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Method for producing pramlintide (PA) by virtue of bombyx mori bioreactor

A technology of Pramlintide and Bombyx mori, which is applied in the field of genetic engineering, can solve the problems of small molecular weight of Pramlintide and the difficulty of high-efficiency soluble expression

Inactive Publication Date: 2016-10-05
JINAN UNIVERSITY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a powerful means, genetic engineering has made it possible to obtain a large number of protein drugs that are difficult to extract or synthesize. However, due to the relatively small molecular weight of Pramlintide compared with conventional protein drugs, efficient soluble expression is difficult.

Method used

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  • Method for producing pramlintide (PA) by virtue of bombyx mori bioreactor
  • Method for producing pramlintide (PA) by virtue of bombyx mori bioreactor
  • Method for producing pramlintide (PA) by virtue of bombyx mori bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Recombinant engineering bacteria E.coli SW106-BmNPV-PAM-PA virus infection of silkworm cell BmN expression protein

[0040] The constructed transfer vectors pFBDM-PAM-IM and pUCDM-PA-IG were introduced into the asd auxotrophic host Escherichia coli SW106 by transformation. During the transformation process, the transfer vectors were transposed, and the target gene fragments were integrated into the viral genome. Constructed into the viral genome BmMultiBacNPV-PAM-IM-PA-IG, the recombinant genetic engineering bacteria E.coli SW106 containing the viral genome BmMulitBacNPV-PAM-IM-PA-IG, in LB-Kan-Tet-Spe-Gm-Cm- Cultivate overnight on DAP solid medium, pick a single colony and inoculate in LB-Kan-Tet-Spe-Gm-Cm-DAP liquid medium for 10-12h, and the OD value reaches 0.8, take 1ml of the bacterial solution into a 1.5ml EP tube, Centrifuge at 3000rpm for 3min, discard the supernatant, resuspend the bacterial solution in sterilized water, centrifuge at 3000rpm for 3m...

Embodiment 2

[0041] Example 2: Bombyx mori larvae were injected with recombinant baculovirus BmNPV-PAM-IM-PA-IG.

[0042] After the successful construction of the recombinant baculovirus BmNPV-PAM-IM-PA-IG, BmN cells were used for blind transmission. After 3 to 4 generations of blind passage, the cell Grace medium was collected directly, centrifuged at 12,000 rpm for 2 minutes, the supernatant was taken, and injected into silkworm larvae (from the 5th instar) at 10 μl / head. After 3 days, hemolymph of silkworm larvae was collected to observe whether red and green fluorescence appeared.

Embodiment 3

[0043] Embodiment 3: Purification of recombinant pramlintide

[0044] Collect silkworm larva hemolymph, add PMSF, centrifuge at 15000rpm for 10 minutes, take supernatant, add 3 times volume of PB solution (ph 6.0). Equilibrate the Ni-NTA affinity column with 20mM PB, 200mM NaCl (ph 6.0) solution 3ml / min, inject the expression supernatant into the Ni-NTA affinity column at 1ml / min, and elute with 20mM PB, 200mM NaCl, 20mM imidazole Miscellaneous protein, 20mM PB, 200mM NaCl, 150mM imidazole eluted protein. The flow-through protein was collected, the impurity protein was eluted with 20mM imidazole, and the target protein was eluted with 150mM imidazole, and the purified product was detected by SDS-PAGE gel and Western Blot.

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Abstract

The invention discloses a method for expressing pramlintide, and belongs to the technical field of genetic engineering. The method is characterized by comprising the following steps: conducting bacterial infection on bombyx mori ovary cells by virtue of a baculovirus multi-gene expression system, so that a baculovirus, which is capable of simultaneously expressing a human-derived amidating enzyme and the pramlintide; and then, injecting the virus into five-instar bombyx mori bodies, so that the amidating enzyme and the pramlintide are co-expressed by the bombyx mori, wherein glycine at the C terminal of the pramlintide is directly amidated by the amidating enzyme, so that the pramlintide, having an activity, is produced, and the pramlintide is purified by virtue of conventional purification means. With the application of the method disclosed by the invention, a plurality of genes can be expressed, which is conducive to co-expression of the human-derived amidating enzyme and the pramlintide; the method disclosed by the invention, which makes use of the bombyx mori bioreactor, is high in protein expression amount and is more complete in modification; and the method disclosed by the invention is short in production time, high in efficiency and low in production cost.

Description

technical field [0001] A method for producing recombinant human amidase and pramlintide belongs to the technical field of genetic engineering. Background technique [0002] α-amidation is an important post-translational processing process of many bioactive peptides in the nervous and endocrine system. Monoxygenase (PAM) is the rate-limiting step in the biosynthetic pathway of amidated polypeptides. A large number of neuropeptides with medicinal value and the C-terminal of active peptides in the endocrine system are amidated. The amidation modification process is very important for their biological activity and stability. If the C-terminal is not modified by amidation, its biological The activity will be reduced by hundreds or even thousands of times. α-amidase is widely found in tissues from amphibians to humans, and has high homology, but Escherichia coli and yeast commonly used in genetic engineering lack this enzyme activity, and the expression product cannot perform po...

Claims

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Application Information

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IPC IPC(8): C12P21/02
Inventor 黄亚东项琪胡浩孙京臣平瑶梁智升
Owner JINAN UNIVERSITY
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