Method for producing antithrombotic medicine by using silk-worm

An antithrombotic drug and the technology of silkworm, which is applied in the direction of drug combination, pharmaceutical formula, and the use of carriers to introduce foreign genetic material, etc., can solve the problems of product inhomogeneity, cumbersome chemical method operation, etc., and achieve good effect and high clinical application value.

Inactive Publication Date: 2003-03-26
浙江中奇生物药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical method has disadvantages such as cumbersome operation and inhomogeneous products.

Method used

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  • Method for producing antithrombotic medicine by using silk-worm
  • Method for producing antithrombotic medicine by using silk-worm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014]Take Chinese kidney cells, grind them at low temperature, add 1ml of Trizol RNA extraction solution produced by GIBCOBRL company, shake gently for 10 minutes, add 500μl of chloroform (Zhejiang Dier Pharmaceutical Co., Ltd.), place at room temperature for 10 minutes, and centrifuge at 12000rpm for 10 minutes. Minutes, take the supernatant, add 2 times the volume of ethanol, after mixing, centrifuge at 12000rpm for 10 minutes, discard the supernatant, add 1000 units of reverse transcriptase (GIBCOBRL company) and 4dNTP (GIBCOBRL company) for reverse transcription, 37 ℃ , 1 hour to obtain cDNA synthesized by reverse transcription of mRNA. Design primers according to the published urokinase gene (Nagai et al., Gene, 1985, 36:183), we designed two pairs of primers P1 and P2: P1: 5'GGGGATCCATGTTAAAATTTCAGTGTGGCAAAAGAC3'P2: 5'GGGCTCGAGATATTTCTTCTGGAATTTCTTCGAAATCGCCGAGGGCCAGGCCATTCTC3', using cDNA as a template, PCR amplification was carried out with P1 and P2 as primers. The ...

Embodiment 2

[0015] The plasmid pUC-UKAV was first digested with BamHI and EcoRI, and cloned into pBaePAK8 (CLONTECH Company) digested with BamHI and EcoRI (GIBCOBRL Company), to construct the fusion gene transfer plasmid pBac-UKAV containing AnnexinV and low molecular weight urokinase. All identification is correct. (Fig. 2) Example 3, the acquisition of recombinant insect baculovirus containing Annexin V and low molecular weight urokinase fusion gene

Embodiment 3

[0016] Take 5ul insect baculovirus transfer plasmid pBac-UKAV containing Annexin V and low molecular weight urokinase fusion gene and 6ul silkworm wild nuclear polyhedrosis virus DNA for co-transfection. Take 6ul Lipofectin (GIBCOBRL company) and add 100ul serum-free TC-100 medium and mix well. The BmN cells previously cultured in a 35mm Dish were washed twice with serum-free TC-100 (GIBCOBRL company) medium, and the transfer plasmid and Lipofectin mixture was added dropwise, cultured at 27°C for 4-5 days, and the supernatant was collected for the second stage. One round of plaque screening. Take 5ul of the supernatant to infect the BmN cells in a 35mm Dish, discard the supernatant after 1 hour and add an equal amount of mixed TC-100 medium and low melting point agarose. Pick plaques after 4-5 days, infect BmN cells for 3-4 days, save the supernatant, lyse the cells with NaOH for Southern hybridization, and take the supernatant of positive clones for the 10th round of plaque ...

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Abstract

The method for producing antithrombotic medicine by using silkworm utilizes the Bombyx noriNuclear polyhedrosis virus expression system to recombine fusion protein gene with human Annexin V and low molecular weight urokinase to obtain recombinant virus. Said recombinant virus is inoculated in bombyx mori larva and chrysalis to make expression, through separation, purification and freeze-drying, the invented antithrombotic oral liquor and injection can be made up. The animal tests show that it possesses obvious antithrombotic action, and is a new type thrombolytic medicine.

Description

technical field [0001] The present invention relates to the method for preparing the fusion protein of Annexin V and low molecular weight urokinase with silkworm larvae and pupae through the silkworm baculovirus expression system, and the present invention also relates to the fusion protein of Annexin V and low molecular weight urokinase produced by silkworm larvae and pupae A method of preparing an antithrombotic drug. Background technique [0002] Thrombosis is one of the diseases that endanger people's life safety. According to statistics, there are about 3 million patients suffering from acute myocardial infarction and cerebral thrombosis in my country every year. Thrombolytic therapy has been successfully used in the treatment of clinical thrombotic diseases such as myocardial infarction. Currently clinically used thrombolytic drugs mainly include urokinase, streptokinase and tissue plasminogen activator, etc. However, due to lack of specificity for thrombus or short h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/49A61P7/02C12N15/62C12N15/85
Inventor 张耀洲金勇丰吴祥甫
Owner 浙江中奇生物药业股份有限公司
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