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Molecular chaperone expression vector and strain for improving secretory expression of phytase in pichia pastoris

A molecular chaperone and expression vector technology, applied in the field of genetic engineering, can solve the problems of insignificant, increased expression, and not significantly increased expression of mannanase, and achieves the effect of overcoming poor effect

Pending Publication Date: 2021-10-22
GUANGDONG VTR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, in the literature reports, the target protein is mainly co-expressed with a single molecular chaperone, and very few are co-expressed with multiple molecular chaperones at the same time, and the expression level is not significantly increased.
Min Qi et al. combined HACI with different molecular chaperones in a simple series and co-expressed them with mannanase. They found that the combination of these molecular chaperones did not significantly increase the expression of mannanase, and the combination of molecular chaperones did not significantly increase the expression of mannanase. The effect is worse than that of a single HACI

Method used

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  • Molecular chaperone expression vector and strain for improving secretory expression of phytase in pichia pastoris
  • Molecular chaperone expression vector and strain for improving secretory expression of phytase in pichia pastoris
  • Molecular chaperone expression vector and strain for improving secretory expression of phytase in pichia pastoris

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Embodiment 1

[0049] The construction of embodiment 1 VectorB, VectorV, VectorH vector

[0050] 1. Construction of VectorB vector

[0051] Using the Pichiapastoris X33 genome and the pPIC9k plasmid as templates, two pairs of primers were designed to amplify the two fragments of the BIP gene and the pPIC9k vector by PCR, and a fusion region was introduced into the primers. Fragment fusion, such as figure 1 As shown, a VectorB circular plasmid was constructed, and the fusion circular plasmid was transformed into E. coli Topl0 competent cells. The recombinant transformants were screened on a plate containing Amp (100 μg / mL) antibiotics, and part of the transformants were picked and transferred to LB+Amp liquid medium Incubate at 37°C for 3 hours, use the verification primers BIP-F11 and BIP-R11 to carry out bacterial liquid PCR verification, the fragment size is about 2kb, and the transformants are sent for sequencing. The construction method of VectorV and VectorH refers to VectorB, and the ...

Embodiment 2

[0093] Application of embodiment 2 vectors VectorBP, VectorVP, VectorHP in Pichia pastoris secreting and expressing thermostable phytase

[0094] (1) Expression of VectorBP, VectorVP, and VectorHP in thermostable phytase expression host V1

[0095] Digest VectorBP, VectorVP, VectorHP, VectorB, VectorV, VectorH, and VectorP vectors into linearized DNA fragments with restriction endonucleases, respectively purify VectorBP, VectorVP, VectorHP, VectorB, VectorV, VectorH, VectorP linearized DNA fragments with purification kits For DNA fragments, the linear DNA fragments of VectorBP, VectorVP, VectorHP, VectorB, VectorV, VectorH, and VectorP were respectively transformed into V1 competent cells containing the high-temperature-resistant phytase gene by electroporation, and V11, V12, and V13 were respectively constructed , V14, V15, V16, V17 high-temperature-resistant phytase expression strains, spread on YPD+G418 plate, culture upside down at 30°C for 3-4 days, pick about 300 single ...

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Abstract

The invention relates to the field of gene engineering, in particular to a molecular chaperone expression vector and a strain for improving secretory expression of phytase in pichia pastoris. The expression vector comprises molecular chaperone genes BIP and PDI which are connected in series, or molecular chaperone genes VHB and PDI which are connected in series, or molecular chaperone genes HACI and PDI which are connected in series. A high-temperature-resistant phytase high-enzyme-activity expression strain V28-VTR-001 which is obtained through genetic engineering modification and screening and contains the expression vector is recombined pichia pastoris, and the total fermentation enzyme activity of 50 L of the recombined pichia pastoris reaches 37239 U / mL.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a molecular chaperone expression vector and bacterial strain for improving the secretion and expression of phytase in Pichia pastoris. Background technique [0002] Crops such as cereals and legumes are the main raw materials for food and animal feed. 50% to 70% of the phosphorus in the raw material exists in the form of phytate phosphorus. Monogastric animals lack the necessary enzymes to decompose phytate phosphorus, and the utilization rate of phosphorus is low, resulting in a large amount of waste and pollution of phosphorus. [0003] Phytase can hydrolyze phytic acid into inositol and phosphoric acid. Adding phytase to feed can not only improve the utilization rate of phosphorus in feed, reduce the pollution of phosphorus in manure to the environment, but also reduce the anti-nutritional effect of phytate. It is an essential feed additive to increase the animal's ability...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/67C12N15/61C12N15/55C12N15/31C12N1/19C12N9/16C12R1/84
CPCC12N15/815C12N9/90C12N9/14C07K14/805C12N9/16C12Y301/03008C12Y301/03026C12Y503/04001C12Y306/01003
Inventor 江民华李阳源黄江何小梅边叶雨贺金玲陈丽芝陈琼银黄佳乐
Owner GUANGDONG VTR BIO TECH
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