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Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein

A kallikrein, recombinant vector technology, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc.

Inactive Publication Date: 2002-12-11
SHENZHEN PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no human pancreatic kallikrein product yet, and the research on the production of recombinant human pancreatic kallikrein by genetic engineering technology has not been reported yet

Method used

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  • Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein
  • Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein
  • Recombinant expression vector expressing human pancreatic tissue kallikrein gene and prepn of human pancreatic tissue kallikrein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Cloning and sequencing of human pancreatic tissue kallikrein gene

[0042] 1 Materials and methods

[0043] 1.1 Materials

[0044] Pancreatic tissue was collected from a part of fresh tissue after pancreatectomy, the blood was washed, and total RNA was extracted immediately.

[0045] 1.2 Plasmids and strains

[0046] Plasmid pBluescript II KS(+) and Escherichia coli XL1-Blue were respectively purchased from STRATAGENE (Jingmei Company).

[0047] 1.3 Tool enzymes and other reagents

[0048] RNA isolation kit and reverse transcription kit were purchased from STRATAGENE (Jingmei Company), restriction enzymes, modification enzymes, pfu high-fidelity Taq DNA polymerase, etc. were purchased from Promega, Gene Company and Shanghai Sangong respectively.

[0049] 1.4 PCR primer design and synthesis

[0050] According to the gene sequence reported by GENBANK, a pair of PCR primers were designed using a computer-aided system, synthesized by Shanghai Sangong and puri...

Embodiment 2

[0062] Example 2 Cloning of Human Pancreatic Tissue Kallikrein Mature Protein Gene

[0063] Using computer software to compare and analyze the amino acid sequences of human (pancreatic tissue), pig, rat, and mouse kallikrein (Figure 3), it is speculated that prokallikrein (the complete protein of kallikrein) is converted into kallikrein (kallikrein mature protein) possible protease cleavage site and the amino acid sequence of kallikrein mature protein, synthesize the PCR primers of the following sequence:

[0064] Upstream (eukaryotic cells): the sequence shown in SEQ ID NO.6;

[0065] Upstream (prokaryotic cells): the sequence shown in SEQ ID NO.7;

[0066] Downstream: the sequence shown in SEQ ID NO.8;

[0067] In the above primer design:

[0068] 1. An ATG is added in front of the mature protein for eukaryotic expression;

[0069] 2. There is no ATG in front of the mature protein, which is used for prokaryotic expression;

[0070] 3. Shared downstream primers;

[0071...

Embodiment 3

[0073] Example 3 Construction of expression vector

[0074] 1. Construction of human pancreatic tissue kallikrein mature protein secretory expression vector pE20mK

[0075] Figure 4 shows the prokaryotic secreted expression vector pE20mK of Chinese pancreatic tissue kallikrein gene. The specific method is to use high-fidelity Taq enzyme to amplify the mature kallikrein gene (SEQ ID NO.3, mKK) from the plasmid KSKK, digest it with restriction endonucleases EcoR I and XhoI, and insert it into the EcoR gene of plasmid pET20b. I. The XhoI site ensures that the reading frame of the kallikrein gene and His is correct, and constructs the secreted expression vector pE20pK. The vector can express tissue kallikrein mature protein in Escherichia coli, and a signal peptide sequence is fused at the N-terminus, so that the expressed target protein tends to distribute in the cytoplasm (periplasmic localization).

[0076] 2. Construction of non-secretory expression vector pE28pK of human pa...

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Abstract

The present invention relates to secretion or non-secretion recombinant vector carrying mature protein gene coding human pancreatic kallikrein and its transformed host cell. The present invnetion also relates to the gene engineering technology method of preparing recombinant human pancreatic kallikrein. The expression vector of the present invention can express effectively protein product with pancreatic kallikrein activity and has fused His affinity sequence and the expression product containing the affinity sequence. During separation and purification, His affinity resin may be used in chromatographic affinity purification. The present invention lays the foundation for developing humanized tissue kallikrein gene engineering product and medicine.

Description

technical field [0001] The invention relates to a recombinant expression carrier carrying human pancreatic kallikrein gene, in particular to a recombinant expression carrier carrying human pancreatic tissue kallikrein mature protein gene. [0002] The present invention also relates to host cells transformed by the recombinant expression vector. [0003] The invention also relates to a method for preparing recombinant kallikrein by applying genetic engineering technology. Background technique [0004] Human kallikreins fall into two major classes, plasma kallikreins and tissue kallikreins. Tissue kallikrein is a group of compounds with similar properties, which can be synthesized by liver, pancreas, cranial nerve, etc. The protein encoded by the tissue kallikrein gene is first expressed to generate a precursor intact protein, which becomes a biologically active mature protein after cleavage and processing by protease in vivo. The tissue kallikrein gene encodes tissue kalli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N9/48C12N15/57C12N15/62C12N15/63C12N15/70C12N15/866
Inventor 李体远戴勇杜珙黄瑞芳石之磷蔡筱彦肖德明
Owner SHENZHEN PEOPLES HOSPITAL
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