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Preparation method of recombinant human plasminogen Kringle 5(hk5)

A technology of human plasminogen and plasminogen, applied in the field of genetic engineering, can solve problems such as unsuitable for industrialization, inactivity, and low expression

Inactive Publication Date: 2009-08-19
SHANGHAI NEWSUMMIT BIOPHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, Escherichia coli is mostly used to express hK5 at home and abroad, and the expression product is either inactive and requires complex denaturation process to obtain a pure product; or the expression level is low, which is not suitable for industrialization

Method used

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  • Preparation method of recombinant human plasminogen Kringle 5(hk5)
  • Preparation method of recombinant human plasminogen Kringle 5(hk5)
  • Preparation method of recombinant human plasminogen Kringle 5(hk5)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Construction of hK5 secretory expression system

[0091] 1. Acquisition of α-K5 fusion gene

[0092] According to the amino acid sequence of natural human plasminogen Kringle 5, and according to the principles of Pichia pastoris codon preference, the target gene was designed and synthesized. Its sequence is the DNA fragment shown in SEQ ID NO:1. The EcoRI site of pThioHisA (purchased from Invitrogen) was inserted to construct the plasmid pThioHisA-hK5 containing hK5 coding sequence. Using the plasmid pThioHisA-hK5 as a template, PCR amplification was performed with the following primers:

[0093] Primer2a: 5'-CCG CTCGAG AAAAGAGTTTTGTTGCCAGACGTTG-3' (SEQ ID NO: 4)

[0094] Primer2b: 5'-CG GAATTCCTGCAG TTAGAAAGAAGGAGCAGCACAT-3' (SEQ ID NO: 5)

[0095] Thus, the K5 gene fused with the leader peptide sequence of Saccharomyces cerevisiae α-factor in the correct frame was obtained.

[0096] At the same time, using the plasmid pPIC9K (purchased from Invitrogen) contain...

Embodiment 2

[0104] Transformation of recombinant plasmids

[0105] The constructed recombinant plasmids pPIC9K-α-K5, pPIC9K-2α-K5, pPIC9K-4α-K5, and pPIC9K-8α-K5 were digested with SalI and linearized, respectively, and transformed into Pichia pastoris GS115 (purchased from Invitrogen 72 hours after the company) cells were coated on the MD plate, the largest colony was inoculated on the YPD plate containing 0.25mg / ml G418, cultivated at 30°C for 48 hours, and then the colonies grown on the plate were treated with 2.0mg / ml G418 Highly resistant engineered bacteria were obtained through YPD plate screening. A batch of highly resistant engineering strains were obtained, and several high-efficiency expressing strains were obtained through expression screening in test tubes.

[0106] Test results: pPIC9K-α-K5, pPIC9K-2α-K5, and pPIC9K-4α-K5 three transformation groups obtained several clones that could grow on the YPD plate of 0.25mg / mlG418, pPIC9K-8α-K5 transformation group Several clones w...

Embodiment 3

[0109] Obtaining high-expression engineered bacteria

[0110] Genomic DNA of different resistant engineering bacteria in Example 2 were extracted respectively, and hK5 gene probes were prepared, and the integrated copy number of hK5 was detected by Dot Blot assay, and positive clones with high copy number were screened.

[0111] test results:

[0112] The highly resistant engineering bacteria in the pPIC9K-8α-K5 transformation group had a relatively high copy number of exogenous genes (more than 30 copies), thereby obtaining engineering bacteria with high copy hK5 genes, while pPIC9K-α-K5, pPIC9K-2α- The clone copy numbers of K5 and pPIC9K-4α-K5 transformation groups were relatively low.

[0113] Small-shake expression detection of different resistant engineering bacteria: Inoculate monoclonal engineering bacteria into YPD medium, take supernatant after methanol induction for 48 hours, and compare gene expression levels of different resistant engineering bacteria.

[0114] t...

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Abstract

The invention provides an optimized coding sequence of recombinant human plasminogen Kringle 5 fragment (hK5) suitable for yeast expression, a method for efficiently producing rhK5, and related engineering cell construction, rhK5 expression and purification techniques. The optimized plasminogen Kringle 5 gene is very suitable for yeast expression (especially multi-copy expression), and has the characteristics of high expression, high stability and high secretion. The invention can obtain the pure product of rhK5 efficiently, conveniently and at low cost.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a production method of recombinant human plasminogen hK5 (HumanPlasminogen Kringle 5), as well as an expression vector and a host strain used in the method. Background technique [0002] Tumor is a common and frequently-occurring disease that poses a great threat to human life and health. About 5 million people die from cancer every year in the world. In some countries, cancer has become the second or even the first cause of death. [0003] Malignant tumor cells, including those of carcinomas and sarcomas, are commonly referred to as cancer cells. Cancer cells are transformed from corresponding normal tissue cells, which are different from normal cells and maintain some inherent characteristics of the source tissue cells to varying degrees. For most malignant tumors, it is easy to miss the opportunity of early detection. When a clinical diagnosis can be made, the cancer cell...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/58C12N15/63C12N1/19C12N9/68
Inventor 王军志袁力勇饶春明史新昌黄阳滨孙九如蒋剑祁晖
Owner SHANGHAI NEWSUMMIT BIOPHARMA
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