Plant expression vector utilizing beer yeast MF-alpha to mediate recombinant protein secretion

Abstract

The invention discloses a plant expression vector utilizing beer yeast MF-alpha to mediate recombinant protein secretion. The plant expression vector contains 35S constitutive promoter, beer yeast MF-alpha gene and exogenous gene. The MF-alpha-exogenous gene in the vector is expressed under control of the 35S constitutive promoter; after the plant expression vector is transferred into wild tobaccoand geranium through agrobacterium mediation, the exogenous gene under control of the 35S constitutive promoter can efficiently express protein coded by the exogenous gene and then secretes the protein out of plants. Experiment results show that yeat MF-alpha signal peptide mediates reporter gene Egfp protein to be secreted out of the plants; when metal sulfur protein MT3 gene of human is used toreplace Egfp gene, it is found that Cd2+ enrichment of transgenic plants is enhanced. Therefore, a genetic engineering operation method can be provided for further building MF-alpha to mediate otherrecombinant proteins to be secreted to the extracellular in the plants.

Description

technical field

[0001] The present invention relates to a plant expression vector that uses brewer's yeast MF-α to mediate the secretion of recombinant protein. The plant expression vector PK-35S-MF-α-Egfp is constructed through Gateway technology, so that the expressed recombinant protein can be secreted outside the plant, and belongs to plant molecules. field of genetic engineering. Background technique

[0002] The signal peptide that guides the secretion of newly synthesized proteins is a specific amino acid sequence present at the N-terminal of secreted proteins. Gunter Blobel put forward the signal peptide hypothesis in the 1970s, and believed that protein synthesis was carried out on ribosomes, and signal peptide, as a fragment of the protein, guided ribosomes to locate on the endoplasmic reticulum channel and pulled the synthesized polypeptide through the endoplasmic reticulum channel , and then the signal peptide is excised by peptidase, the protein is released int...

Examples

Embodiment 1

[0041] Example 1: Obtaining of exogenous α signal peptide and Egfp gene

[0042] The α signal peptide gene is derived from Saccharomyces cerevisiae MF-α, and the Egfp reporter gene is derived from the plant expression vector pKGWFS7,0.

Embodiment 2

[0043] Example 2: Construction of plant expression vector α signal peptide-Egfp

[0044] 1. The subcloning vector of PUC57-MF-α-Egfp synthesized by Shuoqing Biotechnology Co., Ltd.

[0045] Strategies for constructing plant expression vectors such as figure 1 As shown, look for the upstream of the Egfp reporter gene α signal peptide derived from Saccharomyces MF-α and the derived substance expression vector pKGWFS7,0 plus the enzyme cleavage site SalⅠ restriction site (GTCGAC), and the downstream of the Egfp reporter gene plus enzyme cleavage Site EcoRⅤ restriction site (GATATC). Send to Kunming Shuoqing Biological Company to synthesize the subcloning vector of PUC57-MF-α-Egfp, after the synthesis is successful. Plasmid DNA was extracted by alkaline lysis method, and the recombinant plasmid whose size was consistent with the theoretical value was selected by 1% agarose gel electrophoresis for further double enzyme digestion detection.

[0046] 2. Construction of the interm...

Embodiment 3

[0050] Example 3: Plant expression vector PK-35S-MF-α-Egfp transformed into Agrobacterium and screening of transgenic tobacco and geranium

[0051] Take a small amount of the correctly detected plant expression vector PK-35S-MF-α-Egfp plasmid and add it to the Agrobacterium competent cells, mix gently; add the mixture to the cold electroporation cup, tap the cup body to mix to the bottom of the cup; place the electric cup in the chute of the electrotransformer, and conduct electric shocks with the parameters of 200 ohms and 2.5kV / 0.2cm. Centrifuge at 200rpm at 28°C for 3-5h in a centrifuge tube; centrifuge at 7500rpm for 1min at room temperature, discard the supernatant to 100μL to suspend the cells; spread the bacteria on LB solid culture containing spectinomycin (Spe) antibiotic Cultured at 28°C on the base for 2 days; positive clones were selected and tested by bacterial liquid PCR (the primers used were the upstream primer of α signal peptide and the downstream primer of E...

technical field

[0001] The present invention relates to a plant expression vector that uses brewer's yeast MF-α to mediate the secretion of recombinant protein. The plant expression vector PK-35S-MF-α-Egfp is constructed through Gateway technology, so that the expressed recombinant protein can be secreted outside the plant, and belongs to plant molecules. field of genetic engineering. Background technique

[0002] The signal peptide that guides the secretion of newly synthesized proteins is a specific amino acid sequence present at the N-terminal of secreted proteins. Gunter Blobel put forward the signal peptide hypothesis in the 1970s, and believed that protein synthesis was carried out on ribosomes, and signal peptide, as a fragment of the protein, guided ribosomes to locate on the endoplasmic reticulum channel and pulled the synthesized polypeptide through the endoplasmic reticulum channel , and then the signal peptide is excised by peptidase, the protein is released int...

Examples

Embodiment 1

[0041] Example 1: Obtaining of exogenous α signal peptide and Egfp gene

[0042] The α signal peptide gene is derived from Saccharomyces cerevisiae MF-α, and the Egfp reporter gene is derived from the plant expression vector pKGWFS7,0.

Embodiment 2

[0043] Example 2: Construction of plant expression vector α signal peptide-Egfp

[0044] 1. The subcloning vector of PUC57-MF-α-Egfp synthesized by Shuoqing Biotechnology Co., Ltd.

[0045] Strategies for constructing plant expression vectors such as figure 1 As shown, look for the upstream of the Egfp reporter gene α signal peptide derived from Saccharomyces MF-α and the derived substance expression vector pKGWFS7,0 plus the enzyme cleavage site SalⅠ restriction site (GTCGAC), and the downstream of the Egfp reporter gene plus enzyme cleavage Site EcoRⅤ restriction site (GATATC). Send to Kunming Shuoqing Biological Company to synthesize the subcloning vector of PUC57-MF-α-Egfp, after the synthesis is successful. Plasmid DNA was extracted by alkaline lysis method, and the recombinant plasmid whose size was consistent with the theoretical value was selected by 1% agarose gel electrophoresis for further double enzyme digestion detection.

[0046] 2. Construction of the interm...

Embodiment 3

[0050] Example 3: Plant expression vector PK-35S-MF-α-Egfp transformed into Agrobacterium and screening of transgenic tobacco and geranium

[0051] Take a small amount of the correctly detected plant expression vector PK-35S-MF-α-Egfp plasmid and add it to the Agrobacterium competent cells, mix gently; add the mixture to the cold electroporation cup, tap the cup body to mix to the bottom of the cup; place the electric cup in the chute of the electrotransformer, and conduct electric shocks with the parameters of 200 ohms and 2.5kV / 0.2cm. Centrifuge at 200rpm at 28°C for 3-5h in a centrifuge tube; centrifuge at 7500rpm for 1min at room temperature, discard the supernatant to 100μL to suspend the cells; spread the bacteria on LB solid culture containing spectinomycin (Spe) antibiotic Cultured at 28°C on the base for 2 days; positive clones were selected and tested by bacterial liquid PCR (the primers used were the upstream primer of α signal peptide and the downstream primer of E...

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