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Expression equipment and genetically engineered bacterium for expressing foreign proteins in penicillium expansum cells

A technology of genetically engineered bacteria and Penicillium expanded, applied in bacteria, biochemical equipment and methods, fungi, etc., can solve the lack of post-translational processing and folding mechanism, the difficulty of active proteins, and the complex renaturation of inclusion bodies, etc. problems, to achieve the effect of easy preservation and DNA manipulation, high matching degree and low cost

Inactive Publication Date: 2014-04-30
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prokaryotic expression system represented by Escherichia coli lacks post-translational processing and folding mechanisms; therefore, it is easy to produce inclusion bodies when highly expressing foreign proteins, and inclusion body renaturation is an extremely complicated and difficult process
Although the yeast expression system represented by Saccharomyces cerevisiae can translate and fold correctly when expressing foreign proteins, it is prone to excessive glycosylation, so it is difficult to produce active proteins when expressing higher eukaryotic genes

Method used

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  • Expression equipment and genetically engineered bacterium for expressing foreign proteins in penicillium expansum cells
  • Expression equipment and genetically engineered bacterium for expressing foreign proteins in penicillium expansum cells
  • Expression equipment and genetically engineered bacterium for expressing foreign proteins in penicillium expansum cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1, Preparation of Extended Penicillium Heterologous Expression Equipment

[0062] (1) Utilize restriction endonuclease Sac 1, Sal I to convert hygromycin resistance expression cassette from plasmid pV2 (Wang Y, Guo B, Miao Z, Tang K. Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI). FEMS Microbiology letter, 2007, 253-259), the fragment was recovered by gel, and cloned into the corresponding restriction site of pCAMBIA2300 plasmid to obtain the recombinant plasmid pCHAMBIA2300 with hygromycin resistance; The hygromycin expression cassette can also be derived from any other DNA containing the sequence, wherein the nucleotide sequence of the hygromycin phosphotransferase gene is shown in SEQ ID NO:10.

[0063] (2) Take 0.2g of Penicillium expansica mycelium, grind it with liquid nitrogen, add 1mL of CTAB extract, bathe in water at 65°C for 30min, centrifuge at 12000rpm / min for 10min, take the supernatant; add 0...

Embodiment 2

[0078] Embodiment 2, the acquisition of expanding Penicillium genetically engineered bacteria

[0079] The detailed steps are as follows:

[0080] (1) Pick isolated and purified wild-type Penicillium expansica and inoculate it on a PDA plate, culture it at 28°C for about 20 days, and wash the mature spores with sterile water.

[0081] (2) the final vector pCHAMBIA2300 containing in Example 1:: P lip -GUS-T trpC The engineered Agrobacterium EHA105 was inoculated in LB liquid medium containing 100 μg / mL streptomycin and 100 μg / mL kanamycin at 28°C and 200 rpm for overnight culture, and was cultured overnight with 100 μg / mL streptomycin and 100 μg / mL kanamycin The MM culture medium was reactivated, and cultured at 28°C and 220rpm for 48 hours. Draw an appropriate amount of culture and centrifuge at 5000rpm to remove the supernatant, wash with 1M liquid medium, and finally dilute to OD with 1M liquid medium 600 =0.15, then cultured at 28°C, 220rpm for 6-8 hours, until OD 60...

Embodiment 3

[0083] Example 3, Production of GUS protein using Penicillium expanses expression equipment

[0084] Inoculate the genetically engineered Penicillium extensa and the wild-type Penicillium expanse obtained in Example 2 into GM medium respectively, shake and culture at 220 rpm and 26° C. for 2 days, and then filter and recover mycelia. The mycelium was washed three times with distilled water, then put into an eppendorf tube, added GUS dye solution, incubated at 37°C for 3-5h, and the staining of the mycelium was observed. See Figure 4 , Figure 4 Shows transferred into pCHAMBIA2300::P lip -GUS-T trpC The genetic engineering strain of Penicillium expanses presents a remarkable blue color ( Figure 4 II, III, IV in ), while the wild-type Penicillium expanses strain was not stained ( Figure 4 in I). The results show that after using the expression equipment of the present invention, the exogenous protein gene GUS can be highly expressed in the Penicillium expanses cells. ...

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Abstract

The invention discloses expression equipment and genetically engineered bacterium for expressing foreign proteins in penicillium expansum cells. The expression equipment comprises the following components from 5' to 3' in sequence: (a) a lipase gene promoter of penicillium expansum; (b) multiple cloning sites; (c) a terminator of an aspergillus nidulans tryptophan synthetase C gene. The penicillium expansum genetically engineered bacterium obtained by inserting the foreign genes into the expression equipment, transforming agrobacterium tumefaciens with T-DNA binary vectors and combining obtained agrobacterium tumefaciens transformants with penicillium expansum can be used for efficiently expressing the foreign proteins derived from animals, plants, microorganisms and the like to produce plenty of foreign proteins from the foreign proteins. Penicillium expansum grows vigorously, is extensive and cheap in culture conditions, can be subjected to solid culture and submerged fermentation, and is suitable for producing proteins in large industrial demand.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression device and a genetically engineered bacterium for expressing foreign proteins in Penicillium expanses cells. Background technique [0002] Filamentous fungal expression systems have unique advantages over prokaryotic and yeast expression systems in terms of protein recombinant expression. The prokaryotic expression system represented by Escherichia coli lacks post-translational processing and folding mechanisms; therefore, it is easy to produce inclusion bodies when highly expressing foreign proteins, and inclusion body renaturation is an extremely complicated and difficult process. Although the yeast expression system represented by Saccharomyces cerevisiae can translate and fold correctly when expressing foreign proteins, it is prone to excessive glycosylation, so it is difficult to produce active proteins when expressing higher eukaryotic genes. Filament...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N1/21C12N1/15C12P21/00C12R1/83
Inventor 唐克轩张田
Owner SHANGHAI JIAO TONG UNIV
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