New earthworm kinase gene mutant

A technology of lumbrokinase and amino acid, which is applied in the field of new lumbrokinase cDNA mutants, can solve the problems of unclear reasons, unfavorable expression of lumbrokinase cDNA, and no report of lumbrokinase, and achieve the effect of high dissolution activity

Inactive Publication Date: 2004-07-21
MILITARY VETERINARY INST MILITARY SUPPIES PLA
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Problems solved by technology

Due to the existence of various miscellaneous proteins, it is very difficult to purify lumbrokinase for injection from earthworms, and no mature preparation process has been established so far
Researchers in Japan, South Korea and China have published their lumbrokinase cDNA clones in GenBank in recent years, but the research on expressing and preparing lumbrokinase through genetic engineering has not been reported, and the reason is not clear
In the research and analysis of lumbrokinase cDNA, we found that there are many rare eukaryotic codons in the coding sequence of lumbrokinase cDNA, which may be unfavorable to the expression of lumbrokinase cDNA in eukaryotic cells

Method used

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Experimental program
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Effect test

Embodiment 1

[0023] Stable expression of lumbrokinase mutant gene in Chinese hamster ovary cell line

[0024] After the transformation of the lumbrokinase gene was completed by using the multi-site mutation PCR technique, in order to verify the expression ability of the mutant, this experiment constructed a eukaryotic expression vector of the lumbrokinase mutant gene, and transfected Chinese hamsters with liposomes Ovarian cell line (CHO), through G418 screening, obtained a CHO cell line stably expressing lumbrokinase, the expression product has obvious fibrinolytic activity, which can reach 500 tPA units per ml of cell culture supernatant.

[0025] 1. Construction of eukaryotic expression vector

[0026] The plasmid pMD18-LKm2 with the mutated target gene was digested with NotI and BamHI to recover a 0.86kb LKm2 fragment; then the pIRESlneo eukaryotic expression vector was digested with the same enzyme to recover a 5.23kb linearized vector. With a molecular ratio of 4:1, T4 DNA ligase wa...

Embodiment 2

[0036] Comparison of temporal expression of lumbrokinase and its mutants in goat mammary gland

[0037] 1. Construction of eukaryotic expression vector for mammary gland localization

[0038] The plasmid pT-bCP was digested with SpeI+SacII, and the 1.13kb goat β-casein promoter sequence was recovered. The Klenow large fragment was filled in, and connected into the pIRESlneo vector plasmid that was digested with NruI+EcoRV to obtain the intermediate plasmid pI- bCP. Then pMD18-LK and pMD18-LKm2 were digested with NotI+BamHI, and the 0.86kb gene sequence of lumbrokinase and its mutants were respectively recovered, and connected into pI-bCP after the same digestion to obtain the mammary gland localization of lumbrokinase and its mutants Eukaryotic expression vectors pI-bCP-LK and pI-bCP-LKm2.

[0039] 2. Comparison of temporal expression of lumbrokinase and its mutants in goat mammary gland

[0040] After a large number of plasmids pI-bCP-LK and pI-bCP-LKm2 were prepared and p...

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Abstract

A novel vermis kinase gene mutant is obtained by the mutation to the vermis kinase cDNA obtained through reverse transcription by single-step multi-site mutation PCR. It contains 18 mutational bases for high-level expression of vermis kinase cDNA in the tissue and cell of mammal. But the ammino acid sequence coded by it does not change. The expressed vermis fibrin has high dissolving activity.

Description

Technical field: [0001] The present invention relates to a new lumbrokinase gene mutant, in particular to a new lumbrokinase gene mutant based on the cDNA and amino acid sequence of lumbrokinase, in which the rare codons in eukaryotic cells are mutated into a new lumbrokinase gene that is favorable for expression in eukaryotic cells. Kinase cDNA mutants. The lumbrokinase translated from the cDNA mutant can effectively dissolve fibrin, and is widely used in clinical thrombolytic pharmaceutical compositions. Background technique: [0002] Lumbrokinase is a kind of enzyme from earthworms that can specifically dissolve thrombus and improve blood circulation. It can be used as a thrombolytic drug in clinical medicine for the prevention and treatment of thrombosis such as cerebral apoplexy. Compared with other thrombolytic drugs such as tissue plasminogen activator (tPA), its main advantages are that ① it has good therapeutic effect and significant prevention of thrombotic diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/7088A61K38/49A61P7/02C12N15/58
Inventor 张守峰扈荣良涂长春
Owner MILITARY VETERINARY INST MILITARY SUPPIES PLA
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