CN106282278B

CN106282278B - Fermentation method of human papilloma virus L1 protein

Info

Publication number: CN106282278B
Application number: CN201610435356.7A
Authority: CN
Inventors: 贾永峰; 宁荣良; 李得林; 欧晓涛; 黄成潭; 封海生; 孙文正
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Guangdong HEC Pharmaceutical
Priority date: 2015-06-29
Filing date: 2016-06-17
Publication date: 2021-06-08
Anticipated expiration: 2036-06-17
Also published as: CN106282278A

Abstract

The invention belongs to the field of microbial fermentation, and relates to a fermentation method of human papilloma virus L1 protein. According to the invention, the KM71 type pichia pastoris is used as an expression system, and the system comprises a basic growth stage, a glycerol supplementation stage and an induction stage, wherein the induction stage adopts a methanol solution and a sorbitol solution for mixed supplementation, so that the degradation of target protein in the process of expressing the human papilloma virus L1 protein by the pichia pastoris is effectively reduced.

Description

Fermentation method of human papilloma virus L1 protein

Technical Field

The invention relates to the field of biomedicine, in particular to a fermentation method suitable for human papilloma virus L1 protein.

Background

Human Papilloma Virus (HPV) is a small non-enveloped double-stranded circular DNA virus of the polyomaviridae subfamily papuloviridae. Cervical cancer is the most fatal but also most preventable type of cancer in women worldwide, and HPV infection is the chief culprit, resulting in 99% of cervical cancer cases. Therefore, the development of the high-efficiency and low-cost HPV vaccine has very important significance for preventing female cervical carcinoma and sexually transmitted diseases caused by HPV infection.

During the expression of the foreign protein, a certain amount of protease is expressed both intracellularly and extracellularly in host bacteria pichia pastoris, so most of the foreign proteins are degraded. In addition, the HPV vaccine L1 protein has large molecular weight (56KDa) and complex structure, and is easy to have unstable structure in the process of expression modification, which aggravates the degradation degree of the L1 protein. At present, the degradation of HPV L1 protein in the fermentation culture process is an industry difficulty, and the degradation rate is generally 40-60%. Degradation not only causes a decrease in the yield of the target protein, but also the fragments formed by the degradation thereof can cause great difficulty in isolation and purification. In addition, numerous experiments have shown that the degradation of the L1 protein also has a major impact on the later assembly of virus-like particles (VLPs). To avoid degradation by proteases, different strategies have been applied, such as protease deficient strains, addition of protease inhibitors, peptones, etc. For HPV vaccines, it is difficult to identify the relevant protease causing degradation of the L1 protein, and even possibly multiple proteases; in addition, the stability of the L1 protein structure is also an important cause of its degradation. The existing method has no obvious effect on preventing HPV vaccine L1 protein degradation.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a fermentation method capable of reducing the degradation of human papillomavirus L1 protein, the method uses KM71 pichia pastoris as an expression system, and comprises a basic growth stage, a glycerol supplementation stage and an induction stage, wherein the induction stage adopts a methanol solution and a sorbitol solution for mixed feeding.

In some embodiments, the mass ratio of the mixed feed of the methanol solution and the sorbitol solution is 1: 0.85-3. The sorbitol solution is a 50% aqueous solution.

In some embodiments, the methanol solution of the induction stage contains a volume of PTM1 solution, preferably 12ml of PTM1 solution per liter of methanol solution.

In some embodiments, the temperature of the induction stage is controlled between 25 ℃ and 30 ℃.

In some embodiments, the fermentation time is 60-80 h.

In some embodiments, the OD of the induction phase₆₀₀Control is at 250-.

In some embodiments, the substrate culture medium during fermentation is an L-GJY fermentation medium, which comprises the following components: 15-25 g/L of glycerin, 3-7 g/L of yeast powder, 2-5 g/L of urea and CaSO₄·2H₂O 0.4～0.6g/L，MgSO₄3～5g/L，KOH 8～10g/L，H₃PO₄8-12 ml/L, 2-6 ml/L of PTM1 solution and 1-3 ml/L of defoaming agent. In fermentation, the substrate culture medium can be L-GJY fermentation culture medium used in the invention, and can also be BMGY culture medium and BSM culture medium commonly used in the field, and the function of the substrate culture medium is to provide necessary nutrient substances and buffer environment.

The fermentation method of the human papilloma virus L1 protein provided by the invention effectively reduces the problem of L1 protein degradation, reduces the cost for the production of cervical cancer vaccines, and provides an effective method for expressing the L1 protein by using a yeast expression system.

Detailed Description

The embodiment of the invention discloses a fermentation method of human papilloma virus L1 protein. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the product and method described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In the following examples, the media used consisted of:

BMGY seed medium: yeast extract 10g/L, peptone 20g/L, nitrogen source without amino Yeast (YNB)13.4g/L, glycerol 20g/L, dissolved in 0.1mol/L (pH 7.0) phosphate buffer.

L-GJY fermentation medium: 15-25 g/L of glycerin, 3-7 g/L of yeast powder, 2-5 g/L of urea and CaSO₄·2H₂O 0.4～0.6g/L，MgSO₄3～5g/L，KOH 8～10g/L，H₃PO 48-12 ml/L, PTM1 solution 2-6 ml/L, and defoaming agent 1-3 ml/L.

PTM1 solution: CuSO₄·5H₂O 6g/L，KI 0.08g/L，MnSO₄·H₂O 3g/L，Na₂MoO₄·2H₂O 0.2g/L，H₃BO₃0.02g/L，ZnSO₄·7H₂O 20g/L，FeSO₄·7H₂O 65g/L，CoCl₂·6H₂O 0.5g/L，H₂SO₄5ml/L，Biotin 0.2g/L。

Example 1

(1) And (3) strain culture: 2ml of the glycerol stock of KM71/A11-20 was inoculated into a 250ml shake flask containing 100ml of BMGY seed liquid medium and cultured at 30 ℃ and 220r/min for 12 hours. Then 2ml of the cultured seed liquid is respectively taken and transferred into three 500ml shake flasks containing 200ml of BMGY secondary seed liquid culture medium, and the culture is carried out for 18h at 30 ℃ and 220 r/min.

(2) Inoculation: injecting 20L L-GJY fermentation medium into a 50L fermentation tank, and inoculating the cultured secondary seed liquid according to the inoculation amount of 2.5% of the volume ratio.

(3) Fermentation: a basic growth stage: the initial rotation speed is 100r/min, the temperature is controlled at 30 ℃, the ventilation volume is 1vvm, the dissolved oxygen is controlled at more than 50 percent, and the pH value is not required to be controlled. ② glycerin replenishing stage: after the dissolved oxygen rebounds to 80%, the glycerol solution (50%, w/v, containing 12ml/L of PTM1 solution) is supplemented at the rate of 20g/L/h, the temperature is controlled at 30 ℃, the ventilation quantity is 1.3vvm, the dissolved oxygen is controlled at more than 30%, and the pH is controlled at about 5.0 by ammonia water. ③ an induction phase: when OD is reached₆₀₀And (3) stopping supplementing glycerol when the concentration is 250 ℃, beginning induction when dissolved oxygen rebounds to 80%, and supplementing a methanol solution and a sorbitol solution according to the mass ratio of 1:0.85, wherein each liter of the methanol solution contains 12ml of PTM1 solution, and the sorbitol solution is a 50% aqueous solution. The temperature is controlled at 25 ℃, the ventilation rate is 1.5vvm, the dissolved oxygen is controlled at more than 20 percent, and the pH is controlled at 5.0 by ammonia water. The culture is finished when the time is about 60 hours.

(4) And (3) collecting thalli: the fermentation sample was collected during the tank-out, and the expression level of L1 protein was 289mg/L (+ -5 mg/L) after treatment, and the degradation rate was 8% (+ -2%) by SDS-PAGE analysis.

Example 2

(3) Fermentation: a basic growth stage: the initial rotation speed is 100r/min, the temperature is controlled at 30 ℃, the ventilation volume is 1vvm, the dissolved oxygen is controlled at more than 50 percent, and the pH value is not required to be controlled. ② glycerin replenishing stage: after the dissolved oxygen rebounds to 80%, the glycerol solution (50%, w/v, containing 12ml/L of PTM1 solution) is supplemented at the rate of 20g/L/h, the temperature is controlled at 30 ℃, the ventilation quantity is 1.3vvm, the dissolved oxygen is controlled at more than 30%, and the pH is controlled at about 5.0 by ammonia water. ③ an induction phase: when OD is reached₆₀₀And (4) stopping supplementing glycerol when the concentration is 350, beginning induction when dissolved oxygen rebounds to 80%, and supplementing a methanol solution and a sorbitol solution according to the mass ratio of 1:3, wherein each liter of the methanol solution contains 12ml of PTM1 solution, and the sorbitol solution is 50% aqueous solution by mass concentration. The temperature is controlled at 30 ℃, the ventilation rate is 1.5vvm, the dissolved oxygen is controlled at more than 20 percent, and the pH is controlled at 5.0 by ammonia water. The culture is finished when the culture time is about 80 hours.

(4) And (3) collecting thalli: the fermentation sample was collected during the tank-out, and after treatment, the expression level of L1 protein was found to be 295mg/L (+ -5 mg/L), and the degradation rate was calculated to be 6% (+ -2%) by SDS-PAGE analysis.

Example 3

(2) Inoculation: injecting 20LL-GJY fermentation culture medium into a 50L fermentation tank, and inoculating the cultured secondary seed liquid according to the inoculation amount of 2.5% of the volume ratio.

(3) Fermentation: a basic growth stage: the initial rotation speed is 100r/min, the temperature is controlled at 30 ℃, the ventilation volume is 1vvm, the dissolved oxygen is controlled at more than 50 percent, and the pH value is not required to be controlled. ② glycerin replenishing stage: after the dissolved oxygen rebounds to 80%, the glycerol solution (50%, w/v, containing 12ml/L of PTM1 solution) is supplemented at the rate of 20g/L/h, the temperature is controlled at 30 ℃, the ventilation quantity is 1.3vvm, the dissolved oxygen is controlled at more than 30%, and the pH is controlled at about 5.0 by ammonia water. ③ an induction phase: when OD is reached₆₀₀And (3) stopping supplementing the glycerol when the concentration is 300 ℃, beginning to induce when the dissolved oxygen rebounds to 80%, and supplementing a methanol solution and a sorbitol solution according to the mass ratio of 1:1.2, wherein each liter of the methanol solution contains 12ml of PTM1 solution, and the sorbitol solution is a 50% aqueous solution. The temperature is controlled at 28 ℃, the ventilation rate is 1.5vvm, the dissolved oxygen is controlled at more than 20 percent, and the pH is controlled at 5.0 by ammonia water. The culture is finished when the culture time is about 70 hours.

(4) And (3) collecting thalli: the fermentation sample was collected during the tank-out, and after treatment, the expression level of L1 protein was found to be 310mg/L (+ -5 mg/L), and the degradation rate was calculated to be 5% (+ -2%) by SDS-PAGE analysis.

Claims

1. A fermentation method of human papillomavirus L1 protein is characterized in that KM71 pichia pastoris is used as an expression system and comprises a basic growth stage, a glycerol supplementation stage and an induction stage, wherein the induction stage adopts a methanol solution and a sorbitol solution for mixed feeding; the mass ratio of the methanol solution to the sorbitol solution is 1: 0.85-3; the temperature of the induction phase is controlled between 25 ℃ and 30 ℃; the fermentation time is 60-80 h; OD of the Induction phase₆₀₀Control is at 250-.

2. The fermentation process of claim 1, wherein the methanol solution contains 12ml of PTM1 solution per liter.

3. The fermentation method according to claim 1, wherein the sorbitol solution is a 50% aqueous solution by mass concentration.

4. The fermentation method according to claim 1, wherein the substrate culture medium during fermentation is an L-GJY fermentation culture medium, and the components of the L-GJY fermentation culture medium are as follows: 15-25 g/L of glycerin, 3-7 g/L of yeast powder, 2-5 g/L of urea and CaSO₄·2H₂O 0.4~0.6 g/L，MgSO₄ 3~5 g/L，KOH 8~10 g/L，H₃PO₄8-12 ml/L, 2-6 ml/L of PTM1 solution and 1-3 ml/L of defoaming agent.