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Nucleic acid construct and preparation method thereof

A technology of nucleic acid constructs and nucleic acid constructs, which is applied in the field of nucleic acid constructs and preparations, can solve the problems of various protein production or use thresholds, stoppages, etc., and achieve the goal of improving positive cleavage, secretory expression, and extracellular secretory expression Effect

Inactive Publication Date: 2022-03-22
KANGMA SHANGHAI BIOTECH LTD
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Problems solved by technology

[0002] At present, the precise expression of the N-terminus of the peptide chain has always been a flaw in the application of small molecule functional peptide chains (small molecule short-chain polypeptides), especially in clinical applications, and the reason comes from the current academic uncertainty. Simulate the cleavage modification of proteins in cells, so the peptide chain cleavage of small molecule short-chain polypeptides is still only in the prediction stage of in vitro experiments. Even if the prediction is successful, various proteases in cells will still add to the secondary structure of the peptide chain. There are many variables, which make the expression of heterologous proteins still only stay in intracellular expression and extraction processing. This situation will make people have limitations and thresholds for the production or use of various proteins

Method used

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  • Nucleic acid construct and preparation method thereof
  • Nucleic acid construct and preparation method thereof
  • Nucleic acid construct and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0048] In this embodiment, the expression of antibacterial small molecule short-chain peptide is taken as an example for illustration.

[0049] The strain (experimental strain) used in this experiment to express small-molecule short-chain polypeptides is a type of Candida globosa yeast of Kluyveromyces lactis (strain NRRL Y-1140), and it is a crabtreenegative type of yeast.

[0050] In this experiment, the wide-area antimicrobial peptide (small molecule short-chain polypeptide) whose coding sequence is SEQ ID NO: 4 was used as a reporter gene, and the inhibitory effect on Gram-negative bacterial strains (test strains) and Gram-positive bacterial strains (test strains) was used as a reporter gene. Bacteria broth test to detect the intensity of its expression.

[0051] The test strains used in this experiment: the Gram-positive strain is Lactobacillus Acidophilus, and the Gram-negative strain is Escherichia coli.

[0052] (1) Construction of target nucleic acid construct:

[0...

Embodiment 2

[0125] In this example, the culture medium was used to cultivate cells through the K lactis strain, and the RNA content of the strain cells cultured for different periods of time was detected and sequenced to obtain the transcription intensity of the gene. The results are shown in Table 1. In Table 1, CIVTT2118 is Kangma’s A generation strain number.

[0126]

[0127] In Table 1, the sequence number 1 corresponds to the transcription activated by the activation element A of the present invention. It can be seen from Table 1 that within 24 hours, there is the following trend: within 12 hours, compared with the transcription of other genes, it can be seen that the gene with sequence number 1 is strongly transcribed, indicating that the corresponding activation of the gene The sequence number 1 (that is, the promoter element A involved in the embodiment of the present invention) can strongly transcribe the corresponding gene in the early stage, and the transcription intensity ...

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Abstract

The invention provides a nucleic acid construct which comprises a structure as shown in a formula I: A-B-C, in the formula I, A is a first nucleotide sequence for coding a starting element; b is a second nucleotide sequence for coding the signal peptide, and B is connected with the N end of C; and C is a third nucleotide sequence for coding the small molecule short peptide. The method can adapt to extracellular secretion expression of small polypeptides with various sizes, can be practiced without destroying the functionality of the N end of a peptide chain, can accurately cut and maintain a high cutting positive rate, so that a culture system of the small-molecular short-chain polypeptides expressed by cells can be used for producing the small-molecular short-chain polypeptides with high yield and high precision without extraction or additional processing; limitations and thresholds caused by production or use of various proteins are reduced, and clinical application of small-molecule short-chain polypeptides is enlarged; the method can adapt to small polypeptide extracellular secretion expression, can be practiced without destroying the functionality of the N end of a peptide chain, and can be used for accurately cutting and maintaining a high cutting positive rate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid construct and a preparation method. Background technique [0002] At present, the precise expression of the N-terminus of the peptide chain has always been a flaw in the application of small molecule functional peptide chains (small molecule short-chain polypeptides), especially in clinical applications, and the reason comes from the current academic uncertainty. Simulate the cleavage modification of proteins in cells, so the peptide chain cleavage of small molecule short-chain polypeptides is still only in the prediction stage of in vitro experiments. Even if the prediction is successful, various proteases in cells will still add to the secondary structure of the peptide chain. There are many variables, which make the expression of heterologous proteins still only stay in intracellular expression and extraction processing. This situation will make people h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N5/10C12N1/19C12N1/21C12P21/02C12R1/865C12R1/645C12R1/19C12R1/91
CPCC07K14/00C12N15/113C12N15/815C07K2319/02C12R2001/645
Inventor 郭敏胡鼎楷张俊徐丽琼于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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