CN102533655A - CHO-S cell strain for efficient expression type human recombinant protein GLP1/Fc and establishment method thereof - Google Patents

Abstract

The invention provides a cell strain CHO-S for efficient expression type human recombinant protein GLP1/Fc. A plasmid with a human GLP1/Fc gene is transfected into a CHO-S cell, and a CHO-S (GLP1/Fc) monoclonal cell strain for stable and efficient expression of the human recombinant protein GLP1/Fc can be obtained after screening. GLP1/Fc fusion protein can be produced via secretion of the CHO-S (GLP1/Fc) monoclonal cell strain and can directly act on a GLP-1 receptor to promote pancreatic beta-cells, insulin and glucose tolerance can be achieved via secretion, the growth of beta-cells can be promoted, the number of beta-cells can be increased, and the normal levels of the functions of the beta-cells of patients suffering from II type diabetes can be restored.

Description

Efficiently express CHO-S cell strain and the establishment method thereof of people source recombinant protein GLP1/Fc

Technical field

The invention belongs to field of biological pharmacy, relate to CHO-S cell strain and the establishment method thereof of a kind of people of efficiently expressing source recombinant protein GLP1/Fc.

Background technology

Mellitus are to cause one of human main causes of death, seriously jeopardize human health.The World Health Organization that IDF announces shows that to the up-to-date prediction of onset diabetes present situation and development trend whole world type ii diabetes patient has after diagnosing reached 100,013,000 people.Mellitus will be popular in developing countries such as China, India in this century.By 2025, global diabetic subject's number will break through 300,000,000, and diabetic subject's sum of China will account for wherein 1/3rd.So far, diabetic subject's number of China surpasses 50,000,000, and increases the rate increase of 5-6% with year.

Type i diabetes and type ii diabetes are two kinds of principal modes of mellitus.I type and type ii diabetes people show the reduction of progressive pancreatic quality minimizing and beta cell function, and the programmed death of beta cell is the major cause of I type and type ii diabetes islet cells.Insulin treatment is the main means of treatment type i diabetes.The ratio that type ii diabetes accounts for the diabetic subject is about 90-95%.

Type i diabetes people's therapeutic modality mainly is to give injection of insulin or pancreatic islets transplantation.For the type ii diabetes people, using euglycemic agent and insulin secretion augmentor is main pharmacological agent mode.

Traditional treatment of diabetes only lays particular emphasis on to be alleviated like cardinal symptoms such as hyperglycemia, rather than to the real pathogenic factor of type ii diabetes.Present in the world main flow viewpoint thinks that the true cause of onset diabetes is the programmed death of islet cells.

Confirmed that at present multiple hormone can promote the beta cell growth, comprises the enteroglucagon appearance peptide-1 (GLP-1) of growth factor, rhIGF-1, prolactin antagonist, Urogastron and recent findings.

Glucagon-like-peptide-1 (GLP-1) is a kind of incretin, and it has the promotion insulin secretion, suppresses pancreas and rises the plain release of sugar, suppresses the effect of stomach emptying.Its important function is the growth that promotes beta cell, increases the quantity of beta cell, and the beta cell functional rehabilitation that can make the type ii diabetes people is to normal level.

The another one advantage of GLP-1 is that its hypoglycemic activity is that glucose is dependent, therefore can not produce hypoglycemia.In this even be superior to Regular Insulin.In addition, it can also improve the body insulin resistant and improve the body lipid level.Research shows that GLP-1 also maybe be effective to the type i diabetes patient.Such as reducing consumption to exogenous insulin.Because the GLP-1 transformation period in vivo very short (about 1 minute), therefore, endeavouring to seek the long-acting analogue (the plain analogue of intestines hypoglycemic) with GLP-1 same purpose is the focus of attention of international medicine circle.

GLP1/Fc fusion rotein principal feature is that the transformation period is longer, and effect is stronger.It is all that experiment has proved that its injection once can be kept drug effect 4-6, has very strong competitive edge.Because the characteristics of its dimeric structure can significantly strengthen being all the avidity of dimeric GLP-1 acceptor, make drug effect stronger.This fusion rotein directly acts on the GLP-1 acceptor; Promote pancreatic beta-cell regeneration; Excreting insulin and glucose tolerance; Activate cAMP and second messenger's approach that its coupling joins, promote the expression of protein kinase (Akt1 and MAPK) in the beta cell and increase its content, reduce the activity of apoptotic key enzyme caspase-3.Therefore this fusion rotein can fundamentally prevent and treat I type and type ii diabetes.

The production for treating that is established as of recombinant gene has been opened up new way with albumen.Because the limitation of prokaryotic cell prokaryocyte heterologous gene expression system self is difficult to overcome; The eukaryotic cell heterologous gene expression system is widely used in expressing the biological activity protein that needs posttranslational modification; Particularly mammalian expression system has post transcriptional modificaiton function accurately; With procaryotic cell expression system and yeast cell to express system ratio; The glycosylated protein of expressing near the native protein molecule, is the first-selected system that present recombinant protein is produced aspect molecular structure, physicochemical property and biological function.

In the expression system of different mammalian cells, CHO-S Chinese hamster ovary cell exogenous protein expression system uses through practice for many years, has become the mammal cell line of the most successful expression foreign protein.Its advantage is that foreign gene can be stably integrated in the cell, and this clone is easy to the mass-producing cultivation.

Summary of the invention

The object of the present invention is to provide the cell strain CHO-S of a kind of people of efficiently expressing source recombinant protein GLP1/Fc; The plasmid transfection of the present invention through will having people source GLP1/Fc gene is to the CHO-S cell; After screening, obtain to stablize, efficiently express CHO-S (GLP1/Fc) the monoclonal cell strain of people source recombinant protein GLP1/Fc.

For solving the problems of the technologies described above, the present invention adopts following technical scheme to be achieved:

Efficiently express the CHO-S cell strain of people source recombinant protein GLP1/Fc, it is made to the CHO-S host cell by the pMTE2 plamid vector transfection that has the GLP1/Fc gene.

Further, the expression amount of the target protein GLP1/Fc of said CHO-S (GLP1/Fc) monoclonal cell strain reaches 9.25ug/24h10 ⁶Cell.

Further again, the establishment method that efficiently expresses the CHO-S cell strain of people source recombinant protein GLP1/Fc may further comprise the steps:

(1) with cationic-liposome with the pMTE2 plamid vector transfection to the CHO-S cell strain;

(2) use the selection screening of medium that contains Hygromycin B to integrate the stable transfected cells strain of GLP1/Fc gene;

(3) through Dot blot method filter out can expressing human source GLP1/Fc recombinant protein positive colony;

(4) positive colony that obtains is shaken foster in CO2gas incubator, collect secreted protein liquid when waiting to grow to plateau; Said CHO-S cell strain in culturing process secreting, expressing GLP1/Fc recombinant protein in substratum;

(5) detect the relative expression quantity of collecting GLP1/Fc in the protein liquid with Western blot method.

Further again, efficiently express the CHO-S cell strain of people source recombinant protein GLP1/Fc, its purposes is to prepare the application in the medicine of treatment I type and type ii diabetes aspect.

Owing to adopt technical scheme of the present invention; The present invention compared with prior art has the following advantages: CHO-S (GLP1/Fc) monoclonal cell strain secretion produces the GLP1/Fc fusion rotein, and the GLP1/Fc fusion rotein directly acts on the GLP-1 acceptor, promotes pancreatic beta-cell regeneration; Excreting insulin and glucose tolerance; Promote the growth of beta cell, increase the quantity of beta cell, the beta cell functional rehabilitation that can make the type ii diabetes people is to normal level.

Embodiment

Embodiment 1

Concrete preparation method of the present invention is described below:

1. experiment material:

1.1 clone

CHO-S, Chinese hamster ovary cell.

1.2 main agents

1.2.1 CD CHO serum free medium

CD CHO culture medium dry powder 24.3g adds the 950ml aqua sterilisa, is stirred to dissolving fully, regulates pH value between the 6.8-7.2, adds aqua sterilisa to 1000ml, with the degerming of 0.22um membrane filtration, and 4 ℃ of preservations.

1.2.2 transfection reagent

The used reagent of transfection is Lipofectamin2000 (Invitrogen company).

1.2.3 selection substratum

In 10ml liquid CD CHO serum free medium, adding final concentration is the Hygromycin B of 1mg/ml.

1.2.4 one is anti-

Rabbit anti-human igg/HRP, working concentration are dilution in 1: 1000.

2. method and result

2.1 cell recovery

According to conventional cell recovery method: from cell bank, take out a freeze-stored cell; Place rapidly to be preheated to 37 ℃ thermostat water bath, the limit is shaken the limit gently and is melted, wait to melt finish after; The frozen pipe outer wall of sterilizing immediately; In Biohazard Safety Equipment, cell suspension is transferred in the cultivation square vase that contains the 6mlCD CHO serum free medium of having an appointment,, cultivates in the incubator of 8% carbonic acid gas in 36.5 ℃.

2.2 cell amplification

When treating that the CHO-S that cultivates in the square vase grows to 80% left and right sides; According to 1: 1 ratio be transferred to disposable triangle shake shake in the bottle foster; According to cell growth condition, shake in the bottle and can constantly add fresh culture, be added at most shake that bottle amasss 1/3rd; Shake foster rotating speed and adjust, generally between 60rpm-100rpm according to shaking a bottle interior liquid volume.Treat that cell grows to 2-3 * 10 ⁶Individual/as can to divide during ml foster.

2.3 the structure of plasmid vector

The used plasmid vector of transfection is provided by University of Toronto.

2.4 treat the preparation of transfectional cell

Count shaking the cell that grows to logarithmic phase in the bottle, according to count results cell is divided and support to 6 orifice plates, the cell density in each hole is in 8-16 * 10 ⁵Individual/ml.

2.5 the transfection of cell

Liposome method is adopted in the transfection of CHO-S (GLP1/Fc) cell, and the used reagent of transfection is Lipofectamin2000 (Invitrogen company).

2.6 the screening of cell

Behind the cell transfecting 24h, change fresh CD CHO and select substratum (the Hygromycin B that contains 1mg/ml), simultaneously transfectional cell dilution back is added in 96 orifice plates; In 37 ℃; Cultivated for 1～2 week in 8% carbonic acid gas, the centre is changed and is once selected substratum, forms until cell clone.

2.7Dot?blot

About cell screening to the ten days, 96 orifice plates are done Dot blot experiment.Each hole is got the 1ul nutrient solution and is carried out point sample, and with confining liquid room temperature sealing 1h, the back is washed 6 times with the TBST damping fluid with one anti-(rabbit anti-human igg/HRP, dilution in 1: 1000) incubated at room 2h, and each 5min carries out chemoluminescence with ECL under the darkroom.According to exposure status, filter out 6 positive colonies.

2.8 to the positive colony amplification culture

Expand 6 positive colonies that filter out foster respectively.Be transferred at first respectively in 24 orifice plates and cultivate, transfer to 6 orifice plates when waiting to be paved with 24 orifice plates bottom and cultivate, add fresh CD CHO serum free medium simultaneously; Be transferred to disposable triangle after covering with respectively and shake and shake fosterly in the bottle, plateau to be grown to, gather in the crops the protein liquid of each positive colony respectively.

2.9Western?blot

Positive colony to amplification culture is collected protein liquid, adopts the AKTA affinity chromatography to extract the albumen in the cell culture fluid.The protein sample that extracts is done Western blot experiment, identify, analyze its expressing quantity.Protein sample behind the SDS-Page electrophoresis, is transferred on the pvdf membrane, seals 1h with containing 5% skimmed milk; With one anti-(rabbit anti-human igg/HRP, dilution in 1: 1000) incubated at room 2h, wash 6 times then with the TBST damping fluid; Each 5min carries out chemoluminescence with ECL under the darkroom.According to the chemoluminescence situation, calculate the about 5.36-9.25ug/24h10 of expressing quantity ⁶Cell.

The present invention construct can stable transfection people source GLP1/Fc gene mammal cell line, and through drug screening, the monoclonal cell strain of this recombinant protein can be stablized, efficiently expressed to acquisition.The target protein GLP1/Fc of CHO-S (GLP1/Fc) monoclonal cell strain is a kind of incretin, and it has the promotion insulin secretion, suppresses pancreas and rises the plain release of sugar, suppresses the effect of stomach emptying.Important function is the growth that promotes beta cell, increases the quantity of beta cell, and the beta cell functional rehabilitation that can make the type ii diabetes people is to normal level.The another one advantage of target protein GLP1/Fc is that its hypoglycemic activity is that glucose is dependent, therefore can not produce hypoglycemia.

Claims (4)

1. efficiently express the CHO-S cell strain of people source recombinant protein GLP1/Fc, it is characterized in that it is made to the CHO-S host cell by the pMTE2 plamid vector transfection that has the GLP1/Fc gene.

2. the CHO-S cell strain that efficiently expresses people source recombinant protein GLP1/Fc as claimed in claim 1 is characterized in that the expression amount of the target protein GLP1/Fc of said CHO-S (GLP1/Fc) monoclonal cell strain reaches 9.25ug/24h10 ⁶Cell.

3. the establishment method that efficiently expresses the CHO-S cell strain of people source recombinant protein GLP1/Fc as claimed in claim 1, it may further comprise the steps:

(1) with cationic-liposome with the pMTE2 plamid vector transfection to the CHO-S cell strain;

(2) use the selection screening of medium that contains Hygromycin B to integrate the stable transfected cells strain of GLP1/Fc gene;

(3) through Dot blot method filter out can expressing human source GLP1/Fc recombinant protein positive colony;

(4) positive colony that obtains is shaken foster in CO2gas incubator, collect secreted protein liquid when waiting to grow to plateau; Said CHO-S cell strain in culturing process secreting, expressing GLP1/Fc recombinant protein in substratum;

(5) detect the relative expression quantity of collecting GLP1/Fc in the protein liquid with Western blot method.

4. like claim 1 or the 3 described CHO-S cell strains that efficiently express people source recombinant protein GLP/Fc, its purposes is to prepare the application in the medicine of treating I type and type ii diabetes aspect.