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Recombination human endothelium chalene expression strain and solubility expression method

An endostatin and expression method technology, applied in the field of genetic engineering, can solve the problems of low activity recovery rate, low renaturation rate, complicated process and the like, and achieve the effect of being beneficial to purification

Inactive Publication Date: 2011-07-20
SHAN DONG DONG E E JIAO +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Pichia pastoris is mainly secreted expression (Dhanabal, M., Volk, R., Ramchandran, R., Simons, M., and Sukhatme, V.P.1999. Cloning, expression, and in vitroactivity of human endostatin.Biochem. Biophy.Res.Comm.258, 345-352), it is easy to obtain recombinant human endostatin with biological activity, but its production cycle is long, the process is complicated, the cost is high, and the yield is not high, and the expressed exogenous The source protein is prone to partial degradation. For example, Boehm et al. successfully expressed soluble mouse endostatin in a yeast expression system, but the N-terminus of the obtained product was not uniform. The analysis may be caused by protease degradation in yeast (Boehm, T., Pirle-Shephere, S., Trinh, L., Shiloach, J., and Folkman, J.1999. Disruption of the KEX1 gene in Pichia pastoris allows expression of full-length murine and human endostatin. Yeast15, 563-572)
Escherichia coli has the advantages of fast growth, easy control of fermentation conditions, and high expression of foreign proteins. However, the foreign protein expressed in E. coli is easy to form inclusion bodies, and renaturation is required during the purification process, and the renaturation rate tend to be lower
In order to overcome the above-mentioned technical problems, people try to improve the method of preparing recombinant human endostatin from the following two aspects: one is to develop and improve the purification and renaturation method of recombinant endostatin so that it can be obtained from inclusion bodies Obtaining more active proteins (You, W.K., So, S.H., Lee, H., Park, S.Y., Yoon, M.R., Chang, S.I., Kim, H.K., Joe, Y.A., Hong, Y.K., and Chung, S.I. 1999. Purification and characterization of recombinant murine endostatin in E.coli.Exp.Mol.Med.31, 197-202; Violand, B.N., and Harding, E.I.2003.Method of producing mouse and human endostatin.United States Patent 6,653,098), However, due to the complexity of inclusion body renaturation, the activity recovery rate of endostatin is often very low; the second is to increase the soluble expression of recombinant human endostatin in E. coli by changing the cloning strategy and optimizing the culture conditions of the expression strain Quantity (Xu, R., Du, P., Fan, J.J., Zhang, Q., Li, T.P., and Gan R.B. 2002. High-level expression and secretion of recombinant mouse endostatin by Escherichia coli. Protein Expression Purif24, 453-459 ; Zhu Minsheng, Zhigang, Zhu Nianchun, Zhiqiang, Chen Xiaoming, High-efficiency expression method of recombinant human endostatin, CN03155724.4, public date: April 21, 2004)
Chinese patent application CN03155724 discloses a method for high-efficiency expression of human endostatin gene in Escherichia coli. This method mutates the coding sequence of the N-terminal 4 amino acid residues without changing the amino acid sequence of endostatin. The codon preferred by Escherichia coli was used to clone the mutated recombinant human endostatin gene into an expression vector, and lactose was used to induce the expression of recombinant human endostatin. The expression amount could reach 40% of the total bacterial protein, but the expression The product exists in the form of inclusion bodies, and the active human endostatin still has to go through a complex denaturation process

Method used

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  • Recombination human endothelium chalene expression strain and solubility expression method
  • Recombination human endothelium chalene expression strain and solubility expression method
  • Recombination human endothelium chalene expression strain and solubility expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of Recombinant Human Endostatin Expression Plasmid pET-Endo

[0036] According to the gene sequence of human endostatin, the N-terminal and C-terminal primer sequences were synthesized, and the primer sequences were as follows:

[0037] Primer 1: 5'-GCATAGCCATCGTGATTTC-3' (SEQ ID NO: 2) and

[0038] Primer 2: 5'-CGGGATCCCTACTTGGAGGCAGTCAT-3' (SEQ ID NO: 3)

[0039] Using the plasmid pMD-Endo as a template, using the above two primers, the human endostatin gene was amplified by the PCR method, and analyzed by agarose electrophoresis, the amplified fragment was about 550bp ( figure 1 ), which is consistent with the expected. The PCR fragment and the vector pET43.1a were digested with PshAI and BamHI respectively, and ligated using TaKaRa DNA Ligation Kit (TaKaRa DNA Ligation Kit) to obtain the recombinant human endostatin expression vector pET-Endo.

[0040] The above-mentioned vectors were transformed into Escherichia coli Top10 (commercial str...

Embodiment 2

[0041] Example 2 Construction and expression of recombinant human endostatin expression strain

[0042] The recombinant plasmid pET-Endo was transformed into competent Escherichia coli Origami (DE3) by the heat shock method (the operation was the same as in Example 1). On the second day, single clones were selected, transferred to LB culture medium containing 100 mg / L ampicillin, cultured overnight at 37°C with shaking, and plasmids were extracted, identified by restriction enzyme digestion, and induced to express. Cultivate the monoclonal E.coli Origami-Endo containing the expression plasmid in LB medium (containing 100mg / L ampicillin) at 37°C until OD 600 was about 0.5, added IPTG with a final concentration of 1mmol / L to induce expression for 6h, collected the bacteria by centrifugation, and after breaking the bacteria by ultrasonic, carried out SDS-PAGE analysis on the whole bacteria, supernatant and precipitated (inclusion body) proteins respectively, SDS- The concentrati...

Embodiment 3

[0044] Embodiment 3 The influence of temperature on the soluble expression of recombinant human endostatin

[0045] The expression strain E. coli Origami-Endo was inoculated in 5 ml of LB culture medium containing 100 mg / L ampicillin (Amp). In the bacterial incubator, 37°C, 250r / min shaking culture for 15h, then transfer the bacterial solution to LB culture solution containing 100mg / LAmp at a ratio of 1:100, 37°C, 250r / min shaking culture for 3-4h, Make bacterial solution OD 600At about 0.5, add IPTG with a final concentration of 1.0mmol / L to the bacterial solution to induce expression, and the induction temperature is 25°C, 30°C and 37°C, respectively. After 6 hours of induction and expression, collect the bacterial cells, and break the bacteria by ultrasonic. The supernatant and precipitated (inclusion body) proteins were analyzed by SDS-PAGE to detect the soluble expression effect of recombinant human endostatin. After automatic gel scanning density quantitative analysis,...

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Abstract

The invention relates to a method to rebuilding human vivo skin chalone strain and the solubility expression that belongs to gene engineering field. The strain contains expression carrier pET-Endo that could be gained by inserting the human vivo skin chalone into carrier pET43.1a. For cultivating the strain, it is induced by using IPTG at the concentration of 0.1-1.0mmol / L and the temperature in 25-30 degree centigrade, and for 6-10 hours time period; the external albumen solubility expression quantity could reach over 80% of the total expression quantity. And the total expression quantity isabout 50% of the thallus total albumen quantity. Thus, the invention conquers the disadvantage of expressing the human vivo by inclusion body mode. It could be used to rebuild the human vivo skin chalone and the other solubility expression of rebuilding albumen.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant human endostatin expression strain and a method for its soluble expression. technical background [0002] Endostatin (Endostatin) was discovered by O'Reilly et al in 1997 in the cell culture medium of murine hemangioendothelial cell tumor (EOMA) cultured in vitro (O'Reilly, M.S., Boehm, T., and Folkman, J.1997 . Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell88, 277-285). It can inhibit the proliferation of bovine capillary endothelial cells in vitro, and has a molecular weight of 20kDa. Sequence analysis shows that it is a C-terminal fragment of collagen 18 (collagenXVIII). Human endostatin has a molecular weight of 18kDa. Because endostatin can inhibit the growth of vascular endothelial cells, thereby inhibiting the growth of new blood vessels, preventing tumor growth and metastasis, and repeated use without drug resistance (Boehm, T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/70C12N1/21C12N15/12C12R1/19
Inventor 杜翠红张元兴庞甲佩尤金花章安
Owner SHAN DONG DONG E E JIAO
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