Expression equipment for secretory expression of foreign protein by penicillium expansum and genetically engineered bacterium thereof

A technology of genetically engineered bacteria and Penicillium sp. is applied in the field of expression equipment for exogenous protein secreted and expressed by Penicillium sp. and the field of genetically engineered bacteria. Time, strong activity, the effect of optimizing secretion expression ability

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

[0004] The object of the present invention is to aim at the shortage of low expression of exogenous protein and difficulty in separation and purification, to provide a kind of expression equipment and its genetic engineering bacteria which are easy and quick to operate and secrete and express exogenous protein from Penicillium expansum

Method used

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  • Expression equipment for secretory expression of foreign protein by penicillium expansum and genetically engineered bacterium thereof
  • Expression equipment for secretory expression of foreign protein by penicillium expansum and genetically engineered bacterium thereof
  • Expression equipment for secretory expression of foreign protein by penicillium expansum and genetically engineered bacterium thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, Preparation of Extended Penicillium Heterologous Expression Equipment

[0065] (1) Use the restriction endonuclease Sac I, Sal I to convert the hygromycin resistance expression cassette from the 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.

[0066] (2) Take 0.2 g of Penicillium extensa mycelium, grind it with liquid nitrogen, add 1 mL of CTAB extract, bathe in 65 ° C for 30 min, centrifuge at 12000 rpm / min for 10 min, take the supernatant; a...

Embodiment 2

[0081] Embodiment 2, the acquisition of expanding Penicillium genetically engineered bacteria

[0082] The detailed steps are as follows:

[0083] (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.

[0084] (2) The final vector pCHAMBIA2300::P containing in Example 1 lip -Sig-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 ...

Embodiment 3

[0086] Example 3. Production of GUS protein using Penicillium expanses expression equipment

[0087] 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 mycelium. 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 observed the staining situation. See Figure 4 , Figure 4 Shows transfer into pCHAMBIA2300::P lip -Sig-GUS-T trpC The dye solution of Penicillium extensa genetically engineered bacteria showed a remarkable blue color ( Figure 5 B), while the color of the stain solution where the wild-type Penicillium expanses strain was located did not change ( Figure 5 A); The results show that after using the expression equipment described in the present invention, the foreign protein gene GUS is highly exp...

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Abstract

The invention discloses expression equipment for secretory expression of a foreign protein by penicillium expansum and a genetically engineered bacterium thereof. The expression equipment sequentially comprises the following members from 5' to 3': (1) a lipase gene promoter of penicillium expansum; (2) a signal peptide of secretory expression; (3) multiple cloning sites; (4) a terminator of a gene C of aspergillus nidulans tryptophan synthetase. By inserting the foreign gene into the expression equipment, converting into agrobacterium tumefaciens by T-DNA (Transferred-Deoxyribonucleic Acid) and combining with penicillium expansum, the genetically engineered bacterium of penicillium expansum obtained can efficiently secretory-express heterologous genes originated from animals, plants and microorganisms, so that the foreign protein obtained can be produced on a large scale. The penicillium expansum is exuberant to grow, the condition of culture is extensive and cheap, both solid culture and liquid submerged fermentation are feasible, and the extracelluar protein produced is easy to separate and purify, so that the expression equipment is suitable for producing proteins with great industrial demands.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an expression device for exogenous protein secreted and expressed by Penicillium expansum and its genetic engineering bacteria. 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. Filamentous fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N1/21C12N1/15C12P21/00C12N9/20C12N9/18C12N9/26C12N9/88C12N9/42A23K1/16C12R1/83
Inventor 唐克轩张田
Owner SHANGHAI JIAO TONG UNIV
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