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Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2

A technology of secretion expression and Chinese hamster, which is applied to cells modified by introducing foreign genetic material, recombinant DNA technology, and the use of vectors to introduce foreign genetic material, etc., which can solve the problems of unsatisfactory industrial production and low expression of foreign gene products , to achieve strong adaptability and the effect of preventing thrombosis

Inactive Publication Date: 2012-01-18
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 meet the needs of industrialized production, and provides a Chinese hamster ovary genetically engineered cell line that efficiently secretes and expresses AcAPc2

Method used

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  • Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2
  • Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2
  • Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2

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specific Embodiment approach 1

[0028] Specific embodiment one: In this embodiment, the Chinese hamster ovary genetically engineered cell line that efficiently secretes and expresses AcAPc2 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 AcAPc2 was obtained, and the highest expression level reached 10mg / L·72h.

[0029] In this embodiment, the method for constructing a Chinese hamster ovary genetically engineered cell line that efficiently secretes and expresses AcAPc2 is as follows:

[0030] 1. Double enzyme digestion of the target fragment: The AcAPc2 gene sequence with a 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:

[0031]

[0032] Enzyme digestion reaction conditions: place at 37°C for 3 hours, electrophoresis the digested product ...

specific Embodiment approach 2

[0055] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the sequence of the AcAPc2 gene with signal peptide described in Step 1 is shown in SEQ ID NO:1. Others are the same as in the first embodiment.

[0056] The sequence of AcAPc2 gene with signal peptide in this embodiment was synthesized by Shanghai Sangon Company.

[0057] The AcAPc2 gene sequence with signal peptide described in step 1 is based on the original sequence of the mRNA sequence (Aceession: U30793) of Ancylostoma caninum in Genbank, remove the original signal peptide sequence, and select CHO-dhft 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 the original sequence...

specific Embodiment approach 3

[0058] Embodiment 3: The difference between this embodiment and Embodiment 1 is that the gene sequence of the signal peptide added to the AcAPc2 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

The invention discloses a Chinese hamster ovary (CHO) genetic engineering cell line for performing high-level secretory expression on AcAPc2 and relates to a CHO cell line for performing high-level secretory expression on the AcAPc2. The invention aims to solve the problems that the expression level of an exogenous gene product expressed by using CHO is low at present and the industrialized production cannot be realized. The CHO genetic engineering cell line for performing high-level secretory expression on the AcAPc2 is obtained by the following step of: screening dihydrofolate reductase-deficient CHO cells which are taken as host cells, and amplifying to obtain the cell line for performing stable and high-level expression on the AcAPc2. The CHO cell line can perform high-level expression on the AcAPc2; the expression level can be up to 10mg / L.72h; and the CHO cell line can be industrially produced. The cell line is used for resisting blood coagulation and treating tumor, septicaemiaand the like.

Description

technical field [0001] The invention relates to a CHO cell strain that efficiently secretes and expresses AcAPc2. 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. AcAPc2(10KD) is a highly effective and specific inhibitor of factor Xa. According to reports, the antithrombotic effect of Xa inhibitors is superior to that of thrombin inhibitors. AcAPs, as high-efficiency and specific inhibitors of factor Xa, have little effect on platelet aggregation, and the risk of bleeding is small when used for antithrombotic therapy. In 1998, Donnelly et al. reported that AcAPs also had the effect of anti-tumor cell metastasis i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85
Inventor 余琼马龙彪丁海燕张巍
Owner HEILONGJIANG UNIV
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