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Human papilloma virus L1 protein fermentation method

A technology of human papillomavirus and fermentation method, applied in the direction of microorganism-based method, fermentation, virus, etc., can solve the problems of HPV vaccine L1 protein effect is not obvious, it is difficult to determine the protease related to L1 protein degradation, and achieve the reduction of L1 protein Effect of degradation and cost reduction

Active Publication Date: 2017-01-04
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For HPV vaccines, it is difficult to determine the relevant proteases that cause L1 protein degradation, and may even be multiple proteases; in addition, the stability of L1 protein structure is also an important reason for its degradation
Existing methods are not effective in avoiding degradation of HPV vaccine L1 protein

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Strain culture: Take 2ml of KM71 / A11-20 glycerol preservation solution and put it into a 250ml shake flask containing 100ml of BMGY seed solution medium, and culture at 30°C and 220r / min for 12h. Then take 2ml of the above-mentioned cultured seed solution and transfer them to three 500ml shake flasks containing 200ml of BMGY secondary seed solution medium, and cultivate them at 30°C and 220r / min for 18h.

[0019] (2) Inoculation: Inject 20L of L-GJY fermentation medium into a 50L fermenter, and then insert the above-mentioned cultured secondary seed liquid at an inoculation amount of 2.5% by volume.

[0020] (3) Fermentation: ①Basic growth stage: the initial rotation speed is 100r / min, the temperature is controlled at 30°C, the ventilation rate is 1vvm, the dissolved oxygen is controlled above 50%, and the pH does not need to be controlled. ②Glycerol supplementation stage: When the dissolved oxygen rebounds to 80%, add glycerin solution (50%, w / v, containing 12ml / L ...

Embodiment 2

[0023] (1) Strain culture: Take 2ml of KM71 / A11-20 glycerol preservation solution and put it into a 250ml shake flask containing 100ml of BMGY seed solution medium, and culture at 30°C and 220r / min for 12h. Then take 2ml of the above-mentioned cultured seed solution and transfer them to three 500ml shake flasks containing 200ml of BMGY secondary seed solution medium, and cultivate them at 30°C and 220r / min for 18h.

[0024] (2) Inoculation: Inject 20L of L-GJY fermentation medium into a 50L fermenter, and then insert the above-mentioned cultured secondary seed liquid at an inoculation amount of 2.5% by volume.

[0025] (3) Fermentation: ①Basic growth stage: the initial rotation speed is 100r / min, the temperature is controlled at 30°C, the ventilation rate is 1vvm, the dissolved oxygen is controlled above 50%, and the pH does not need to be controlled. ②Glycerol supplementation stage: When the dissolved oxygen rebounds to 80%, add glycerin solution (50%, w / v, containing 12ml / L ...

Embodiment 3

[0028] (1) Strain culture: Take 2ml of KM71 / A11-20 glycerol preservation solution and put it into a 250ml shake flask containing 100ml of BMGY seed solution medium, and culture at 30°C and 220r / min for 12h. Then take 2ml of the above-mentioned cultured seed solution and transfer them to three 500ml shake flasks containing 200ml of BMGY secondary seed solution medium, and cultivate them at 30°C and 220r / min for 18h.

[0029] (2) Inoculation: Inject 20LL-GJY fermentation medium into a 50L fermenter, and then insert the above-mentioned cultured secondary seed liquid at an inoculation amount of 2.5% by volume.

[0030](3) Fermentation: ①Basic growth stage: the initial rotation speed is 100r / min, the temperature is controlled at 30°C, the ventilation rate is 1vvm, the dissolved oxygen is controlled above 50%, and the pH does not need to be controlled. ②Glycerol supplementation stage: When the dissolved oxygen rebounds to 80%, add glycerin solution (50%, w / v, containing 12ml / L PTM1 ...

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Abstract

The invention belongs to the field of microbial fermentation and relates to a human papilloma virus L1 protein fermentation method. The human papilloma virus L1 protein fermentation method uses KM71 type pichia pastoris as an expression system and comprises a basic growth stage, a glycerin supplementing stage and an induction stage, wherein a methanol solution and a sorbitol solution are mixed for material supplementation at the induction stage. The target protein degradation in the human papilloma virus L1 protein expression process of pichia pastoris is effectively reduced.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a fermentation method suitable for human papillomavirus L1 protein. Background technique [0002] Human papillomavirus (HPV) is a non-enveloped small double-stranded circular DNA virus belonging to the subfamily Polyomaviridae of the family Papovaviridae. Cervical cancer is the deadliest but most preventable type of cancer in women worldwide, and HPV infection is to blame, causing 99% of cervical cancer cases. Therefore, the development of efficient and cheap HPV vaccine is of great significance for the prevention of female cervical cancer and sexually transmitted diseases caused by HPV infection. [0003] In the process of exogenous protein expression, a certain amount of proteases are expressed inside and outside the cell of the host Pichia pastoris, so most of the exogenous proteins are faced with the problem of being degraded. In addition, the HPV vaccine L1 protein has a large m...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12R1/84
CPCC07K14/005C12N2710/20022C12P21/02
Inventor 贾永峰宁荣良李得林欧晓涛黄成潭封海生孙文正
Owner SUNSHINE LAKE PHARM CO LTD