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Efficient expression method for novel anticoagulant and thrombolytic dual-function fusion protein

A high-efficiency expression and fusion protein technology, applied in the direction of fusion polypeptides, protease inhibitors, microbial-based methods, etc., can solve the problems of inability to investigate interactions, surface analysis, and large influence of accidental factors

Inactive Publication Date: 2015-05-06
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The single-factor method takes a long time, and the accidental factors in the operation have a great influence. In addition, the interaction between various factors cannot be investigated, resulting in low accuracy of the test results; Due to the inability to fit the functional relationship between the influencing factors and the response value, it is impossible to conduct surface analysis on all factors, so it is generally difficult to obtain the optimal condition; the response surface method has a small number of experiments and a short cycle, which can fully examine the interaction between various factors , the optimal parameter value can also be obtained through the analytical regression equation, which greatly improves the accuracy of the experiment and can make up for the shortcomings of the above optimization methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] 1. The method for expressing hirudin 12 peptide and reteplase fusion protein by recombinant Escherichia coli engineering bacteria:

[0013] ① Shake flask fermentation: Freshly prepare LB liquid medium with distilled water, add 100ug / ml Amp solution, and then inoculate the bacteria stored in 15% glycerol tube into the medium with 1% inoculum amount, and culture at 37°C and 200rpm 12h to activate the bacteria. Then transfer the activated bacterium solution to the shake flask according to the inoculation amount of 5%. In the shake flask, it is the LB medium prepared by distilled water, which contains Amp100ug / ml, Ca 2+ 1mmol / L, Mg 2+ 20mmol / L, continue culturing for 3h, add 1.5mmol / L IPTG as an inducer, and induce for 4h at 39°C and 200rpm.

[0014] ②Collect bacteria: After the induction, centrifuge at 8000rpm for 3min at 4°C, blow and wash the obtained bacterial pellet with PBS (pH7.4), and then centrifuge under the same conditions to obtain bacterial cells.

[0015] ...

Embodiment 2

[0018] 1. The method for expressing hirudin 12 peptide and reteplase fusion protein by recombinant Escherichia coli engineering bacteria:

[0019] ① Shake flask fermentation: Freshly prepare LB liquid medium with distilled water, add 100ug / ml Amp solution, and then inoculate the bacteria stored in 15% glycerol tube into the medium with 1% inoculum amount, and culture at 37°C and 200rpm 12h to activate the bacteria. Then transfer the activated bacterium solution to the shake flask according to the inoculation amount of 5%. In the shake flask, it is the LB medium prepared by distilled water, which contains Amp100ug / ml, Ca 2+ 1mmol / L, Mg 2+ 20mmol / L, continue culturing for 3h, add 1mmol / L IPTG as an inducer, and induce for 4h at 38°C and 200rpm.

[0020] ②Collect bacteria: After the induction, centrifuge at 8000rpm for 3min at 4°C, blow and wash the obtained bacterial pellet with PBS (pH7.4), and then centrifuge under the same conditions to obtain bacterial cells.

[0021] ③B...

Embodiment 3

[0024] 1. The method for expressing hirudin 12 peptide and reteplase fusion protein by recombinant Escherichia coli engineering bacteria:

[0025] ① Shake flask fermentation: Freshly prepare LB liquid medium with distilled water, add 100ug / ml Amp solution, and then inoculate the bacteria stored in 15% glycerol tube into the medium with 1% inoculum amount, and culture at 37°C and 200rpm 12h to activate the bacteria. Then transfer the activated bacterium solution to the shake flask according to the inoculation amount of 5%. In the shake flask, it is the LB medium prepared by distilled water, which contains Amp100ug / ml, Ca 2+ 1.05mmol / L, Mg 2+ 20mmol / L, continue to culture for 3h, add 1mmol / L IPTG as inducer, induce 4h at 38.06℃, 207.84rpm.

[0026] ②Collect bacteria: After the induction, centrifuge at 8000rpm for 3min at 4°C, blow and wash the obtained bacterial pellet with PBS (pH7.4), and then centrifuge under the same conditions to obtain bacterial cells.

[0027] ③Bacteria...

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PUM

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Abstract

The invention belongs to the field of biological pharmacy and particularly relates to an efficient method for a novel anticoagulant and thrombolytic dual-function fusion protein-hirudin 12 peptide and reteplase fusion protein (HV12p-rPA). According to the method, seven major factors (preparation of a culture medium, inoculation quantity, induction time, rotation speed, induction temperature, a feed supplementing mode and trace elements) in shake-flask culture conditions of escherichia coli engineering bacteria are screened and optimized to determine three most important factors of the rotation speed, temperature and calcium ion concentration, which affect target protein expression amount, and the expression amount of target protein HV12p-rPA is up to 36.15%. The efficient expression method is simple, feasible, efficient, and relatively low in cost, and has a good application value.

Description

technical field [0001] The present invention relates to the field of biopharmaceuticals, in particular to the high-efficiency expression of a novel anticoagulant and thrombolytic bifunctional fusion protein—hirudin 12 peptide and reptease fusion protein (HV12p-rPA) in pET21a-HrP / BL21 Escherichia coli engineering bacteria method. Background technique [0002] The high-efficiency expression target in the present invention is hirudin 12 peptide and reteplase fusion protein (HV12p-rPA), which is achieved through a (Gly) 3 The flexible peptide group fuses the C-terminal 12 peptide of recombinant hirudin HV3 with the rPA gene to form a new protein with both anticoagulant and thrombolytic functions (patent number: ZL 2006 1 0022457.8). The protein is expressed through Escherichia coli, the most commonly used expression system at present, and its expression level directly affects the progress of subsequent research and industrialization, but the expression level of the fusion prote...

Claims

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Application Information

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IPC IPC(8): C12N9/64C07K14/815C12R1/19
CPCC12N9/6483C07K14/815C07K2319/00
Inventor 余蓉刘立平许小枫邓璇
Owner SICHUAN UNIV
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