CN101525597B - New hepatitis A inactivated vaccine virus strain and method for culturing same - Google Patents

Abstract

The invention provides a new hepatitis A virus JS-4 strain for preparing hepatitis A inactivated vaccine and a method for culturing the same. The strain is separated from deject a of a patient infected with acute hepatitis A and is transmitted to a Vero cell for adapted culture; through neutralization tests and other methods, the strain is proved to be hepatitis A virus; in the adaptation process, the subculture cycle of early generation is 28 days; when the strain is transmitted to the tenth generation, the cycle is shortened to 21 days; the strain is continuously cultured for ten generations; the culture temperature is between 35 and 36 DEG C; the cycle for virus propagation is shortened from 28 to 21 days; the antigen titer can reach 1:640-1:1,280; and the infectious titer is between 106.67 and 107.50 CCID50/mL. The marmoset virulence test proves that the strain has weak virulence, is reliable in safety and has good immunogenicity and protective effect when the strain is used for producing the hepatitis A inactivated vaccine and is an ideal strain for producing the hepatitis A inactivated vaccine.

Description

New hepatitis A inactivated vaccine virus strain and cultural method thereof

Technical field

The present invention relates to a kind of new hepatitis A virus JS-4 strain and cultural method thereof for preparing hepatitis A inactivated vaccine.

Background technology

Hepatitis A is to be infected the acute infectious disease of a kind of serious harm human health that causes by hepatitis A virus.In a developing country especially important public health problem, show as higher sickness rate and localized epidemics, even outburst causes death.Therefore the preventing and controlling of hepatitis A occupy an important position in the prevention and control of diseases in the whole world.

China is the high popular district of hepatitis A, has at least every year 240000 people to suffer from hepatitis A and causes enormous economic loss and social influence.The Premier Wen Jiabao proposes in five the meeting government work report of ten National People's Congress the immunity of bringing into the orbit of state planning of transmissible diseases such as hepatitis A, epidemic meningitis.

China has three manufacturer production Attenuated HAVs now; Two manufacturer production hav inactivated vaccines, but owing to strain, the cell matrix of each family's use are different, output and quality is all variant; China is populous, and hepatitis A vaccine still far can not satisfy the requirement of home market.

Summary of the invention

One aspect of the present invention relates to a kind of new hepatitis A virus strain, called after JS-4 strain, and this strain is preserved in Chinese typical culture collection center on January 25th, 2008, and deposit number is CCTCC NO:V200802.This virus stain antigen titre can reach 1: 640-1: 1280; Infection titer is 106.67-107.50CCID50/mL; A little less than the marmoset virulence test proves that this strain virulence; Produce hepatitis A inactivated vaccine not only more reliable aspect the security with it, have good immunogenicity and protection effect, and this strain has the characteristic that proliferating cycle is short on the Vero cell, reproductive efficiency is high; Being suitable for doing the hepatitis A inactivated vaccine seed culture of viruses of large-scale industrial production, is the desirable strain of producing hepatitis A inactivated vaccine.

The present invention relates to the cultural method of HAV on the other hand, and this method may further comprise the steps:

(a) from hepatitis A acute infection patient ight soil, separate, identify through biological characteristics and confirm as hepatitis A virus;

(b) with on the Vero cell, cultivating after the activated carbon treatment, early the generation culture cycle is 28 days, foreshortens to 21 days after reaching for 10 generations, continues to cultivate for 10 generations, and culture temperature is 35 ℃-36 ℃, and incubation time shortens from 28 days that to cultivate up to cultivating the peak be 21 days;

(c) select the virus after step (b) cell cultures adapts to test the back as the strain of producing vaccine.

The invention still further relates to the antigenic method of a kind of preparation hepatitis A inactivated vaccine virus, it is characterized in that comprising the steps:

(a) static or three-dimensional absorption Cultivation of Vero in nutrient solution makes cell concn reach 10 ⁶-10 ⁷/ mL;

(b) being 0.2-0.5 with infection multiplicity (M.O.I) is inoculated into the hepatitis A virus strain JS-4 strain of claim 1 in the Vero cell culture fluid of above-mentioned cell concn;, 37 ℃ of cells with suspension are inoculated in three layers of bottle of 3L after adsorbing 30-50min; Put 35-36 ℃ of cultivation; Changed liquid once in per during this time seven days, received malicious in 21 days;

(c) obtain virus-cell cultures suspension with Digestive system digestion;

(d) after broken extracting, concentrated and purified, deactivation, obtain hepatitis A inactivated vaccine virus antigen.

Than prior art; Strain provided by the invention is used for the production of hav inactivated vaccine; Can improve the output and the quality of vaccine greatly, the hepatitis A strain that is adapted to the Vero cell at home and abroad all maintains the leading position, and is more suitable for large-scale industrial production than human diploid cell.

Below in conjunction with embodiment the present invention is done further detailed explanation.

The cultural method of embodiment 1 hepatitis A virus strain JS-4 strain

The acute phase ight soil of gathering from clinical hepatitis A patient is prepared into 20% suspension, handles the centrifugal 30min of 10000rpm afterwards with 5% vegetable active charcoal, resets and add an amount of microbiotic in the absorption, put 4 ℃ subsequent use.

After treating that little square vase Vero cell grows up to individual layer, wash the cell face once, every bottle graft kind 2ml fecal suspension, 37 ℃ of absorption 2 hours, additional cell maintenance medium is put 36 ℃ and is cultivated and to receive poison in 28 days, during changed liquid once in per seven days.

Separate the sample that goes down to posterity by (b) step and proceed the cultivation of the cell adapted property of Vero.Different is every bottle of cell inoculation 1mL sample, and 37 ℃ of absorption 2 hours was cultivated 28 days for 36 ℃, reaches to reduce incubation time to 21 day after 10 generations, continues to pass 10 generations again, passes for 5 generations at Kolle flask earlier, and the 3L monolayer bottles passed for 3 generations, and three layers of bottle of 3L passed for 2 generations.

Wherein, each cultivate expiration after, with the virus-cell cultures suspension of pancreatin EDTA digestion acquisition, it is subsequent use to put-80 ℃ of preservations.With ultrasonic disruption appearance smudge cells, percentage of damage reaches more than 95%, is that 0.2-0.5 inoculates with infective dose (M.O.I) before the inoculation.

The final strain hepatitis A virus strain that obtains, called after JS-4 strain, this strain is preserved in Chinese typical culture collection center on February 25th, 2008, and deposit number is CCTCC-V200802.

Being characterized as of this hepatitis A inactivated vaccine virus strain JS-4 strain:

Virion diameter: 27-32nm;

Acid resistance: under the pH3.0 condition, 2-8 ℃ acts on 18 hours, and infection titer does not have obvious influence;

Ether-resistant property: through ether 2-8 ℃ of effect 18 hours, infection titer did not have obvious influence;

Neutralization test: neutralized by the HAV specific antibody;

Gene sequencing: belong to same gene hypotype up to 98.2% with hepatitis A virus gene sequence homology among the NCBI Gene Bank;

Antigen titre: 1: 640-1: 1280;

Infection titer: 106.67-107.50CCID50/mL;

Bred peak period: 21-28 days;

The righttest culture temperature: 35 ℃-36 ℃;

Breeding position: in the cell bag slurry;

Has the HAV characteristic.

The antigenic method of embodiment 2 preparation hepatitis A inactivated vaccine virus

Take out the cell cryopreservation pipe from Vero cell work seed bank, put into 40 ℃ of water rapidly and dissolve, be added to then in 199 nutrient solutions of 37 ℃ of preparatory temperature, plant the cell bottle, put 37 ℃ of cultivations, after growing up to individual layer in 3-4 days, in amplification number generation, is until the regulation generation of production usefulness.

Above-mentioned cell is pressed 0.2MOI add the hepatitis A virus strain JS-4 strain that embodiment 1 obtains; 37 ℃ with the cells absorption 40min that suspends after be inoculated in three layers of bottle of 3L, put 36 ℃ of cultivations, during changed liquid once in per seven days; Received poison in 21 days; Wherein the nutrient solution composition is 199 substratum, contains the 2-8% calf serum and cultivates virus-cell cultures suspension that the expiration back obtains with Digestive system digestion, after broken extracting, concentrated and purified, deactivation, obtains hepatitis A inactivated vaccine virus antigen.

The related experiment data are seen following tabulation among the present invention.Proof JS-4 strain has the characteristic of efficient stable propagation on the Vero cell, a little less than the marmoset virulence test proves that this strain virulence, produces hepatitis A inactivated vaccine not only more reliable aspect the security with its, has good immunogenicity and protection effect.

Different generation antigen titres of table 1 JS-4 strain and infection titer

Generation	Antigen titre (EU/0.1mL)	Infection titer (lgCCID 50/mL)
			10 15 18 20	320 640 640 1280	4.50 6.00 6.67 7.50

Table 2 JS-4 strain 19 generations propagation peak period test-results

Generation time (my god)	12	16	20	21	22	23	24	25	26	28	30
												Antigen titre (EU/0.1 mL)	?12 ?80	?12 ?80	?25 ?60	?25 ?60	?256 ?0	?256 ?0	?128 ?0	?128 ?0	?128 ?0	?128 ?0	640

Table 3 JS-4 strain virulence situation is (marmoset test) relatively

Group

The inoculation strain

The marmoset numbering

Anti--HAV titre (week)

Serum enzyme labeled compound assay (before the inoculation)

Serum enzyme labeled compound assay (inoculation back)

The ight soil toxin expelling

The hepatic tissue pathology inspection

?

?

?

Before the inoculation

After the inoculation

ALT

?AST

ALT

AST

?

?

?

?

?

The experimental group control group

JS-4 strain JS-12 HAV virulent strain

7 8 ?9 ?10

--?-?-

1∶64(8) 1∶1024(8) ?1∶4(8) ?1∶2(3)

- - ?- ?-

- - ?- ?-

- - ?+ ?+

- - ?+ ?+

- - ?+ ?+

- - ?++ ?++

Claims (2)

1. hepatitis A virus strain, called after JS-4, this strain deposit number is CCTCC-V200802.

2. one kind prepares the antigenic method of hepatitis A inactivated vaccine virus, it is characterized in that comprising the steps:

(a) static or three-dimensional absorption Cultivation of Vero in nutrient solution makes cell concn reach 10 ⁶-10 ⁷/ mL;

(b) being 0.2-0.5 with infection multiplicity (M.O.I) is inoculated into the hepatitis A virus strain JS-4 strain of claim 1 in the Vero cell culture fluid of above-mentioned cell concn;, 37 ℃ of cells with suspension are inoculated in three layers of bottle of 3L after adsorbing 30-50min; Put 35-36 ℃ of cultivation; Changed liquid once in per during this time seven days, received malicious in 21 days;

(c) obtain virus-cell cultures suspension with Digestive system digestion;

(d) after broken extracting, concentrated and purified, deactivation, obtain hepatitis A inactivated vaccine virus antigen.