Chlamydomonas reinhardtii Se1K expression vector as well as construction method and expression method thereof

An expression vector and construction method technology, applied in the field of genetic engineering, can solve the problems of unsatisfactory, single, unable to achieve large-scale expression of selenoprotein, and achieve the effects of good singleness and directionality, and improved efficiency

Inactive Publication Date: 2015-04-29
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Application Information

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Problems solved by technology

However, the production efficiency of selenoproteins is very low, and it is impossible to achieve large-scale expression of selenoproteins, nor can it be purified to protein
Therefore, the in vitro expression and synthesis of selenoproteins cannot meet the single, efficient, and large-scale application requirements.

Method used

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  • Chlamydomonas reinhardtii Se1K expression vector as well as construction method and expression method thereof
  • Chlamydomonas reinhardtii Se1K expression vector as well as construction method and expression method thereof
  • Chlamydomonas reinhardtii Se1K expression vector as well as construction method and expression method thereof

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Experimental program
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Embodiment 1

[0065] S10, using the human fetal brain library (stored in the laboratory) as a template, carry out PCR amplification through upstream primer F1 and downstream primer R1, and amplify the ORF fragment (301bp) of human SelK;

[0066]

[0067] The PCR reaction conditions were pre-denaturation at 94°C for 5min; 94°C for 30s; 55°C for 30s; 72°C for 40sec; and 72°C extension for 7min after 30 cycles. Then all PCR amplification products were taken for agarose gel electrophoresis. PCR product gel recovery;

[0068] The PCR product electrophoresis result of the ORF fragment of human SelK is as follows image 3 As shown, wherein M is the standard molecular weight of DL2000, and lanes 1 and 2 are the RT-PCR amplification products of the ORF fragment of human SelK.

[0069] S20, amplifying the SECIS gene fragment of Chlamydomonas reinhardtii SelW1, specifically according to the following steps:

[0070] S21, extract total RNA from Chlamydomonas reinhardtii, inoculate cell wall-defec...

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Abstract

The invention provides a construction method of a chlamydomonas reinhardtii Se1K expression vector. The construction method comprises the following steps: amplifying an ORF gene segment of human Se1K to obtain an ORF gene amplification product; amplifying an SECIS gene segment of chlamydomonas reinhardtii Se1W1 to obtain an SECIS gene amplification product; recombining the ORF gene amplification product with the SECIS gene amplification product to obtain a recombinant Se1K gene; and introducing the recombinant Se1K gene into a plasmid vector which can be expressed in the chlamydomonas reinhardtii to form a recombinant plasmid product. According to the chlamydomonas reinhardtii Se1K expression vector constructed by the method, a novel plasmid vector capable of expressing an exogenous target selenoprotein K in the chlamydomonas reinhardtii is constructed; compared with the existing in-vitro expression vectors such as a prokaryotic expression system, and a method, the product is generated by a similar expression mechanism, so that the product has good monotonicity and directionality; the efficiency is greatly improved; the chlamydomonas reinhardtii can be adopted as a reactor for producing selenoprotein during expression; and the construction method is portable and efficient, and can completely meet the bases of research on structure functions of the selenoprotein and development of selenoprotein anti-cancer drugs.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Chlamydomonas reinhardtii SelK expression vector, a construction method and an expression method thereof. Background technique [0002] As an essential trace element for humans and animals, selenium plays an important role in many physiological functions and is closely related to many diseases, such as cancer, neurodegenerative diseases and cardiovascular diseases. Selenium mainly exerts biological functions through various selenoproteins. For example, among the selenoproteins discovered so far, SelK selenoprotein participates in coping with oxidative stress in cardiomyocytes and in regulating endoplasmic reticulum stress in human liver cancer cells. Promote calcium flow in the process of immune cell activation mediated by body; participate in the degradation of endoplasmic reticulum-related proteins, participate in the regulation of CD36 palmitoylation ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/66C12R1/89
Inventor 张仁利李瑞敏
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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