Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation

A technology of hepatitis B surface antigen and brewer's yeast, applied in anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, fermentation, recombinant DNA technology, etc., can solve low cell concentration, low productivity of recombinant HBsAg, unfavorable recombination HBsAg expression level and other issues, to achieve the effect of improving tolerance and improving productivity by weight ratio

Active Publication Date: 2004-12-08
BEIJING BIOLOGICAL PROD INST CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] One of the main problems in the current batch fermentation process is that since the medium of batch fermentation contains three components of natural yeast extract powder, natural soybean peptone and glucose, which are fed at one time, it cannot be adjusted during the production process.
Among them, the quality of yeast extract powder has the greatest impact on the productivity of recombinant HBsAg; even if imported raw materials are purchased at high prices, the productivity of recombinant HBsAg in different fermentation batches is unstable due to large batch differences in yeast extract powder
Sometimes although the cell concentration is high, the productivity of recombinant HBsAg is very low
The second is that in batch fermentation, the growth phase of the cells is about 15-20 hours; and in the antigen expression phase of the next 20-25 hours, because the glucose has been exhausted, the cells stop growing, resulting in the final cell concentration is too low, and there is no Conducive to improving the expression level of recombinant HBsAg

Method used

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  • Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation
  • Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation
  • Brewer's yeast recombined hepatitis B virus surface antigen stream plus addition femrentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047]Various media were prepared according to the formula shown in Table 1 below, wherein the composition of the basic media was 1.65 g / 53 ml of yeast extract powder, 1.925 g / 53 ml of soybean peptone, and 3.75 ml of 25% glucose. Its preparation method is: take 1.65g of yeast extract powder produced by DIFCO company, 1.925g of soybean peptone produced by Sheffield company, add water to dissolve 49.25ml to constant volume (increased yeast powder and adenine are added in this step). 121°C, 20 minutes for disinfection. After cooling, 3.75 ml of 25% glucose sterilized at 121° C. for 20 minutes was added (increased glucose was added at this step).

[0048] With the same inoculum size, the recombinant HBsAg engineering strain 2150-2-3 (MATa, adel, leu2-04, cir 0 ) in the culture medium of various formulations shown in the following table 1 in the shake flask, 31,38,48,53 hours sampling survey the OD value of 660nm, the data are summarized in the following table 1:

[0049] ...

Embodiment 2

[0052] The seed culture step was basically carried out according to the method described in the "Cell growth" section of Cheryl A. Schulman et. al., Journal of Biotechnology, 21(1991): 109-126). Specifically, the stored strains were thawed, inoculated into 250 ml shake flasks containing 50 ml of 5XLEU selective medium, cultured at 28°C at 350 rpm for 18 hours, and then transferred to two cultures containing 500 ml each. 5XLEU-selective medium in a 2-liter shake flask, and then cultured at 28° C. at 350 rpm for 18 hours. The composition of the 5XLEU-selective medium is the same as that described in the literature Cheryl A. Schulman et.al., Journal of Biotechnology, 21 (1991): 109-126).

[0053] 30 liters of fermentor continuous feeding culture adopts YEPD medium to carry out as follows: Weigh imported natural yeast extract powder (DIFCO company product, article number is 0127-01) 371.2 grams (content in fermented liquid is 32 grams per liter ), 417.6 grams of imported natural ...

Embodiment 3

[0055] The cultivation step of seed is with embodiment 2.

[0056] The production and fermentation steps are carried out in the following manner, wherein the source of the materials used, the feeding device, the fermentation temperature, the ventilation rate, the rotation speed of the fermenter, the method for measuring the retention rate of leucine prototroph cells, and the average exogenous gene of the cells The method for the number of plasmid copies and the method for measuring the HBsAg content per liter of fermentation broth are the same as those described in Example 2.

[0057] Continuous feeding culture in 30-liter fermenter: imported natural yeast extract powder 32 g / liter, imported natural soybean peptone 36 g / liter, add water to 9.9 liters, sterilize at 121°C for 30 minutes, after cooling, add sterilized 25 % glucose solution 700 ml, adjust the pH to 6.0 with sterilized 9N phosphoric acid solution. Insert 1 liter of mature seeds, cultivate for 14 hours, and start f...

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Abstract

A hepatitis B surface antigen is produced with beer yeast engineering bacteria. The process is carried out by: 1) fermenting seeds of the beer yeast engineering bacteria, 2) fermenting by the said seeds, and 3) separating and purifying fermented mixture to obtain hepatitis B surface antigen, specially, flow adding adenine, adenosine, adenosine monophosphate, adenosine diphosphate or adenosine triphosphate at the rate of 14mg/L.h by weight into its culture medium in the said step 2. It achieves in higher expression level of recombined HBsAg.

Description

technical field [0001] The invention relates to the field of microbial fermentation. Specifically, the present invention relates to a fermentation process using yeast to produce hepatitis B surface antigen. Background technique [0002] The total number of people affected by hepatitis B in the world reaches 2 billion people, and 350 million people are carriers of hepatitis B virus surface antigen (HBsAg). Infection with hepatitis B results in a lifelong carrier state; serious sequelae develop as cirrhosis and primary hepatocellular carcinoma. Hepatitis B kills millions of people every year worldwide. [0003] China is a high-incidence area of ​​hepatitis B and is a major hepatitis country in the world. More than 600 million people have been infected with hepatitis B, and the HBsAg carrier rate is about 10%. Among them, 12 million patients with chronic hepatitis have lost or partially lost the ability to work. They struggle with the disease all day long and have no good me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N1/19C12N15/13C12N15/63C12N15/81C12P21/02
Inventor 马民强汪和睦周根喜张德友
Owner BEIJING BIOLOGICAL PROD INST CO LTD
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