CN102041246A

CN102041246A - Human embryo retina transformed cell line for producing biological products

Info

Publication number: CN102041246A
Application number: CN2009101972546A
Authority: CN
Inventors: 梁旻; 石长信
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-10-16
Filing date: 2009-10-16
Publication date: 2011-05-04

Abstract

The invention constructs a human embryo retina transformed cell line SL-2 by transforming adenovirus gene fragments into human embryo retina cells. The cell line can be used for producing biological products such as adenovirus and the like, and has obvious advantages. The cell line can also be used for producing influenza virus, herpes simplex virus, measles virus, rotavirus and other viruses, polypeptides and other biological products.

Description

Be used for people's embryo retina transformation cell lines that biological products are produced

Technical field

The present invention relates to biological technical field, particularly utilize gene recombination technology to make up in order to producing the clone of biological products such as virus, vaccine and recombinant polypeptide medicine, and the purposes of this clone in biological products preparation process such as virus.

Background technology

The production of a certain specific virus needs specific host cell system, usually, exists coadapted relation between virus and its host cell.For example, be widely used in the research adenovirus carrier in fields such as the treatment of heredopathia, malignant tumour and novel gene engineered vaccine, host cell is the HEK293 cell the most widely in vitro culture adenovirus process.In recent years, along with the application of adenovirus carrier is increasingly extensive, particularly along with a large amount of, the deep clinical trial that is applied as various diseases of adenovirus carrier, it is found that when utilizing HEK293 cell cultures first-generation adenovirus carrier (the E1 district is or/and E3 district disappearance), be integrated in adenoviral sequence (the adenovirus DNA fragment that comprises 4.3kb length in the HEK293 cellular genome, this segment DNA contains complete adenovirus E 1 a and E1b sequence and E1a promoter region) can recombinate with first-generation adenovirus carrier, thus produce reverse mutation wildtype adenovirus virus.This phenomenon is a great potential safety hazard for the clinical application of adenovirus carrier.

This defective at the HEK293 cell, Holland Crucell company has developed a kind of new clone PER.C6, this cell strain derives from people's embryo retinoblast (retinoblasts) of adenovirus E 1 a/E1b gene transformation, uses the PER.C6 cell can significantly reduce or stop reverse mutation wildtype adenovirus virus as host cell generation in the process of scale operation adenovirus carrier.Research subsequently and application have enlarged the use range of this cell strain, this cell strain are applied to also obtain good effect in the production of antibody producing and influenza vaccines at present.

Summary of the invention

The purpose of this invention is to provide a kind of improved people's embryo retina transformation cell lines SL-2, this clone has the advantages that to be better than the HEK293 cell.Another object of the present invention provides the purposes of virus, vaccine and recombinant polypeptides such as utilizing SL-2 clone production adenovirus, influenza virus, hsv, Measles virus and rotavirus.

People's embryo retinoblast is suitable for making up the cell strain of production virus product, vaccine and recombinant polypeptide kind biological product, and in order to be applied to the production of extensive biological products, need to build up and can stablize the transformation cell lines that goes down to posterity.The present invention adopts method different and the Per.C6 cell construction, utilizes suitable adenoviral gene fragment to transform people's embryo retina cell, has successfully constructed people's embryo retina transformation cell lines SL-2.

In the present invention, we have at first made up plasmid pSL28, and this plasmid has lacked the gene fragment of 100 the base length in adenovirus left side, has kept adenovirus promoter and E1a/E1b gene.We transform people's embryo retina cell with this plasmid subsequently, have obtained transformation cell lines SL-2.We test and assess the characteristic of this clone, found that, this clone has good growth characteristics, its stand density is 2.7 times of HEK293 cell, the viral yield of individual cells and HEK293 cell be (11000--15000 virion/every cell) quite, the virus overall yield is 2-3 a times of HEK293 cell, and the generation probability of reverse mutation wildtype adenovirus virus is below 1/1000 of HEK293 cell.

Result to sum up, people's embryo retina transformation cell lines SL-2 has good characteristic, can be applied to the production of biological products such as virus product, vaccine and polypeptide drugs.

Description of drawings

Fig. 1. the transformant structure collection of illustrative plates of plasmid pSL28

Fig. 2 .SL-2 cell and HEK293 cell E1a expression of gene, the E1a expression level of visible SL-2 cell is higher than the HEK293 cell among the figure.

Embodiment

The structure of embodiment 1pSL28 plasmid

The pXC1 plasmid contains the adenovirus hominis gene order, and from bp22 to bp5790 (McKinnon et.al.Gene.1982,19,33-42).We have at first deleted the Bsa BI fragment in the pXC1 plasmid, obtain plasmid pSL26.Utilize PCR method to amplify adenovirus left side bp100 to xbaI (bp1339) fragment, amplimer is:

Positive-sense strand primer: 5 '-AGA ATT CCC CTT CCA GCT CTC TGC CCC-3 '

Antisense strand primer: 5 '-ACA GCT ATC CGT ACT ACT AT-3 '

The fragment cloning that amplifies is gone into T carrier pCST, can get plasmid pSL27 thus.

EcoRI-XbaI fragment with in the displacement of the EcoRI-XbaI fragment in the pSL27 plasmid pXC1 plasmid obtains plasmid pSL28.(pSL28 plasmid structure collection of illustrative plates is seen accompanying drawing 1)

Embodiment 2 uses the conversion of pSL28 plasmid to people's embryo retina cell

Preparation pSL28 plasmid under the situation of adding the Invitrogen conversion reagent Lipofectmine of company, utilizes the pSL28 plasmid to transform people's embryo retina cell, obtains the strain of people's embryo retina transformant.We obtain 9 transformed cell clonings altogether, and process is to the detection of the E1a genetic expression of each cell clone, and picking wherein efficiently expresses a strain cell clone of E1a gene, called after SL-2 cell strain.Compare with the HEK293 cell, the SL-2 cell can higher levels of expression E1a gene.Detected result is seen accompanying drawing 2.As seen, in the test that parallel triplicate detects, the E1a gene expression dose of SL-2 cell is higher than the HEK293 cell among the figure.The SL-2 cell is people's embryo retina transformation cell lines, this clone has been preserved in (address: Chinese Wuhan Wuhan University 430072, Chinese typical culture collection center, preservation date: on October 13rd, 2009, deposit number: CCTCC C200960, the specific name of this biomaterial: people's embryo retina transformation cell lines (Transformed Human Embryo Retina Cell).

Embodiment 3SL-2 cell strain production adenovirus Characteristics Detection

We produce virus characteristic to the SL-2 cell strain and carry out a series of detections.In the cell strain Detection of Stability, we are with SL-2 cell strain continuous passage 40 times, and the cell of all back generations all can stably express E1a gene product.Utilize Ad5LacZ adenovirus carrier (disappearance E1 district gene) to infect the SL-2 cell of each generation, the Ad5LacZ adenovirus carrier can be in the SL-2 of all generations cell smooth growth, after testing, the SL-2 cell of each generation, infect through the Ad5LacZ adenovirus carrier, the results progeny virus on average can produce the 11000--15000 virion in each cell.In cultivating to the counting of unit culture area inner cell quantity, and with the HEK293 cell relatively, find that the quantity of SL-2 cell is 2.5 times of HEK293 cell quantity in the unit culture area.The viral sum that produces in the unit culture area is compared, find viral load that the SL-2 cell produces be the viral load that produces of HEK293 cell 2-3 doubly.

To producing the detection of reverse mutation wildtype adenovirus virus levels: utilize in the SL-2 cell of cultivating respectively at equal amts through the Ad5LacZ adenovirus carrier of plaque purification and HEK293 cell, infect after three days, harvested cell, adopt ultracentrifugation method purifying to obtain progeny virus, and the level of reverse mutation wildtype adenovirus virus in the detection progeny virus, the result shows that through the HEK293 cell cultures, reverse mutation wildtype adenovirus virus levels is 1/10 in the filial generation Ad5LacZ adenovirus carrier ⁶, through the SL-2 cell cultures, reverse mutation wildtype adenovirus virus levels is 1/10 in the filial generation Ad5LacZ adenovirus carrier ⁹This shows that use SL-2 cell strain production adenovirus, the generation probability of reverse mutation wildtype adenovirus virus is below 1/1000 of HEK293 cell.

SL-2 cell strain among the present invention also is applicable to other virus of preparation and biological products, as host cell as virus production such as influenza virus, hsv, Measles virus and rotaviruss, and as the engineering cell in polypeptide drugs such as the antibody producing.

Although above describe the present invention in detail with embodiment, this area those skilled in the art will appreciate that, can carry out any change and not depart from scope of the present invention the present invention.

Claims

1. people's embryo retina transformant is characterized in that having utilized the adenovirus hominis gene fragment that people's embryo retina cell is transformed, and the people's embryo retina transformant that obtains thus can specific expressed adenovirus Ela gene.

2. the clone described in the claim 1 is SL-2 clone (preserving number: CCTCC-C200960).

3. the clone described in the claim 1 or 2, it is as the purposes of the host cell in the adenovirus preparation process.

4. the clone described in the claim 1 or 2, it is as the purposes of the host cell in the viral preparation process such as influenza virus, hsv, Measles virus and rotavirus.

5. the clone described in the claim 1 or 2, it is as the purposes of the host cell in polypeptide and the other biological goods preparation process.