CN110669739A - Preparation method of novel hepatitis A virus antigen - Google Patents
Preparation method of novel hepatitis A virus antigen Download PDFInfo
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Abstract
The invention discloses a preparation method of a novel hepatitis A virus antigen, which comprises the following steps: step one, HAV contained in the culture supernatant of the attenuated strain of the hepatitis A virus strain L-A-1 with the preservation number of CCTCC No. V92004 is cloned, purified and screened to obtain an L-A-1M strain; step two, inoculating the L-A-1M strain to a human embryo lung diploid cell, culturing in a culture solution, changing a maintenance solution once in 10-17 days, collecting a supernatant, changing the maintenance solution, reserving a sample, and collecting the culture supernatant for 5 times in total; step three, collecting the supernatant at the 5 th time, digesting the supernatant by trypsin, and collecting the virus harvest containing the hepatitis A virus by using Hank's solution with 1/5 liquid maintaining amount; and step four, processing the virus harvest to obtain the hepatitis A virus antigen.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a novel hepatitis A virus antigen.
Background
Hepatitis a is an acute and often self-limiting infectious disease caused by Hepatitis A Virus (HAV), which is transmitted via the intestinal tract and is transmitted in the human population mainly by the fecal oral route. Numerous practices have shown that vaccination with hepatitis a vaccines is the most effective measure for preventing and controlling hepatitis a epidemics.
Hepatitis A Virus (HAV) is a small non-enveloped RNA virus, designated Enterovirus type 72 in 1982, but it has been found by further studies to differ from other small RNA viruses (enteroviruses) in at least 6 ways: (1) HAV differs in nucleotide and amino acid sequence characteristics from other viruses; (2) HAV adapts, grows, replicates slowly in cell culture; HAV has a long replication cycle, requiring weeks for proliferation in cell culture, whereas enteroviruses require only days; (3) do not normally cause Cytopathic effects (CPE); (4) stable to heat and low pH, resistant to organic solvents, and insensitive to chloroform; HAV is stable at 60 ℃ while enteroviruses are unstable; (5) HAV is propagated in liver cells, i.e., hepatotrophic; (6) HAV has multiple genotypes, but only one serotype. Therefore, in 1991 a new genus was established in the picornaviridae family, called hepacivirus (Hepatovirus). HAV is classified as the only species of this genus, Hepatovirus, and Hepatovirus is also translated by humans as a hepatotropic virus.
HAV genome RNA can be directly used as a template for coding synthetic protein, namely mRNA, and belongs to positive single-stranded RNA (+ ssRNA) during the replication process of HAV genome, virus genome RNA is used as the template to replicate to form complementary negative single-stranded RNA (-ssRNA), then-ssRNA is synthesized into offspring + ssRNA, newly synthesized offspring + ssRNA has 3 functions, ① is used as mRNA to translate to generate more virus protein, ② is used as model synthesis-ssRNA, ③ is used as progeny virus genome to package, HAV genome is replicated and capsid protein is synthesized, and then virus particles are assembled and released outside cells.
HAV can be propagated and passaged using primary African monkey kidney cells, passaged monkey kidney cells, human fibroblasts, human hepatoma cells, human embryonic kidney cells, human embryonic lung diploid cells, and the like.
The existing hepatitis A virus antigen main preparation method comprises the following steps: (1) the culture solution mainly comprises MEM or Earle's solution containing appropriate amount of de-energized newborn calf serum; (2) inoculating the virus seeds into cells according to the MOI of 0.05-1.0, and culturing at an appropriate temperature, wherein a maintenance solution is replaced according to the growth condition of the cells during the culture period, and the maintenance solution is a liquid such as MEM (MEM) or Earle's solution containing newborn bovine serum with an appropriate concentration; (3) culturing until the virus propagation peak (generally 21-28 days), digesting cells containing the hepatitis A virus by adopting trypsin with proper concentration or other proper methods, and collecting the cells as a virus harvest by a centrifugation or filtration method; (4) after the qualified virus harvest is processed by freeze thawing and/or ultrasonic wave or other suitable methods to harvest the virus, the hepatitis A virus is extracted by methods such as trichloromethane extraction and the like.
The defects of the prior art mainly comprise: (1) most of the used hepatitis A virus seeds are slowly proliferated, and the proliferation period is longer by about 4 weeks; (2) most of virus culture maintenance liquid uses bovine serum, so that the cost is high, and the later extraction and purification of the hepatitis A virus antigen are not facilitated; (3) only hepatitis A virus contained in cells or virus antigen in culture supernatant is collected, so that the amount of the harvested virus is small, and resources are wasted; (4) most hepatitis A virus antigens in culture supernatant are only harvested once, the harvested virus amount is small, and the advantages of continuous and multiple harvesting and liquid changing of virus culture cannot be exerted.
Disclosure of Invention
Based on the problems in the prior art, the invention designs and develops a novel hepatitis A virus antigen preparation method, and aims to solve the problems that most of the used hepatitis A virus seeds are slow in proliferation, the virus culture maintenance solution is high in cost, the virus yield is low, and the advantages of continuous and repeated virus culture harvest and solution change cannot be exerted.
The technical scheme provided by the invention is as follows:
a method for preparing a novel hepatitis A virus antigen comprises the following steps:
step one, HAV contained in the culture supernatant of the attenuated strain of the hepatitis A virus strain L-A-1 with the preservation number of CCTCC No. V92004 is cloned, purified and screened to obtain an L-A-1M strain;
step two, inoculating the L-A-1M strain to a human embryo lung diploid cell, culturing in a culture solution, changing a maintenance solution once in 10-17 days, collecting a supernatant, changing the maintenance solution, reserving a sample, and collecting the culture supernatant for 5 times in total;
step three, collecting the supernatant at the 5 th time, digesting the supernatant by trypsin, and collecting the virus harvest containing the hepatitis A virus by using Hank's solution with 1/5 liquid maintaining amount;
and step four, processing the virus harvest to obtain the hepatitis A virus antigen.
Preferably, in the step one, the clone purification screening process is performed 3 times.
Preferably, in the second step, the culture medium is a MEM culture medium containing 8% to 12% newborn bovine serum.
Preferably, in the second step, the culture solution is a low serum culture solution containing a proper amount of newborn bovine serum.
Preferably, in the second step, the maintaining solution is a MEM culture solution containing 0% to 1% of hydrolyzed milk protein.
Preferably, in the second step, the human embryo lung diploid cell culturing process comprises:
recovering 1 or more cells in the working cell bank according to a ratio of 1:2, digesting the cells into a monolayer by using 0.25% trypsin after the cells grow into the monolayer, carrying out amplification passage according to a ratio of 1:4, and standing or rotating the cells at a temperature of between 37 and plus or minus 0.5 ℃ for culture.
Preferably, in the second step, the L-A-1M strain is inoculated at an MOI of 0.05 to 1.0.
Preferably, in step three, the concentration of trypsin is 0.25%.
Compared with the prior art, the invention has the following beneficial effects:
1. the used virus seeds are L-A-1M strains of hepatitis A virus with independent intellectual property rights, the proliferation cycle is relatively short and about 14 days, HAV is contained in both culture supernatant and cultured cells, serum-free maintenance liquid can be used for culture, a mode of continuously harvesting and changing the liquid for multiple times can be adopted for culture, a cell factory is adopted for static and large-scale production process for culture, the production efficiency is improved, and the culture progress is accelerated;
2. the production cell is a human embryonic lung diploid cell (2BS strain) which is humanized, can be subcultured in limited generations, has no exogenous substances and tumorigenicity, and has good safety;
3. the hepatitis A virus culture is carried out by utilizing static culture of a cell factory and adopting a mode of continuously harvesting and changing liquid for multiple times, thereby being beneficial to saving the production cost, improving the production efficiency and reducing the risk of pollution;
4. the virus culture maintenance solution adopts a bovine serum-free culture solution, which is beneficial to reducing the production cost, reducing the introduction opportunity of exogenous substances, being beneficial to later-stage extraction and purification of the hepatitis A virus antigen and reducing the pressure of the subsequent purification process;
5. the hepatitis A virus antigen in the culture supernatant is continuously harvested for multiple times, the yield per unit and the production efficiency are improved, and the operation is simple, convenient and feasible;
6. meanwhile, hepatitis A virus antigens contained in the cells are collected, so that the yield of the virus antigens is ensured, and waste is avoided;
7. respectively extracting hepatitis A virus antigens from the harvested materials collected by different ways by different and appropriate methods; after the hepatitis A virus antigen in the culture supernatant is clarified, the hepatitis A virus antigen is extracted by adopting technologies such as ultrafiltration, molecular sieve chromatography and the like; releasing HAV antigen from hepatitis A virus antigen contained in cells by freeze thawing, ultrasonic or cracking, and extracting by PEG precipitation, centrifugation, chloroform extraction, ultrafiltration, ion exchange chromatography, molecular sieve chromatography, etc.; is beneficial to improving the production process, reasonably utilizing resources and simplifying the production operation steps.
Drawings
Fig. 1 is a schematic structural diagram of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
As shown in FIG. 1, the present invention provides a method for preparing a novel hepatitis A virus antigen, which is characterized by comprising the following steps:
step one, HAV contained in the culture supernatant of the attenuated strain of the hepatitis A virus strain L-A-1 with the preservation number of CCTCC No. V92004 is cloned, purified and screened to obtain an L-A-1M strain;
step two, inoculating the L-A-1M strain to a human embryo lung diploid cell, culturing in a culture solution, changing a maintenance solution once in 10-17 days, collecting a supernatant, changing the maintenance solution, reserving a sample, and collecting the culture supernatant for 5 times in total;
step three, collecting the supernatant at the 5 th time, digesting the supernatant by trypsin, and collecting the virus harvest containing the hepatitis A virus by using Hank's solution with 1/5 liquid maintaining amount;
and step four, processing the virus harvest to obtain the hepatitis A virus antigen.
Examples
1. Cell and virus seed
The human embryonic lung diploid cell (2BS strain) and the hepatitis A virus L-A-1M strain are prepared and provided by two chambers of vaccine of the Limited liability company of the research institute of biological products of Changchun.
The human embryonic lung diploid cell (2BS strain) is from Beijing institute of biological products; the cell bank is a secondary management, namely a main cell bank and a working cell bank; the diploid cell 2BS strain is from 11 generations, the main cell bank is from 18 generations, and the working cell bank is from 24 generations.
The preparation process of the hepatitis A virus L-A-1M strain comprises the following steps: first, HAV contained in culture supernatant of attenuated strain of hepatitis A virus strain L-A-1 was subjected to 104、105、106、107Diluting, adding into 2BS cells grown into a single layer in a 24-well plate for culture, adding 5 wells for each dilution, collecting the solution every two weeks, supplementing the maintenance solution, and detecting the antigen content of the collected solution; secondly, the liquid with high antigen content is selected for 106、107、108、109Diluting, adding into 2BS cells grown into a single layer in a 24-well plate for culture, adding 5 wells for each dilution, collecting the solution every two weeks, supplementing the maintenance solution, and detecting the antigen content of the collected solution; thirdly, the liquid with high antigen content is selected to be 106、107、108、109Diluting, adding into 2BS cells grown into a single layer in a 24-well plate for culture, adding 5 wells for each dilution, collecting the solution every two weeks, supplementing the maintenance solution, and detecting the antigen content of the collected solution; finally, the harvest solution with high antigen content is selected to be subjected to amplification culture in the 2BS cells which grow into a monolayer, and the L-A-1M strain is obtained
2. Main reagent and instrument
25cm2And 225cm2Cell culture flasks were purchased from corning, usa; cell factories, 10-and 40-layer, were purchased from denmark NUNC and corning, usa; MEM medium was purchased from Nikkiso pharmaceutical Co; TC hydrolyzed milk protein was purchased from BD, usa; trypsin was purchased from GIBCO; newborn bovine serum was purchased from bio-engineering ltd of national sea, langzhou; the hepatitis A virus Ag detection kit (ELISA method) is purchased from Beijing Wantai biological pharmaceutical industry, Inc.
3. Cell preparation
Taking 1 or more cells in a working cell bank, recovering the cells according to the ratio of 1:2, growing a monolayer, digesting the monolayer with 0.25% trypsin, carrying out amplification passage according to the ratio of 1:4, standing the cell at the temperature of 37 +/-0.5 ℃ or carrying out rotary culture to prepare a certain number of cells for inoculating the virus, and taking the cells as a cell batch to carry out parallel experiments, wherein the cell batch is recorded as a first batch, a second batch, a third batch and a fourth batch respectively.
4. Culture solution
The culture solution is MEM culture solution containing 8-12% newborn calf serum or low serum culture solution containing a proper amount of newborn calf serum.
The maintenance liquid is MEM culture liquid containing 0 to 1 percent of hydrolyzed milk protein.
5. Virus inoculation and culture
Inoculating the diploid cells of human embryo lungs with the production virus seeds according to the MOI of 0.05-1.0, carrying out rotary or static culture at 35 +/-0.5 ℃, changing the maintenance solution once in 10-17 days, collecting culture supernatant, changing the solution, reserving samples, and collecting the culture supernatant for 5 times. After the fifth culture supernatant was collected, it was digested with 0.25% trypsin, and cells containing hepatitis A Virus were collected as a virus harvest using 1/5 holding solution of Hank's solution. The virus harvest is subjected to freeze-thawing and/or sonication or other suitable methods to harvest the virus.
According to the attached drawing, the harvested materials collected by different ways are respectively extracted by different and proper methods.
1. Hepatitis a virus antigen in culture supernatant: after being clarified by centrifugation for 30 minutes at 4000r/min, an ultrafiltration membrane pack with the molecular weight cut-off of 100kD is adopted for ultrafiltration concentration and washing filtration, and Sepharose 6 Fast Flow is taken as a gel medium for molecular sieve chromatography for extraction.
2. Hepatitis a virus antigen contained in cells: cracking cells by using a sodium deoxycholate cracking agent with the final concentration of 0.1-0.5% for more than 1 hour to release HAV antigen; precipitating at 2-8 ℃ for more than 12 hours by using PEG6000 with the final concentration of 6-8%, centrifuging at 4000r/min for more than 30 minutes, and collecting the centrifugal precipitate by using PBS buffer solution with the liquid amount of 1/10 liquid; extracting the collected liquid for 3-5 times by using chloroform with the volume of 1/2, and collecting supernatant; then an ultrafiltration membrane package with the molecular weight cutoff of 100kD is adopted to carry out ultrafiltration concentration and washing filtration on the collected supernatant; then DEAE Sepharose Fast Flow is used as gel medium to carry out anion exchange chromatography and/or Sepharose 6 Fast Flow is used as gel medium to carry out molecular sieve chromatography for extraction.
Test results
The hepatitis A virus detection kit (ELISA method) is adopted to detect the hepatitis A virus contained in the virus culture supernatant and the cells. The results of the detection of the presence of hepatitis A virus in the virus culture supernatant and the cells (the highest positive dilution) are shown in Table 1.
TABLE 1 hepatitis A Virus test results (highest positive dilution factor)
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (8)
1. A method for preparing a novel hepatitis A virus antigen is characterized by comprising the following steps:
step one, HAV contained in the culture supernatant of the attenuated strain of the hepatitis A virus strain L-A-1 with the preservation number of CCTCC No. V92004 is cloned, purified and screened to obtain an L-A-1M strain;
step two, inoculating the L-A-1M strain to a human embryo lung diploid cell, culturing in a culture solution, changing a maintenance solution once in 10-17 days, collecting a supernatant, changing the maintenance solution, reserving a sample, and collecting the culture supernatant for 5 times in total;
step three, collecting the supernatant at the 5 th time, digesting the supernatant by trypsin, and collecting the virus harvest containing the hepatitis A virus by using Hank's solution with 1/5 liquid maintaining amount;
and step four, processing the virus harvest to obtain the hepatitis A virus antigen.
2. The method for preparing a novel hepatitis A virus antigen according to claim 1, wherein in the first step, the cloning purification screening process is performed 3 times.
3. The method for producing a novel hepatitis A virus antigen according to claim 2, wherein in the second step, the culture medium is a MEM culture medium containing 8% to 12% newborn bovine serum.
4. The method for producing a novel hepatitis A virus antigen according to claim 3, wherein in the second step, the culture medium is a low serum culture medium containing a suitable amount of newborn bovine serum.
5. The method for producing a novel hepatitis A virus antigen according to claim 3 or 4, wherein in the second step, the maintenance solution is an MEM culture solution containing 0% to 1% of hydrolyzed milk protein.
6. The method for preparing the novel hepatitis A virus antigen according to claim 5, wherein in the second step, the human embryonic lung diploid cell culture process comprises:
recovering 1 or more cells in the working cell bank according to a ratio of 1:2, digesting the cells into a monolayer by using 0.25% trypsin after the cells grow into the monolayer, carrying out amplification passage according to a ratio of 1:4, and standing or rotating the cells at a temperature of between 37 and plus or minus 0.5 ℃ for culture.
7. The method for producing a novel hepatitis A virus antigen according to claim 6, wherein in the second step, the L-A-1M strain is inoculated at an MOI of 0.05 to 1.0.
8. The method for producing a novel hepatitis A virus antigen according to claim 6 or 7, wherein the concentration of trypsin in step three is 0.25%.
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| CN114350624A (en) * | 2021-12-28 | 2022-04-15 | 艾美康淮生物制药（江苏）有限公司 | Method for preparing hepatitis A inactivated vaccine |
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