRFP gene

[0029] Referring to the TurboRFP gene sequence (GenBank: MW560964, bases 5851 to 6546), the TurboRFP gene was synthesized, which was specifically handed over to General Biological Gene (Anhui) Co., Ltd. for synthesis. The synthesized TurboRFP gene is completely consistent with the TurboRFP sequence published by GenBank (GenBank: MW560964, bases 5851-6546). In order to facilitate cloning, when synthesizing the TurboRFP gene sequence, a 5'-AGCAAGCTT-3' sequence was introduced at the 5' end, where AGC was a protective base, AAGCTT was a HindIII restriction site, and 5'-ATAGCGGCCGC-3' was introduced at the 3' end. , wherein ATA is the protective base, and GCGGCCGC is the Not I restriction site.

[0030] (2) Construction of expression vector containing TurboRFP sequence

[0031] The PCR amplification product of TurboRFP double digested with HindIII and Not I, and the...

Embodiment 2

[0065] 1. Construction of a monocistronic vector containing EGFP

[0066] Amplify EGFP according to the method of Example 1, and the method for constructing the EGFP monocistronic vector is the same as the method for constructing the TurboRFP gene monocistronic vector, except that the EGFP gene is used to replace the TurboRFP gene to obtain the vector pIRES-C3-EGFP, which The structure diagram is as figure 1 shown in F.

[0067] 2. Construction of pIRES-EGFP-TurboRFP gene bicistronic vector

[0068] (1) PCR amplification of TurboRFP gene

[0069] The primers P3 and P4 were designed with reference to the pIRES-C3-TurboRFP sequence constructed in Example 1, and SmaI and XbaI restriction sites were introduced into the 5' ends of the primers, respectively. The primer sequences are as follows (underlined are restriction sites):

[0070] P3: 5'-CCGCCCGGGATGAGCGAGCTGATCAAGGAGAAC-3' (as shown in SEQ ID NO. 6);

[0071] P4: 5'-CTATCTAGATCATCTGTGCCCCAGTTTGCTA-3' (as shown in SEQ ID ...

Embodiment 3

[0086] In order to verify whether the leader sequence can improve the expression of Fluc and in order to increase the fold accurately and accurately, the pIRES-EGFP-Fluc bicistronic vector and pIRES-EF-L13 containing the leader sequence (SEQ ID NO. 1) were constructed in this example. vector.

[0087] 1. Construction of pIRES-EGFP-Fluc bicistronic vector

[0088] The Fluc gene was artificially synthesized with reference to the gene sequence of Firefly luciferase (Fluc) (GenBank: MK484108.1, bases 648-2300), which was specifically handed over to General Biogene (Anhui) Co., Ltd. for synthesis. The synthesized Fluc gene is consistent with GenBank: MK484108.1, the 648-2300th nucleotide sequence.

[0089] The pIRES-EGFP-Fluc bicistronic vector was constructed with reference to the method for constructing the pIRES-EGFP-TurboRFP vector in Example 2, and the difference from Example 2 was that the TurboRFP gene in Example 2 was replaced with the Fluc gene. The schematic diagram of ...

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Abstract

The invention provides a leader sequence for improving non-cap-dependent translation efficiency and application of the leader sequence, and belongs to the technical field of genetic engineering. The leader sequence is as shown in SEQ ID NO. 1. According to the present invention, with the leader sequence, the expression quantity of the target gene mediated by the non-cap-dependent internal ribosome entry site (IRES) element in the transient mammalian cell expression system can be effectively improved;

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a leader sequence for improving cap-independent translation efficiency and its application. Background technique [0002] In eukaryotes, translation can be initiated by different mechanisms. Most eukaryotic mRNAs are translated by a mechanism called cap-dependent translation. Some cellular and viral mRNAs are translated by a different mechanism in which ribosomes are recruited by RNA structures at internal ribozyme entry sites (IRES). Since IRES elements are capable of initiating translation of genes without a cap structure, they have been used in bicistronic and polycistronic vectors for co-expression of proteins of interest. In these vectors, a viral promoter directs the synthetic reading frame of a single mRNA strand containing an IRES element. The first gene is translated by a cap-dependent ribosome scanning mechanism, while translation of subsequent genes is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/12C12N15/53C12N15/13
CPCC12N15/113C12N15/85C07K14/43595C12N9/0004C07K16/241C12N2800/107C12N2840/105C12N2840/203
Inventor 王天云马凯米春柳林艳耿少雷孙秋丽王小引张俊河
Owner XINXIANG MEDICAL UNIV
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