CN101302496A - Stable and effective-expressing transgene CHO cell strain of hepatitis B surface antigen for vaccin - Google Patents
Stable and effective-expressing transgene CHO cell strain of hepatitis B surface antigen for vaccin Download PDFInfo
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- CN101302496A CN101302496A CNA2007101071131A CN200710107113A CN101302496A CN 101302496 A CN101302496 A CN 101302496A CN A2007101071131 A CNA2007101071131 A CN A2007101071131A CN 200710107113 A CN200710107113 A CN 200710107113A CN 101302496 A CN101302496 A CN 101302496A
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Abstract
The invention provides a CHO cell strain which can express HBV surface antigen for vaccine stably and efficiently. A CHO cell strain which can express hepatitis B surface antigen stably and efficiently is obtained by adopting the method of pore plate screening and gene expansion screening the CHO cell strain which can express against Epitope of HBV already.
Description
Technical field:
The present invention relates to a kind of stable high expression level vaccine hepatitis B virus surface antigen (Hepatitis Bsurface antigen, Chinese hamster ovary celI strain HBsAg).
Background technology:
Hepatitis B virus (HBV) is a kind of dna virus, and hepatitis b virus infected be a global public health problem, at the infection rate of China's hepatitis B about 60%, the carrying rate of hepatitis B surface antigen about 10%, annual have 300,000 people finally will to die from the relevant chronic disease of hepatitis B approximately, is dead first of transmissible disease.
The envelope protein of hepatitis B virus is called HBsAg, it is the composition that decision virus is adsorbed to the permissive cell acceptor, the gene of encoded packets membranin can be divided into S, Pre-S1 and Pre-S2 (HBsAg often refers to the albumen by the S genes encoding traditionally), and envelope protein is made up of three kinds of albumen:
1, small protein (S albumen) is by the S genes encoding.
2, middle albumen (M albumen) is by S and Pre-S2 gene regions coding.
3, large protein (L albumen) is by S, Pre-S1 and Pre-S2 gene regions coding.
The virion that 3 kinds of different shapes are arranged among the HBV infected person anteserum: pellet shapes particle (diameter 22nm), tubular particle (diameter 22nm, length 100-1000nm), Dane particle do not exist independent pre-s1 protein or pre-s2 protein.
The situation difference that these 3 kinds of albumen of small protein (S), middle albumen (M) and large protein (L) exist on the variable grain surface, among the hepatitis B infected person, most is the small spherical particles of 22nm in patient's blood flow of the infected, only by small protein or 5% the middle albumen of having an appointment simultaneously form; Virus replication phase patient has simultaneously that minority is different in size, the cast particle of diameter 22nm, is made up of albumen among small protein, the 2%-5% and 5%-10% large protein.At the non-replicative phase of virus, large protein is very low or nothing in serum, is trapped in the liver cell in a large number.Therefore can judge whether the HBV virus in the virus carrier liver is duplicating according to detecting having or not of large protein in the blood.
General random was inserted in the genomic dna after the expression member entered cell, and the expression level of target gene can have very big difference because of different insertion sites.Often need a large amount of screening operations of wasting time and energy in order to obtain a more satisfactory stable expression cell strain.
Hepatitis B (hepatitis B) treatment can be divided into two big classes: a class is an antiviral therapy.Antiviral therapy mainly is a Interferon, rabbit (IFNa) and nucleoside medicine, but curative effect is not satisfied.Another kind of is immunotherapy, mainly be the host at be infected by the virus hepatocellular immune response and cause inflammation cytokine mediated, immunotherapy can be broken immune tolerance state, induce effective immunity of organism, suppress and removing virus, therefore, the inoculation Hepatitis B virus vaccine is one of effective measures of control hepatitis B.
At present, the hepatitis B vaccine of using in the world mainly is that S albumen with yeast or expressing cho cell is as vaccine.Reorganization (yeast) Hepatitis B virus vaccine is the principal item of present domestic and international application, and Hepatitis B Vaccine Prepared From Yeast Recombinanted is single antigen; The reorganization of pasteur (CHO) Hepatitis B virus vaccine contains PreS2 and S antigen; Expressed the hepatitis B surface antigen that contains S and PreS1 gene in the Chinese hamster ovary celI of Virology Inst., China Academy of Preventive Medicine Sciences's development.
The recombinaant CHO cell Hepatitis B virus vaccine, since going into operation, supply falls short of demand always, but have the difficult problem that expression amount is low, cost is high always.Domestic production of vaccine is generally less than 5mg/L because of the cell strain expression amount is too low, causes yielding poorly, cost can be in any more.At present, research at home also mainly rests on how to utilize Chinese hamster ovary celI manufacturing Hepatitis B virus vaccine, does not also rise to the research in the screening field of stablizing the high expressing cell strain.
ELISA is that enzyme connects the abbreviation that immunosorbent is measured (Enzyme-Linked ImmunosorbnentAssay).It is a kind of immunoenzyme technics that grows up after immunofluorescence and radioimmunoassay technique.This technology has developed very rapidly since coming out the beginning of the seventies, has been widely used in biology and medical science applied many fields at present.
ELISA is based on immunological response, the very high experimental technique of a kind of susceptibility that the specific reaction of antigen, antibody and enzyme are combined to the efficient catalytic effect of substrate.Owing to carry out in the hole that is reflected at a kind of solid phase carrier-polystyrene microtiter plates of antigen, antibody, after a kind of reagent of every adding is hatched, can remove unnecessary free reactant by washing, thus warranty test result's specificity and stability.In actual applications, by different designs, concrete method steps can have multiple.Be used to detect antibody indirect method, be used to detect antigenic double antibody sandwich method and be used to detect small molecules antigen or haptenic antigenic competition method or the like.Relatively more commonly used is ELISA double antibody sandwich method and ELISA indirect method.
Summary of the invention:
The objective of the invention is to filter out a kind of Chinese hamster ovary celI strain that efficiently expresses hepatitis B surface antigen for vaccin that has in order to solve the deficiency that the above-mentioned background technology exists.
Purpose of the present invention realizes by following scheme: a kind of Chinese hamster ovary celI strain of hepatitis B surface antigen for vaccin has obtained the Chinese hamster ovary celI strain of its stably express first through orifice plate screening and gene amplification method.
The present invention identifies the specificity and the product output of above-mentioned cell strain product.Biological characteristics to above-mentioned cell strain adopts the ELISA method.
Embodiment:
One, the selection of high expression level strain
1. utilize orifice plate to filter out resistant cell clone's alive hole.
2. use the concentration of the full S gene protein of its supernatant of elisa assay.
3. the cell that expression amount is high changes in the orifice plate to be cultivated.
4. use the supernatant of the full S gene protein of ELISA methods analyst.
5. keep the concentration of expressing purpose antibody and meet the requirements of the clone.
Two, gene amplification
1. inoculating cell in the wave and culture bottle.
2. culturing cell in the substratum of screening pressure is arranged.
3. when cell density reaches density, collect substratum and be used for elisa assay.
4. remove those can not increase output with the pressure extension clone.
5. the repeat amplification protcol circulation arrives desired point up to output.
Three, preparation master cell bank
1. when each orifice plate filtered out the higher cell strain of expression amount, pair cell was counted, and frozen density is 5 * 10
6Individual viable cell/ml ,-80 ℃ are spent the night, and go in the liquid nitrogen then and preserve.
2. when filtering out the higher cell strain of expression amount in shaking bottle, freeze-stored cell screens frozen term harmonization with orifice plate.
3. pair cell carries out the check of mycoplasma contamination before the cell warehouse-in.
Claims (2)
1, a kind of stability and high efficiency is expressed the Chinese hamster ovary celI strain of vaccine with hepatitis B virus surface antigen.
2, a kind of by the expressed vaccine of the described cell strain of claim 1 HBV surface antigen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101071131A CN101302496A (en) | 2007-05-08 | 2007-05-08 | Stable and effective-expressing transgene CHO cell strain of hepatitis B surface antigen for vaccin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101071131A CN101302496A (en) | 2007-05-08 | 2007-05-08 | Stable and effective-expressing transgene CHO cell strain of hepatitis B surface antigen for vaccin |
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| Publication Number | Publication Date |
|---|---|
| CN101302496A true CN101302496A (en) | 2008-11-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101071131A Pending CN101302496A (en) | 2007-05-08 | 2007-05-08 | Stable and effective-expressing transgene CHO cell strain of hepatitis B surface antigen for vaccin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323436A (en) * | 2012-03-20 | 2013-09-25 | 无锡药明康德生物技术有限公司 | Method for screening high-output stable cell strain by HTRF |
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2007
- 2007-05-08 CN CNA2007101071131A patent/CN101302496A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323436A (en) * | 2012-03-20 | 2013-09-25 | 无锡药明康德生物技术有限公司 | Method for screening high-output stable cell strain by HTRF |
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Open date: 20081112 |