CN103387957B

CN103387957B - The application of human embryonic lung fibroblast SV-4

Info

Publication number: CN103387957B
Application number: CN201310359523.0A
Authority: CN
Inventors: 吕哲; 李雅静; 王巍巍; 高强; 尹卫东
Original assignee: Sinovac Research & Development Co Ltd
Current assignee: Sinovac Research & Development Co Ltd
Priority date: 2013-08-16
Filing date: 2013-08-16
Publication date: 2016-06-08
Anticipated expiration: 2033-08-16
Also published as: CN103387957A

Abstract

Does the present invention provide preserving number to be CGMCC? the human embryonic lung fibroblast SV-4 of No.6957 and application thereof, SV-4 cell characteristic is obvious, grow vigorous, life cycle is long, and average population doubling level was 68 generations, and pure pollution-free, cultural method is simple, multiple virus is had adaptability, relatively strong particularly with rubella virus adaptability, can be applicable to the research and development of rubella virus vaccine and preparation.

Description

The application of human embryonic lung fibroblast SV-4

Technical field

The present invention relates to biological technical field, specifically, relate to the application of human embryonic lung fibroblast SV-4.

Background technology

Human diploid cell derives from normal people tissue, it is possible to carries out the Secondary Culture of limited generation in vitro and sets up stable cell bank system, having normal people's diploid chromosome number order, without potential tumorigenicity. For vaccine for man, use human diploid cell is substrate, it is not necessary to consider the impact of foreign cell albumen and foreign DNA residual quantity. Mainly there are WI-38, MRC-5,2BS, KMB-17 etc. in current home and abroad for the human diploid cell of production of vaccine. The Clinical practice of more than 40 year experience have shown that human diploid cell possesses good safety and effectiveness as production of vaccine cellular matrix.

There is some shortcoming in human diploid cell liquid, affects its extensive use in production of vaccine, for instance the breeding generation of cell is limited, condition of culture requires higher, incubation time is longer, and viral yield is relatively low, and the subject matter existed in using human diploid cell process both at home and abroad includes:

(1) primordial seed is deficient, and the cell threshold life-span is shorter, and viral yield is low. Aforesaid 4 kinds of domestic and international general human diploid cell ultimate lives are about 50 generations, within production and application generation was limited to for 40 generations. The germinal cell seed generation obtained from official or other regular channels is close to 20 generations, being difficult to reach cell amplification desirable production scale, the more high cultivation difficulty of cell generation is more big simultaneously, and the growth conditions of cell is more poor, these all can directly influence the yield of virus, increases production cost.

(2) restriction of the use authority of cell and scope. The obtainable above-mentioned cell strain quantity for producing is extremely limited, if for producing, needs to pay high use license fee, also defines the kind producing vaccine, seriously hinder enterprise development and produce more multi items.

As can be seen here, existing human diploid cell strain cannot meet the demand of current production of vaccine, developing and develop biological nature good, virus wide accommodation, producing virus easier human diploid cell substrate becomes the urgent needs in production of vaccine field.

Summary of the invention

It is an object of the invention to provide the application of human embryonic lung fibroblast SV-4.

The human embryonic lung fibroblast SV-4 of the present invention, now it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, Datun Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, preservation date December in 2012 7 days, deposit number CGMCCNo.6957.

The present invention provides the extracorporeal culturing method of SV-4, is inoculated in by SV-4 cell in MEM, M199, DMEM or BME culture medium containing 5-10% tire cattle or calf serum, is placed in 35-37 DEG C, 5%CO₂Cultivate under condition.

The present invention also provides for SV-4 application in Virus culture, or utilizing the method that SV-4 cell prepares virus liquid: SV-4 cell is through amplification and Secondary Culture, virus inoculation on SV-4 cell after going down to posterity, cultivates and gathers in the crops metainfective SV-4 cell, it is thus achieved that viral suspension. Viral suspension after purification can be used for vaccine to be prepared.

In aforementioned applications or method, described virus is rubella virus and other virus.

In aforementioned applications or method, first SV-4 cell is cultured to cell confluency degree 90-100%, in point kind of the ratio Secondary Culture of 1:2-4, virus inoculation when cell reaches 90-100% degree of converging.

In aforementioned applications or method, by virus inoculation on 0.01-0.1MOI SV-4 cell after going down to posterity, it is placed in 31-38 DEG C, 5%CO₂Cultivate under condition.

In aforementioned applications or method, the culture medium cultivating the use of SV-4 cell is MEM, M199, DMEM or BME culture medium containing 5-10% tire cattle or calf serum.

The human embryo lung (HEL) that the present invention uses becomes fiber diploid cell SV-4, under current detection level, is not detected by intraor extracellular source virokine and pollutes, for pure free of contamination human diploid fibroblasts.

The human embryo lung (HEL) that the present invention uses becomes fiber diploid cell SV-4 to be proved in nude mice animal experiment, and this cell strain does not have tumorigenicity.

The human embryo lung (HEL) that the present invention uses becomes fiber diploid cell strain SV-4 to have following biological characteristics:

(1) cell is cultivated simple

In MEM, M199, DMEM or BME culture medium of the tire cattle containing 5-10% or calf serum, in 35-37 DEG C, 5%CO₂Cultivate SV-4 cell under condition, after fine and close monolayer to be formed, carry out continuous passage according to the ratio 1:2-4 of going down to posterity.

(2) rubella virus sensitivity is higher

Study by the SV-4 cell of the present invention being carried out the adaptability of multiple virus, find that it is stronger to rubella virus adaptability, with same virus inoculation condition and condition of culture, the rubella virus titre mean height 0.5lgCCID that the rubella virus that SV-4 cell is cultivated is cultivated compared with MRC-5 cell₅₀/mL��

Accompanying drawing explanation

Fig. 1 is the rubella virus titre of continuous 21 days that SV-4 cell is cultivated.

The virus titer of absorption 1 hour and direct inoculation when Fig. 2 is inoculation rubella virus.

Fig. 3 is the titre that SV-4 cell and MRC-5 cell cultivate rubella virus respectively.

Fig. 4 is the titre result of variations that the rubella virus that SV-4 cell is cultivated is saved in 2-8 DEG C.

Fig. 5 is the titre result of variations that the rubella virus that MRC-5 cell is cultivated is saved in 2-8 DEG C.

Detailed description of the invention

Following example are used for illustrating the present invention, but are not limited to the scope of the present invention. If not specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art, raw materials used it is commercial goods.

Embodiment 1 rubella virus adaptability in SV-4

Cultivate SV-4 cell to 2 �� 10⁷Individual/175cm², being worked by rubella virus, (ATCC is numbered VR-135q in seed RA27/3 strain^Tm) it is 0.01��0.1 inoculating cell according to MOI, add the cell maintenance medium of 2% tire cattle or calf serum, put 32.0 �� 1.0 DEG C or 37.0 �� 1.0 DEG C cultivations; sample every three days and detect virus titer; gathered in the crops whole virus liquid at the 6th, 12,18 days, change cell maintenance medium, co-culture 21 days.Result is shown in Fig. 1. Cultivate SV-4 cell to 2 �� 10⁷Individual/175cm², being worked by rubella virus, (ATCC is numbered VR-135q in seed RA27/3 strain^Tm) it is 0.01��0.1 inoculating cell according to MOI, add the cell maintenance medium of 2% tire cattle or calf serum, adsorb 1h at 32.0 �� 1.0 DEG C or do not adsorb; sample every three days and detect virus titer; gathered in the crops whole virus liquid at the 6th, 12,18 days, change cell maintenance medium, co-culture 21 days. Result is shown in Fig. 2. Result shows that the vaccination ways of rubella virus can adopt direct inoculation and absorption inoculating two kinds method. Cultivate SV-4 nucleus and MRC-5 cell to 2 �� 10⁷Individual/175cm², being worked by rubella virus, (ATCC is numbered VR-135q in seed RA27/3 strain^Tm) it is 0.01��0.1 inoculating cell according to MOI, add the cell maintenance medium of 2% tire cattle or calf serum, put 32.0 �� 1.0 DEG C; cultivate, sample every three days and detect virus titer, gathering in the crops whole virus liquid at the 6th, 12 days; change cell maintenance medium, co-culture 14 days. Relatively two kinds of cells are the virus titer of substrate, and result is shown in Fig. 3. The rubella virus mean height 0.5lgCCID that the rubella virus that SV-4 cell is cultivated is cultivated than MRC-5 cell₅₀/ mL, it was shown that SV-4 cell is to well adapting to property of rubella virus. With the rubella virus that identical results condition results SV-4 cell and MRC-5 cell are cultivated, and add 1%PVP, 2%PVP and 3%PVP respectively as protective agent. Place the same time for 2-8 DEG C, the change of detection virus titer. Fig. 4 is the titre result of variations of the rubella virus that SV-4 cell is cultivated. Fig. 5 is the titre result of variations of the rubella virus that MRC-5 cell is cultivated. Result shows that the viral 2-8 DEG C of titre on average declined after placing 14 days that two kinds of cells are cultivated is basically identical.

Although, above the present invention is described in detail with a general description of the specific embodiments, but on basis of the present invention, it is possible to it is made some modifications or improvements, and this will be apparent to those skilled in the art. Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Claims

1. the cultural method of a rubella virus, it is characterized in that, deposit number be CGMCCNo.6957 human embryonic lung fibroblast SV-4 cell through amplification and Secondary Culture, on the SV-4 cell after going down to posterity by 0.01-0.1MOI inoculate rubella virus, it is placed in 31-38 DEG C, 5%CO₂Cultivate under condition and gather in the crops metainfective SV-4 cell, it is thus achieved that rubella virus suspension.

2. method according to claim 1, it is characterised in that SV-4 cell is first cultured to cell confluency degree 90-100%, in point kind of the ratio Secondary Culture of 1:2-1:4, virus inoculation when cell reaches 90-100% degree of converging.

3. method according to claim 1, it is characterised in that by virus inoculation on 0.01-0.1MOI SV-4 cell after going down to posterity, be placed in 32 DEG C, 5%CO₂Cultivate under condition.

4. method according to claim 1, it is characterised in that the culture medium cultivating the use of SV-4 cell is MEM, M199, DMEM or BME culture medium containing 5-10% tire cattle or calf serum.