CN108715815A - A method of based on recombination Hansenula yeast cell technology high-efficient culture, expression hepatitis B surface antigen - Google Patents
A method of based on recombination Hansenula yeast cell technology high-efficient culture, expression hepatitis B surface antigen Download PDFInfo
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- CN108715815A CN108715815A CN201810537430.5A CN201810537430A CN108715815A CN 108715815 A CN108715815 A CN 108715815A CN 201810537430 A CN201810537430 A CN 201810537430A CN 108715815 A CN108715815 A CN 108715815A
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Abstract
The present invention relates to a kind of based on the method for recombinating Hansenula yeast cell technology high-efficient culture, expressing hepatitis B surface antigen, belongs to medical bioengineering technical field.The method of the invention includes first order seed incubation step, secondary seed incubation step, three-level seed culture step, fermented and cultured step.Cell density in the present invention after the amplification of recombinant hepatitis B vaccine (Hansenula yeast) cell, culture expression expands than existing Recombinant hepatitis B vaccine cell, cultivates the cell density higher of expression, HBsAg expression quantity highers, and HBsAg grain structures are complete, cell granulations size is uniform.
Description
Technical field
The present invention relates to a kind of based on the side for recombinating Hansenula yeast cell technology high-efficient culture, expressing hepatitis B surface antigen
Method belongs to medical bioengineering technical field.
Background technology
Virus B hepatitis (viral hepatitis type B, abbreviation hepatitis B) system is caused by hepatitis B (HBV),
Using weak, anorexia, Nausea and vomiting, oil, hepatomegaly and dysfunction of liver are detested as main clinical manifestation.Some cases have fever
And jaundice；A small number of case protracted courses switch to it is chronic, or develop into hepatic sclerosis even liver cancer；Severe one disease progression can rapidly develop
For heavy type hepatitis；Other the infecteds then become asymptomatic virus carrier.
Hepatitis type B virus abbreviation hepatitis B.It is a kind of DNA virus, belongs to Hepadnaviridae
(hepadnaviridae).According to current known, HBV just only has neurological susceptibility to people and orangutan, causes virus B hepatitis disease
Disease.Complete hepatitis B can also be referred to as red Na particle (Dane) at graininess.Nineteen sixty-five finds by red Na, a diameter of 42
Nanometer, particle are divided into shell and core two parts.
Hepatitis type B virus (HBV) belongs to Hepadnaviridae (hepadnaviridae), and genome is about 3.2kb, is portion
Divide double-stranded cyclic DNA.The resistance of HBV is stronger, but 65 DEG C of 10h, boils 10min or high pressure steam and can inactivate HBV.Containing chlorine system
Agent, ethylene oxide, glutaraldehyde, Peracetic acid and Iodophor etc. also have preferable inactivating efficacy.
The hepatitis B vaccine of prevalence is Recombinant hepatitis B vaccine in the world at present.Recombinant hepatitis B vaccine is divided into as lactation
The vaccine and Hepatitis B Vaccine Prepared From Yeast Recombinanted of animal cell expression.Recombinant hepatitis B vaccine is that transgenic technology, structure is utilized to contain
The recombinant plasmid for having hepatitis B HBsAg genes is transferred to yeast (brewer's yeast Pichia pastoris or Hansenula yeast) or recombination China
The hepatitis B surface antibody of hamster ovary cell (CHO) expression.
The hepatitis B vaccine cell disadvantage of existing gene recombination technology structure see the table below 1：
The hepatitis B vaccine cell disadvantage of 1 existing gene recombination technology of table structure
Invention content
The present invention is inoculated with through first order seed, cultivates, secondary seed inoculation, culture, the inoculation of three-level seed, culture, and fermentation connects
Kind, amplification cultivation express to obtain fermentation culture medium, can be expressed by this method, formed it is big with natural hepatitis B virus particles
Small consistent viruslike particle, and cell fermentation density is high, antigen presentation amount is high.
The present invention provides a kind of based on the side for recombinating Hansenula yeast cell technology high-efficient culture, expressing hepatitis B surface antigen
Method, the method includes first order seed incubation step, secondary seed incubation step, three-level seed culture step, fermented and cultured steps
Suddenly；
The fermented and cultured step is：25L three-level inoculums are taken to be inoculated in 100-150L fermentation mediums, 28-
32 DEG C of culture 88-95h；
The fermentation medium is made of following components by weight：
The mixing speed in growth period increases to 500rpm by 200rpm, and pH value 4.85-5.25, air mass flow is by 100L/
Min increases to 300L/min, and dissolved oxygen is not less than 10%；
The feed supplement phase keeps the fermentation condition in growth period constant, when dissolved oxygen rises to maximum value, 8-10L glycerine is added, repeatedly
6-8 times；
The mixing speed of induction period is reduced to 450rpm by 500rpm, and pH value 5.35-5.75, air mass flow is by 150L/
Min is reduced to 20L/min, dissolved oxygen 60-90%, also, when dissolved oxygen rises to maximum value after last time glycerine is added, and starts
First stream plus volume ratio are 1：The glycerine methyl alcohol mixed liquor 25-30L of 4-6, then flow and add methanol 70-80L, it is 10- to flow the speed added
40mL/min。
The present invention is preferably that the three-level seed culture step is：3L secondary seed cultures are taken to be inoculated in 30-45L three-levels
In seed culture medium, 28-32 DEG C of culture 13-15h, mixing speed increases to 400rpm, pH value 4.85-5.25 by 200rpm,
Air mass flow is 10-15L/min, and dissolved oxygen is not less than 20%；
The three-level seed culture medium is made of following components by weight：
7.50 parts of ammonium sulfate
10.05 parts of yeast nitrogen culture medium
0.038 part of glycerine.
The present invention is preferably that the secondary seed incubation step is：200mL first order seed cultures are taken to be inoculated in 3-4L bis-
In grade seed culture medium, 29-31 DEG C of culture 19.5-20.5h, shaking speed 190-210rpm；
The secondary seed medium is made of following components by weight：
7.50 parts of ammonium sulfate
10.05 parts of yeast nitrogen culture medium
0.038 part of glycerine.
The present invention is preferably that the first order seed incubation step is：1mL is recombinated into Hansenula yeast working seed lots engineering bacteria
Kind is inoculated in 300-350mL first cell culture mediums, 29-31 DEG C of culture 19.5-20.5h, shaking speed 190-210rpm；
The first cell culture medium is made of following components by weight：
Cell density in the present invention after the amplification of recombinant hepatitis B vaccine (Hansenula yeast) cell, culture expression is than existing
The cell density higher of the amplification of Recombinant hepatitis B vaccine cell, culture expression, HBsAg expression quantity highers, and HBsAg particle knots
Structure is complete, and cell granulations size is uniform.
Description of the drawings
6 width of attached drawing of the present invention,
Fig. 1 is fermentation process cell culture, expression figure when repeating embodiment 1 for the first time；
Fermentation process cell culture, expression figure when Fig. 2 is second of repetition embodiment 1；
Fig. 3 is that third time repeats fermentation process cell culture when embodiment 1, expression figure；
Fig. 4 is the HBsAg grain structures that recombinant hepatitis B vaccine (Hansenula yeast) is expressed when repeating embodiment 1 for the first time
Electronic Speculum detection figure；
Fig. 5, which is second, repeats the HBsAg grain structures that recombinant hepatitis B vaccine (Hansenula yeast) is expressed when embodiment 1
Electronic Speculum detection figure；
Fig. 6 is the HBsAg grain structures that third time repeats that recombinant hepatitis B vaccine (Hansenula yeast) is expressed when embodiment 1
Electronic Speculum detection figure.
Specific implementation mode
Following non-limiting embodiments can make those skilled in the art be more fully understood the present invention, but not with
Any mode limits the present invention.
The recombination Hansenula yeast engineered strain of following HBsAg expressions with DNA recombinant techniques structure, strain number are
HBsAgU35-16-9, quoted from《Chinese Pharmacopoeia version in 2015》Page 163.
Following first cell culture mediums are made of following components by weight：
Following secondary seed mediums are made of following components by weight：
7.50 parts of ammonium sulfate
10.05 parts of yeast nitrogen culture medium
0.038 part of glycerine.
Following three-level seed culture mediums are made of following components by weight：
7.50 parts of ammonium sulfate
10.05 parts of yeast nitrogen culture medium
0.038 part of glycerine.
Following fermentation mediums are made of following components by weight：
Embodiment 1
A method of based on recombination Hansenula yeast cell technology high-efficient culture, expression hepatitis B surface antigen, the method
Include the following steps：
First order seed incubation step：
1mL recombination Hansenula yeast working seed lots engineering strains are inoculated in 350mL first cell culture mediums, 30 DEG C of cultures
20h, shaking speed 200rpm obtain first order seed culture；
Secondary seed incubation step：
200mL first order seed cultures are taken to be inoculated in 3L secondary seed mediums, 30 DEG C are cultivated 20h, and shaking speed is
200rpm obtains secondary seed culture；
Three-level seed culture step：
Take 3L secondary seed cultures to be inoculated in 30L three-level seed culture mediums, 30 DEG C culture 13h, mixing speed by
200rpm increases to 400rpm, pH value 5.05, air mass flow 10L/min, and dissolved oxygen is not less than 20%, obtains the training of three-level seed
Support object；
Fermented and cultured step：
25L three-level inoculums are taken to be inoculated in 140L fermentation mediums, 30 DEG C of culture 92h；
The mixing speed in growth period increases to 500rpm by 200rpm, and pH value 4.85-5.25, air mass flow is by 100L/
Min increases to 300L/min, and dissolved oxygen is not less than 10%；
The feed supplement phase keeps the fermentation condition in growth period constant, and when dissolved oxygen rises to maximum value, glycerine is added, 6 times repeatedly, preceding
It is separately added into 8L glycerine 2 times, is separately added into 10L glycerine latter 4 times；
The mixing speed of induction period is reduced to 450rpm by 500rpm, and pH value 5.35-5.75, air mass flow is by 150L/
Min is reduced to 20L/min, dissolved oxygen 60-90%, also, when dissolved oxygen rises to maximum value after last time glycerine is added, and starts
First stream plus volume ratio are 1：5 glycerine methyl alcohol mixed liquor 30L, then flow and add methanol 70L, it is 10-40mL/min to flow the speed added.
It is repeated 3 times according to embodiment 1.
Test case 1
The cell density detection that embodiment 1 obtains：It takes 1mL uniform fermentation liquid, 13000rpm to centrifuge 4min, removes supernatant
Liquid claims solids, that is, zymotic fluid cell density, as a result see the table below 2.
Table 2 is repeated 3 times the cell density detected value that embodiment 1 obtains
| Cell density detected value (g/mL) | |
| It repeats for the first time | 0.5250 |
| Second of repetition | 0.5326 |
| Third time repeats | 0.5210 |
Test case 2
Antigenic content detects：HBsAg in serum level is measured using double-antibody method, microwell plate is coated with using one plant of monoclonal antibody
Solid phase antibody is made, enzyme labelled antibody is marked using another plant of monoclonal antibody.HBsAg calibrations are first added in the micropore of coating microwell plate
Product or test serum add enzyme marker, form solid matrix antibody-antigen-hrp-antibody complex after incubation, fully wash
After Chemoluminescent substrate is added, then measure its luminous value (RLU), content in sample can be calculated according to calibration curve, tie
Fruit see the table below 3.
Table 3 is repeated 3 times the cellular antigens content detection value that embodiment 1 obtains
| HBsAg detection of expression value (mg/L) | |
| It repeats for the first time | 613.00 |
| Second of repetition | 588.00 |
| Third time repeats | 597.98 |
Claims (4)
1. a kind of based on the method for recombinating Hansenula yeast cell technology high-efficient culture, expressing hepatitis B surface antigen, it is characterised in that：
The method includes first order seed incubation step, secondary seed incubation step, three-level seed culture step, fermented and cultured steps；
The fermented and cultured step is：25L three-level inoculums are taken to be inoculated in 100-150L fermentation mediums, 28-32 DEG C
Cultivate 88-95h；
The fermentation medium is made of following components by weight：
The mixing speed in growth period increases to 500rpm, pH value 4.85-5.25 by 200rpm, and air mass flow is increased by 100L/min
300L/min is added to, dissolved oxygen is not less than 10%；
The feed supplement phase keeps the fermentation condition in growth period constant, when dissolved oxygen rises to maximum value, 8-10L glycerine is added, repeatedly 6-8
It is secondary；
The mixing speed of induction period is reduced to 450rpm, pH value 5.35-5.75, air mass flow to be dropped by 150L/min by 500rpm
Down to 20L/min, dissolved oxygen 60-90%, also, when dissolved oxygen rises to maximum value after last time glycerine is added, start first to flow
It is 1 to add volume ratio：The glycerine methyl alcohol mixed liquor 25-30L of 4-6, then flow and add methanol 70-80L, it is 10-40mL/ to flow the speed added
min。
2. according to the method described in claim 1, it is characterized in that：The three-level seed culture step is：Take 3L secondary seeds
Culture is inoculated in 30-45L three-level seed culture mediums, and 28-32 DEG C of culture 13-15h, mixing speed is increased to by 200rpm
400rpm, pH value 4.85-5.25, air mass flow 10-15L/min, dissolved oxygen are not less than 20%；
The three-level seed culture medium is made of following components by weight：
7.50 parts of ammonium sulfate
10.05 parts of yeast nitrogen culture medium
0.038 part of glycerine.
3. according to the method described in claim 2, it is characterized in that：The secondary seed incubation step is：Take 200mL level-one kinds
Sub- culture is inoculated in 3-4L secondary seed mediums, 29-31 DEG C of culture 19.5-20.5h, shaking speed 190-
210rpm；
The secondary seed medium is made of following components by weight：
7.50 parts of ammonium sulfate
10.05 parts of yeast nitrogen culture medium
0.038 part of glycerine.
4. according to the method described in claim 1, it is characterized in that：The first order seed incubation step is：The 1mL recombination Chinese is inferior
Yeast working seed lots engineering strain is inoculated in 300-350mL first cell culture mediums, 29-31 DEG C of culture 19.5-20.5h, shaking table
Rotating speed 190-210rpm；
The first cell culture medium is made of following components by weight：
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2207374C1 (en) * | 2002-05-14 | 2003-06-27 | Закрытое акционерное общество "Медицинские технологии - "МТХ" | Recombinant plasmid encoding hepatitis b virus surface antigen (hbsag), its preparing and yeast strain hansenula polymorpha as producer of hepatitis b virus surface antigen (hbsag) |
| RU2586511C1 (en) * | 2015-04-24 | 2016-06-10 | Закрытое акционерное общество научно-производственная компания "Комбиотех" | RECOMBINANT Hansenula polymorpha YEAST STRAIN - PRODUCER OF HEPATITIS B VIRUS SURFACE ANTIGEN SEROTYPE "ayw" |
| CN105734066A (en) * | 2016-03-11 | 2016-07-06 | 安徽智飞龙科马生物制药有限公司 | Method for constructing eukaryon Hansenula polymorpha engineering bacteria with recombinant hepatitis B virus genes and method for producing hepatitis B surface antigens |
| CN105797151A (en) * | 2016-03-25 | 2016-07-27 | 汪和睦 | High-dose hepatitis b vaccine based on recombination hansenula polymorpha |
| CN108047316A (en) * | 2018-01-04 | 2018-05-18 | 南京赛威信生物医药有限公司 | The isolation and purification method of recombination hepatitis B core antigen |
-
2018
- 2018-05-30 CN CN201810537430.5A patent/CN108715815A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2207374C1 (en) * | 2002-05-14 | 2003-06-27 | Закрытое акционерное общество "Медицинские технологии - "МТХ" | Recombinant plasmid encoding hepatitis b virus surface antigen (hbsag), its preparing and yeast strain hansenula polymorpha as producer of hepatitis b virus surface antigen (hbsag) |
| RU2586511C1 (en) * | 2015-04-24 | 2016-06-10 | Закрытое акционерное общество научно-производственная компания "Комбиотех" | RECOMBINANT Hansenula polymorpha YEAST STRAIN - PRODUCER OF HEPATITIS B VIRUS SURFACE ANTIGEN SEROTYPE "ayw" |
| CN105734066A (en) * | 2016-03-11 | 2016-07-06 | 安徽智飞龙科马生物制药有限公司 | Method for constructing eukaryon Hansenula polymorpha engineering bacteria with recombinant hepatitis B virus genes and method for producing hepatitis B surface antigens |
| CN105797151A (en) * | 2016-03-25 | 2016-07-27 | 汪和睦 | High-dose hepatitis b vaccine based on recombination hansenula polymorpha |
| CN108047316A (en) * | 2018-01-04 | 2018-05-18 | 南京赛威信生物医药有限公司 | The isolation and purification method of recombination hepatitis B core antigen |
Non-Patent Citations (1)
| Title |
|---|
| 江宁: "《微生物生物技术》", 31 May 2008 * |
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Application publication date: 20181030 |