Method for constructing secretory expression carrier and secretory expression system of Chlammydomonas reinhardtii

An expression vector and construction method technology, applied in the biological field, can solve the problems of no Chlamydomonas reinhardtii, no Chlamydomonas reinhardtii secreted and expressed exogenous gene construction method and application, etc. Effect

Active Publication Date: 2011-09-14
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no Chlamydomonas reinhardtii secreted expression vector on the domestic market, and there is no construction method and application for Chlamydomonas reinhardtii to secrete and express foreign genes

Method used

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  • Method for constructing secretory expression carrier and secretory expression system of Chlammydomonas reinhardtii
  • Method for constructing secretory expression carrier and secretory expression system of Chlammydomonas reinhardtii
  • Method for constructing secretory expression carrier and secretory expression system of Chlammydomonas reinhardtii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Embodiment 1: Construction of the expression vector pHsg1 of the present invention

[0085] According to the known amino acid sequence of Chlamydomonas reinhardtii PHOX protein (GenBank accession number: XP_001703098.1), using the signal peptide analysis software SignalP V3.0, the signal peptide that can guide the secretion of synthesized protein from the nucleus to the outside of the cell was predicted. According to the nucleotide sequence of the predicted signal peptide, add upstream Nhe I restriction enzyme site, the nucleotide sequence point mutation is carried out downstream to form Tsp The E I enzyme cutting site was sent to Shanghai Biological Engineering Co., Ltd. for synthesis by artificial synthesis, and the nucleotide sequence was named as Sg1 , signal peptide gene Sg1 After synthesis, it was cloned into the commercial vector pUC57, and the insertion site was Sma I, the constructed vector containing the sequence was named pUCSg1. Sg1 The signal pe...

Embodiment 2

[0089] Embodiment 2: Construction of the expression vector pHsg2 of the present invention

[0090] In order to facilitate the connection of the target gene and the secreted Chlamydomonas reinhardtii expression vector, the signal peptide gene Sg1 Based on the addition of Bgl II. Afe I. Age I. Mlu I. Pmac I restriction enzyme cutting site, sent to Shanghai Bioengineering Co., Ltd. to synthesize by artificial synthesis, the nucleotide sequence named Sg2 , signal peptide Sg2 After synthesis, it was cloned into the commercial vector pUC57, and the insertion site was Sma I, the constructed vector containing the sequence was named pUCSg2. Sg2 The signal peptide gene sequence is shown in SEQ ID NO.3.

[0091] restriction endonuclease Nhe I digested plasmid pUCSg2, recovered it with TaKaRa Fragment Purification Kit kit, and then used Sma I digested the enzyme cutting site, cut the gel to recover the Sg2 fragment (144bp), and inserted it into the Nhe I and ...

Embodiment example 3

[0092] Example 3: Construction of the expression vector pHSgFe-1 of the present invention

[0093] According to the known amino acid sequence of Chlamydomonas reinhardtii Fe-assimilating protein 2 (GenBank accession number: ABB04462.1), using the signal peptide analysis software SignalP V3.0, the signal peptide that can guide the secretion of the synthesized protein from the nucleus to the outside of the cell was predicted. According to the nucleotide sequence of the predicted signal peptide, add upstream Nhe I restriction enzyme site, downstream addition Bgl II. Pst I. Afe I. Age I. Mlu I. Pmac The I restriction enzyme cutting site was sent to Shanghai Bioengineering Co., Ltd. for synthesis by artificial synthesis. The nucleotide sequence was named SgFe. After the signal peptide gene SgFe was synthesized, it was cloned into the commercial vector pUC57. The insertion site was Sma I. The constructed vector containing this sequence was named pUCSgFe. The seque...

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Abstract

The invention discloses a method for constructing a secretory expression carrier and secretory expression system of Chlammydomonas reinhardtii. In the invention, the method comprises the steps of predicating a signal peptide gene sequence by using an amino acid sequence from a secretory protein of the Chlammydomonas reinhardtii through signal peptide predicating software; artificially synthesizing a signal peptide gene obtained by predicating; and inserting the signal peptide gene between a promoter and a cloning site of the Chlammydomonas reinhardtii expression carrier to construct the secretory expression carrier of the Chlammydomonas reinhardtii; meanwhile, detecting the expression efficiency of the constructed secretory expression carrier of the Chlammydomonas reinhardtii by using an exogenous gene; converting the secretory expression carrier of the Chlammydomonas reinhardtii, containing the exogenous gene, to the Chlammydomonas reinhardtii by using a Chlammydomonas reinhardtii genetic conversion method; and screening the Chlammydomonas reinhardtii to obtain a transgene engineering strain capable of expressing the exogenous gene in a secretory way. By using the invention, the basis is laid for using the Chlammydomonas reinhardtii as the secretory expression exogenous gene of a bioreactor.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a Chlamydomonas reinhardtii secreted expression vector and a secreted expression system. Background technique [0002] Chlamydomonas reinhardtii is a single-celled eukaryotic alga belonging to the genus Chlamydomonas ( Chlamydomonas ). The individuals of Chlamydomonas reinhardtii are oval, spherical or elliptical, with a slightly pointed front and two equal-length flagella on both sides, which can swim freely. Generally, the diameter of Chlamydomonas individuals of the spherical type is 5-17mm, and the length of the oval type is 6-27mm, and the width is 27mm. Chlamydomonas reinhardtii can grow both photoautotrophically and heterotrophically depending on acetate. The growth rate is fast, the cell number doubling time is 5-6 hours, and a large number of genetic progeny can be obtained in a short time for genetic analysis. At present, the sequencing of its ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12R1/89
Inventor 胡章立吴锦霞陈俊
Owner SHENZHEN UNIV
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