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Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode

A secretory expression, Chinese hamster technology, applied in the direction of genetic engineering, cells modified by introducing foreign genetic material, plant gene improvement, etc. strong effect

Inactive Publication Date: 2012-02-22
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the problem that the expression level of exogenous gene products expressed by CHO is low at present, and cannot satisfy industrialized production, and provides a Chinese hamster ovary genetically engineered cell line that efficiently secretes and expresses AcAP5

Method used

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  • Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode
  • Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode
  • Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode

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specific Embodiment approach 1

[0027] Specific embodiment one: In this embodiment, the Chinese hamster ovary genetically engineered cell line that efficiently secretes and expresses AcAP5 uses dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO-dhfr - ) as a host cell, after screening and amplification, a cell line with stable and high-efficiency expression of AcAP5 was obtained, and the highest expression level reached 12mg / L·72h.

[0028] In this embodiment, the method for constructing a Chinese hamster ovary genetically engineered cell line that efficiently secretes and expresses AcAP5 is as follows:

[0029] 1. Double enzyme digestion of the target fragment: The AcAP5 gene sequence with signal peptide synthesized by Shanghai Sangong Company was cloned into the T vector, and the T vector was double digested with BamHI and NotI. The reaction system is as follows:

[0030]

[0031] Enzyme digestion reaction conditions: place at 37°C for 3 hours, electrophoresis the digested product with 1...

specific Embodiment approach 2

[0054] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the sequence of the AcAP5 gene with signal peptide described in Step 1 is shown in SEQ ID NO:1. Others are the same as in the first embodiment.

[0055] The sequence of the AcAP5 gene with signal peptide in this embodiment was synthesized by Shanghai Sangon Company.

[0056] In this embodiment, the sequence of the AcAP5 gene with the signal peptide is based on the original sequence of the mRNA sequence (Aceession: U30795) of Ancylostoma caninum in Genbank, the original signal peptide sequence is removed, and CHO-dhft is selected according to the characteristics of the expression system - Favored codons. In order to maximize the efficiency of transcription and translation, and at the same time make the expression product secreted outside the cell, add Kozak sequence and signal peptide at the 5' end of the original sequence, and add restriction endonuclease BamHI and NotI sites at both ends of ...

specific Embodiment approach 3

[0057] Embodiment 3: The difference between this embodiment and Embodiment 1 is that the gene sequence of the signal peptide added to the AcAP5 gene with signal peptide in Step 1 is shown in SEQ ID NO:2. Others are the same as in the first embodiment.

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Abstract

A Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode relates to a Chinese hamster ovary (CHO) cell line capable of efficiently expressing the AcAP5 in secretion mode. The cell line resolves the problem that at present, expression amount is low by using CHO to express exogenous gene products, and requirements of industrial production cannot be met. The Chinese hamster ovary genetic engineering cell line for the efficient secretion expression AcAP5 utilizes dihydrofolic acid reductase defect type Chinese hamster ovary cells as host cells, and the cell line capable of stably and efficiently expressing AcAP5 is obtained after the host cells are screened and amplified. The CHO cell line can efficiently express the AcAP5, can be produced in industrialization mode and is used for resisting blood coagulation and thrombus. Furthermore, expression amount can reach to 12 mg / L / 72h.

Description

technical field [0001] The invention relates to a CHO cell strain that efficiently secretes and expresses AcAP5. Background technique [0002] When hookworm sucks blood, its head gland secretes a substance with anticoagulant activity called anticoagulant peptides (AcAPs). The essence of this substance is a proteolytic enzyme, which has the functions of prolonging plasma prothrombin time (pt), inhibiting blood coagulation and promoting fibrinolysis. AcAPs currently have three recombinant proteins, AcAP5, AcAP6, and AcAPc2. AcAP5 is composed of 77 amino acids and is an efficient and specific inhibitor of factor Xa. The reaction site with Xa is in peptide 37-42 (-C-R-S-R-G-C-), and the action site is between the 40th arginine and the 41st glycine between. The anticoagulant and antithrombotic effects of AcAPs will become a new clinical anticoagulant and antithrombotic drug. [0003] At present, the highest expression level of AcAP5 expressed by CHO is 6 mg / L, which cannot me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/57C12N9/64C12N15/63C12N15/66
Inventor 余琼张巍付海燕刘威
Owner HEILONGJIANG UNIV
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