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Method for expressing Reteplase in Escherichia coli by using dual plasmid system

A technology of Escherichia coli and recombinant Escherichia coli, applied in the field of biomedicine, can solve the problems of lack, verification of thrombolytic activity, inability to ensure clinical application, etc., and achieve the effects of low cost, simple production process, and efficient soluble expression

Inactive Publication Date: 2011-11-23
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some problems in this method: both pRSETa and pREP4 plasmids lack elements to assist the formation of disulfide bonds. Although LacI expressed by pREP4 plasmid can activate the high expression of rPA in the pRSETa-rPA plasmid, it cannot assist in the formation of rPA activity. essential disulfide bond, so the patent only identified the obtained rPA recombinant protein by western blot, and failed to verify its thrombolytic activity, which could not ensure the correct clinical application

Method used

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  • Method for expressing Reteplase in Escherichia coli by using dual plasmid system
  • Method for expressing Reteplase in Escherichia coli by using dual plasmid system
  • Method for expressing Reteplase in Escherichia coli by using dual plasmid system

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Experimental program
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Embodiment 1

[0031] Construction of rPA genetically engineered bacteria

[0032] 1. Using the pMD18T-tPA plasmid constructed in the previous research of our laboratory as a template (Zhen Jie, Jiang Chengying, Du Lianxiang, etc.. Reteplase gene cloning and expression in Pichia methanolica. Journal of South China University of Technology Natural Science Edition, 2006, 34(12): 25-29).

[0033] Using upstream primers:

[0034] 5’-CGC GGATCC ATCTTACCAAGGAAACAGTGACTGCTAC-3’ (NO3);

[0035] Downstream primer:

[0036] 5’-GCC AAGCTT CAATGTCTTACCAAGGAAACAGTGACTGC-3’ (NO4);

[0037] Carry out PCR amplification, the amplified PCR products are detected by agarose gel electrophoresis, and then purified and recovered by gel purification to obtain rPA gene fragments.

[0038] In order to facilitate subsequent operations, the upstream and downstream primers were introduced Bam HⅠand Hin dⅢ restriction site (underlined part).

[0039] The sequence comparison confirmed that the rPA coding gene sequence obtained by ...

Embodiment 2

[0046] Induced expression of rPA in E. coli

[0047] 1. Inoculate the recombinant E. coli co-transformed with pET22b-rPA and pET40b plasmids into 5 mL of LB liquid medium containing a final concentration of 50 μg / mL Amp and 30 μg / mL Kan, and cultivate overnight at 37°C and 220 rpm.

[0048] 2. Transfer the overnight cultures obtained in the previous step to 200 mL of LB liquid medium at a volume ratio of 1%, and then add IPTG (final concentration 0.6 mM) to induce induction at 25°C when the OD600 is 0.6 Express for 3 h.

[0049] 3. After the induction of expression, take 3 mL of bacterial solution and centrifuge at 12000 rpm to collect the bacterial cells, add SDS loading buffer, and boil to collect total protein. The remaining bacterial liquid was collected by centrifugation at 12000 rpm and broken by ultrasonic method. After centrifugation at 12000 rpm, the supernatant (soluble protein) and precipitate (protein in the form of inclusion bodies) were detected by SDS-PAGE...

Embodiment 3

[0052] Recombinant protein thrombolytic activity detection

[0053] 1. Preparation of fibrin plate: Weigh 0.02 g of fibrinogen and add it to a small conical flask containing 5 mL of PBS, mix well and place it at 37°C for 10 min to preheat it to dissolve it. At the same time, take another appropriate amount of thrombin into an EP tube containing 1mL PBS, blow gently, and put it in 37℃ to preheat.

[0054] 2. Weigh out 0.07 g of agarose and dissolve it with 7 mL PBS, heat to dissolve, and then cool to an appropriate temperature. The thrombin solution was quickly mixed into the fibrinogen solution, mixed immediately, and then added to the agarose solution. After shaking, the mixture was poured into a plate to cool and solidify.

[0055] 3. Use a puncher to punch holes in the prepared fibrin plate, add 30 μL of the sample to be tested, and incubate at 37°C for 12 h to detect the size of the thrombolytic circle. In the experiment, the induced expression products of pET22b and pET40b tra...

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Abstract

The invention relates to a method for expressing Reteplase (rPA) in Escherichia coli by using a dual plasmid system. The method comprises the following steps of: performing polymerase chain reaction (PCR) amplification to obtain gene segment of the rPA, and cloning the gene segment into a vector pET22b to construct a recombinant plasmid pET22b-rPA; co-transforming the pET22b-rPA and pET40b into Escherichia coli BL21 (DE3) by using different resistances of the pET22b-rPA and the pET40b, and screening engineering bacteria under ampicillin (Amp) and kanamycin (Kan) double resistance selection pressure; and performing inducible expression on the engineering bacteria by using isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to obtain the soluble rPA target protein. Through detection by a fibrin plate method, the rPA protein obtained in the method is not needed to be subjected to renaturation and has obvious thrombolytic activity.

Description

Technical field [0001] The invention belongs to the preparation technology of recombinant protein drugs in the field of biomedicine, and specifically relates to a method for directly expressing and producing a drug Reteplase (rPA) with thrombolytic activity in E. coli using a double plasmid system. Background technique [0002] Thrombosis complications caused by cardiovascular disease seriously threaten human life and are one of the main causes of death and sickness. There are as many as 13 million cardiovascular disease patients worldwide each year, among which the mortality rate of acute myocardial infarction (AMI) is as high as 30%. Therefore, the research and development of thrombolytic drugs with high efficiency, specificity, safety and low side effects has always been a hot topic worldwide. [0003] rPA is a single-chain protein with a molecular weight of 39.6 kD, composed of 355 amino acids. It is a deletion variant of human tissue type plasminogen activator (tPA) that lacks...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/58C12N15/70C12N1/21C12N9/48
Inventor 罗学刚张同存倪萌
Owner TIANJIN UNIV OF SCI & TECH
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