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Pichia pastoris strain for expressing osteopontin

A Pichia pastoris, osteopontin technology, applied in fungi, microorganism-based methods, polypeptides containing localization/targeting motifs, etc.

Pending Publication Date: 2022-06-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is difficult to achieve large-scale marketization of OPN through fresh milk extraction, and the cost is high

Method used

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  • Pichia pastoris strain for expressing osteopontin
  • Pichia pastoris strain for expressing osteopontin
  • Pichia pastoris strain for expressing osteopontin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of embodiment 1 expression plasmid

[0034] (1) Using the artificially synthesized OPN gene as a template, use the primers OPN-F and OPN-R in Table 1 to amplify osteopontin, use the plasmid pPICZαA stored in the laboratory as a template, and use the primers aOPN- The α-signal peptide was amplified from Z-F and aOPN-Z-R. The amplified osteopontin and α-signal peptide were connected to the pPICZαA plasmid digested with EcoRI and NotI to obtain the recombinant plasmid pPICZαA-αfactor-OPN. Plasmid map such as figure 1 shown.

[0035] The PCR system is:

[0036] Table 1 PCR reaction system

[0037]

[0038]

[0039] The PCR conditions are:

[0040] Table 2 PCR amplification conditions

[0041]

[0042] Design the pPICZαA-αfactor-OPN recombinant plasmid, the plasmid map and primer sequences are as follows figure 1 and shown in Table 3.

[0043] Table 3 pPICZαA-αfactor-OPN primer sequence

[0044]

Embodiment 2

[0045] The construction of embodiment 2 recombinant Pichia pastoris

[0046]The recombinant plasmid pPICZαA-αfactor-OPN in Example 1 was linearized with Dra I, and 80 μL of Pichia pastoris X33 competent cells were mixed with 10 μL of the linearized recombinant plasmid, and transferred to a 0.2 cm electrotransformation cup, and ice-bath the electrotransformation cup containing the mixed solution for 5 min. Adjust the parameters of the electrotransformer, put it in the Pichia pastoris gear, the voltage is 1.5 kV, the capacitance is 25 microfarads, the resistance is 200 ohms, and the time is about 5 milliseconds. After the electric shock, quickly add 1mL of 1M sorbitol solution pre-cooled on ice to the transformation cup, gently pipette to mix, and transfer the mixture to a centrifuge tube. Incubate at 30°C for 1-2h, and centrifuge at 2000g for 5min. Discard 800 μL of the supernatant, blow the remaining bacteria evenly, spread it on a YPD plate, and culture it in a 30°C incubat...

Embodiment 3

[0047] Example 3 Fermentative production of osteopontin in recombinant Pichia pastoris

[0048] Pick a single colony from the YPD plate in Example 2 and inoculate it into a round-bottomed test tube containing 2mL of YPD medium for overnight culture. Take 1mL of the overnight culture and inoculate it into 25mL of BMGY medium for shake-flask fermentation at 30°C and 220rpm cultured to OD 600 At about 90, 4°C, 3500g, centrifuge for 10min, collect the cells, discard the supernatant, and wash twice with sterile water, centrifuge under the same conditions after each wash, and take the supernatant. The cells were resuspended in BMMY medium, and the expression was induced at 30°C and 220rpm, and methanol was added every 24h to a final concentration of 1%. After being induced by methanol for 7 days, the supernatant was collected by centrifugation, concentrated by centrifugation, and quantitatively analyzed for osteopontin by SDS-PAGE and Western Blot. The result is as figure 2 , ...

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Abstract

The invention discloses a pichia pastoris strain for expressing osteopontin, and belongs to the field of genetic engineering. According to the invention, an osteopontin expression cassette containing a PAOX1 promoter, an alpha signal peptide and osteopontin of which the amino acid sequence is as shown in SEQ ID NO.1 is successfully integrated on an AOX1 site on a pichia pastoris genome. Fermentation experiments find that the pichia pastoris genetic engineering strain provided by the invention can successfully express osteopontin and partially secrete the osteopontin out of cells, but the protein secretion is delayed in the later period of fermentation, and most of the protein enters vacuoles. Through Western Blot verification and ELISA quantitative determination, it is determined that successful expression of the osteopontin is achieved, and the expression quantity of the osteopontin reaches 32.2 mg / L.

Description

technical field [0001] The invention relates to a Pichia yeast strain expressing osteopontin, which belongs to the field of genetic engineering. Background technique [0002] Osteopontin (OPN), also known as sialoglycoprotein, is a highly phosphorylated acidic protein with O-glycosylation modification, which can be synthesized or secreted by various tissue cells in the body. Therefore, it was initially considered that OPN is an important bone matrix protein, which is closely related to the formation and development of bone. In recent years, studies have found that OPN is very high in breast milk, especially colostrum, and is a very important type of immune active protein in breast milk. There are significant differences in the OPN content of breast milk among different species. For example, the OPN content in bovine milk is about 18 mg / L, while the OPN concentration in human milk is as high as 138 mg / L. At the same time, breast milk OPN also shows obvious regional differen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/62C12R1/84
CPCC12N15/815C07K14/47C07K2319/02
Inventor 刘龙陈坚吕雪芹堵国成李江华刘延峰张智航陶孟瑞孙丹
Owner JIANGNAN UNIV
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