CN109266616A - A kind of humanization mouse podocytes model of stable expression AQP2 albumen and its construction method and application - Google Patents

A kind of humanization mouse podocytes model of stable expression AQP2 albumen and its construction method and application Download PDF

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CN109266616A
CN109266616A CN201810956096.7A CN201810956096A CN109266616A CN 109266616 A CN109266616 A CN 109266616A CN 201810956096 A CN201810956096 A CN 201810956096A CN 109266616 A CN109266616 A CN 109266616A
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李荣山
黄波
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Shanxi Provincial Peoples Hospital
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Abstract

The invention discloses a kind of humanization mouse podocytes model of stable expression AQP2 albumen and its construction method and applications, the cell model is to be preserved in China General Microbiological culture presevation administrative center (CGMCC), and deposit number is the cell or its progeny cell of CGMCC NO.16201.The present invention also provides the humanization mouse podocytes models of aforementioned stable expression AQP2 albumen to study in AQP2 albumen, the application of the assessment of preclinical drug effect pharmacological property or drug screening of antibody etc..The present invention constructs plasmid AQP2-DYK using suitable amplimer and identification primer, the cell model that mouse podocytes after its transfection construct after being screened by G418 screening and positive colony, it can not only stablize, high efficient expression AQP2 albumen, and construct quick, at low cost.

Description

It is a kind of it is stable expression AQP2 albumen humanization mouse podocytes model and its building side Method and application
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of humanization mouse foot of stable expression AQP2 albumen Cell model and its construction method and application.
Background technique
Existing research proves at present, and it is autosomal inheritance that hereditary diabetes insipidus, which has a kind of hereditary form, by Aquporin2 (AQP2) caused by gene mutation, mutational site 1731A > C.Thus, theoretically, establish the expression of humanization AQP2 albumen height Cell model is feasible.Usually, we verify monoclonal antibody by the animal of humanization or other are special Property targeting source of people molecule drug drug effect and other related pharmacological properties assessments, wherein the mouse of humanization is common experiment Animal, and mainly realized by transgene method.This kind of methods occur in preclinical pharmacological property evaluation process Very important effect, but there is also many deficiencies, primary is a little exactly to establish stable, high efficient expression target protein Not only time-consuming but also expense is also relatively high for humanization mouse cell model.Therefore, building is quick at low cost, for related egg White research, the stabilization of the preclinical drug effect pharmacological property assessment of antibody, humanizing cells' model of high expression AQP2 albumen are very necessary, Also there is quite high application value.
Summary of the invention
The purpose of the present invention is to provide the humanization mouse podocytes models and its structure of a kind of stable expression AQP2 albumen Construction method can not only be stablized, high efficient expression AQP2 albumen with solving the problems of the prior art, and construct quick, cost It is low.
To achieve the above object, the technical solution of the embodiment of the present invention is as follows:
The embodiment of the present invention provides a kind of humanization mouse podocytes model of stable expression AQP2 albumen, the cell membrane Type is to be preserved in China General Microbiological culture presevation administrative center (CGMCC), and deposit number is CGMCC NO.:16201's Cell or its progeny cell.
The classification naming of cell model described in the embodiment of the present invention is mouse podocytes AQP2 cell strain, and depositary institution is China General Microbiological culture presevation administrative center (CGMCC), address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation Date is on July 19th, 2018, and deposit number is CGMCC NO.:16201.
Stablize the construction method of the humanization mouse podocytes model of expression AQP2 albumen described in the embodiment of the present invention, The method includes the steps as follows:
I, AQP2-DYK carrier is constructed, is specifically comprised the following steps:
(1) using the cDNA of people as template, (alternatively referred to as with SEQ ID No:1 (alternatively referred to as A1F) and SEQ ID No:2 A1R primer amplification shown in) introduces two restriction enzyme restriction enzyme site HindIII by PCR amplification, obtains SEQ ID The amplified production of purpose AQP2 segment shown in No:3, recycles purpose AQP2 for the amplified production after agarose gel electrophoresis Segment；
(2) the purpose AQP2 segment of recycling is connect with plasmid PUC19, after primer identification effectively, through alkaline lysis method Plasmid DNA is extracted, the plasmid PUC19-hAQP2 with purpose AQP2 segment is obtained；
(3) again with two restriction enzyme HindIII and Kpn1 digestions, by the purpose of the plasmid PUC19-hAQP2 AQP2 segment is cloned into the corresponding restriction enzyme site of ANXA11-DYK carrier, after being identified with primer, obtains plasmid AQP2-DYK；
II, the humanization mouse podocytes model for constructing AQP2 albumen, specifically comprise the following steps:
(4) mouse podocytes are subjected to cell culture；
(5) plasmid AQP2-DYK made from step (3) is transfected in the mouse podocytes cultivated to step (4)；
(6) Screening of Media for being again 400~700ug/ml with G418 concentration, individual cells continue to cultivate shape packed cell Group carries out positive colony cell screening to cell mass, obtains the humanization mouse podocytes strain for stablizing expression AQP2 albumen.
Preferably, in the step (2) with SEQ ID No:4 (alternatively referred to as A2F) and SEQ ID No:5 (alternatively referred to as A2R primer shown in) is identified.
Preferably, in the step (3) with SEQ ID No:6 (alternatively referred to as A3F) and SEQ ID No:7 (alternatively referred to as A3R primer shown in) is identified.
It should be noted that can be carried out to primer under the premise of understanding the object of the invention and primer sequence as above Various conventional modifications, for example, addition restriction enzyme site, addition protection base etc..
In the present invention, G418 described in step (6) (Geneticin, Geneticin) is a kind of aminoglycoside antibiosis Element is the most common resistance screening reagent of stable transfection in molecular genetic test.
It is highly preferred that being used for the culture of screening in step (6) to guarantee that the individual cells filtered out form group's cell mass G418 concentration is 400~700ug/ml in base.
The inventors found that in step (6) first with the Screening of Media of 400~700ug/ml of G418 concentration after, it is single A cell continues to cultivate the group's of being capable of forming cell mass, when G418 concentration too low (such as 300ug/ml or so), after culture 5-7 days, carefully Born of the same parents' death rate reaches 80% or more, and individual cells can not the group's of being formed cell mass；When G418 excessive concentration (such as 800ug/ml or so), After culture 1 day, cell mortality just reaches 80% or more, and individual cells also can not the group's of being formed cell mass.
It is highly preferred that continuing G418 in the culture medium of culture in step (6) to guarantee that individual cells form group's cell mass Concentration is 400~900ug/ml.
It is highly preferred that continuing to need every 2- in incubation in step (6) to guarantee that individual cells form group's cell mass 4 days one subcultures of replacement.Most preferably, every 3 days one subcultures of replacement.
The present invention also provides the humanization mouse podocytes models for stablizing expression AQP2 albumen of above method building.
Preferably, the cell model can stablize expression AQP2 albumen at least 40 generations.
Preferably, it is 15-22pg/cell/ that the cell model, which passes on the specific production rate for remaining to express AQP2 after 50 generations, day。
The present invention also provides the humanization mouse podocytes models of aforementioned stable expression AQP2 albumen to study in AQP2 albumen, The application of the assessment of preclinical drug effect pharmacological property or drug screening of antibody etc..
The present invention has the advantage that
The present invention constructs plasmid AQP2-DYK using suitable amplimer and identification primer, the mouse foot after its transfection Cell can not only be stablized, high efficient expression AQP2 albumen by the cell model constructed after G418 screening and positive colony screening, And building is quick, at low cost.
Detailed description of the invention
Fig. 1 Western blot determines that AQP2 albumen is expressed in mouse podocytes model
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
The experimental materials used in the following example, biological agent etc. are unless otherwise specified commercially available.Also, such as nothing Specified otherwise, cell culture as used in the following examples, molecular genetics, nucleic acid chemistry, Immunology Lab operating procedure It is widely used conventional steps in corresponding field.
Embodiment 1 constructs AQP2-DYK carrier
(1) using the cDNA of people as template, design primer sequence A1F and A1R introduce two restriction enzyme restriction enzyme sites HindIII obtains the amplified production of the AQP2 segment of 0.8kb mesh, by the amplification by the protein-coding region PCR amplification AQP2 Product recycles purpose AQP2 segment after 2% agarose gel electrophoresis, wherein primer sequence and purpose AQP2 fragment sequence are such as Under:
A1F:TAAGCTTGGATGTGGGAGCTCCGCTCCAT
A1R:AGGTGTTCGAAGGCCTTGGTACCCCGTGGCA
Purpose AQP2 segment:
TAAGCTTGGATGTGGGAGCTCCGCTCCATAGCCTTCTCCAGGGCTGTGTTCGCAGAGTTCCTGGCCAC ACTCCTCTTCGTCTTCTTTGGCCTCGGCTCTGCCCTCAACTGGCCACAGGCCCTGCCCTCTGTGCTACAGATTGCC ATGGCGTTTGGCTTGGGTATTGGCACCCTGGTACAGGCTCTGGGCCACATAAGCGGGGCCCACATCAACCCTGCCG TGACTGTGGCCTGCCTGGTGGGCTGCCACGTCTCCGTTCTCCGAGCCGCCTTCTACGTGGCTGCCCAGCTGCTGGG GGCTGTGGCCGGAGCCGCTCTGCTCCATGAGATCACGCCAGCAGACATCCGCGGGGACCTGGCTGTCAATGCTCTC AGCAACAGCACGACGGCTGGCCAGGCGGTGACTGTGGAGCTCTTCCTGACACTGCAGCTGGTGCTCTGCATCTTCG CCTCCACCGATGAGCGCCGCGGAGAGAACCCGGGCACCCCTGCTCTCTCCATAGGCTTCTCTGTGGCCCTGGGCCA CCTCCTTGGGATCCATTACACCGGCTGCTCTATGAATCCTGCCCACTCCCTGGCTCCAGCTGTCGTCACTGGCAAA TTTGATGACCACTGGGTCTTCTGGATCGGACCCCTGGTGGGCGCCATCCTGGGCTCCCTCCTCTACAACTACGTGC TGTTTCCGCCAGCCAAGAGCCTGTCGGAGCGCCTGGCAGTGCTGAAGGGCCTGGAGCCGGACACCGATTGGGAGGA GCGCGAGGTGCGACGGCGGCAGTCGGTGGAGCTGCACTCGCCGCAGAGCCTGCCACGGGGTACCAAGGCCTTCGAA CACCT；
(2) the purpose AQP2 segment of recycling is connect with plasmid PUC19, then with after primer A2F and A2R identification effectively, passed through Alkaline lysis method extracts Plasmid DNA, obtains the plasmid PUC19-hAQP2 with purpose AQP2 segment；
(3) by two restriction enzyme HindIII and Kpn1 digestions, by the purpose of the plasmid PUC19-hAQP2 AQP2 segment is cloned into the corresponding restriction enzyme site of ANXA11-DYK carrier, after primer A3F and A3R identification, obtains plasmid AQP2-DYK；
The humanization mouse podocytes strain of expression AQP2 albumen is stablized in the building of embodiment 2
(4) mouse podocytes AQP2 is incubated in 3 6cm culture dishes, culture solution RPMI-1640 (10%FBS, 1% PS, 50-100U/ml Y-IFN), PH 7.2,35-37 degree, 5% carbon dioxide incubator culture 20h, cell density 20%；
(5) by 1 μ g plasmid AQP2-DYK obtained in embodiment 1, by FuGENE6 Transfection, (Promega is public Department) reagent transfects into above-mentioned mouse podocytes, and culture is for 24 hours；
(6) it is cultivated again with G418 concentration 500ug/ml culture medium, the death rate is observed after 1 day and cell death occurs, every 3 It is replaced the culture medium that 1 G418 concentration is 500ug/ml and continues to cultivate, former unicellular formation group cell mass, the group of selecting after 15 days Under small cell scraper, digested 5 minutes with 0.25% pancreatin, the culture for being 500ug/ml with 10vol.% fetal calf serum G418 concentration Base plants the cell concentration 1% in 6 new hole culture dishes respectively after neutralizing, large stretch of death no longer occurs in cell.It replaces 1 time within every 3 days G418 high concentration is that the culture medium of 800ug/ml selected pellet cellular after 15 days for the second time, is carried out using antibody A QP2 (C-17) Immunoblotting determines protein expression (such as Fig. 1), filters out the positive colony that can stablize expression AQP2 albumen, then by above-mentioned positive gram Grand cell is diluted to 6/ml, and after cell passed on for 40 generations, the specific production rate for being still able to maintain AQP2 is the cell of 22pg/cell/day Strain, after cell passed on for 50 generations, the specific production rate for being still able to maintain AQP2 is the cell strain of 20pg/cell/day.
The humanization mouse podocytes strain of the building expression AQP2 albumen of embodiment 3
Step is cultivated again with G418 concentration for the culture medium of 500ug/ml in (6), and G418 concentration is replaced after 3 days and is The culture medium of 800ug/ml continues to cultivate, and other conditions are same as Example 2, after cell passed on for 40 generations, is able to maintain AQP2's Specific production rate is 18pg/cell/day, and after cell passed on for 50 generations, the specific production rate for being still able to maintain AQP2 is 15pg/cell/ day。
The humanization mouse podocytes strain of expression AQP2 albumen is stablized in the building of comparative example 1
Step (4) and step (5) are trained in step (6) with the culture medium of G418 concentration 300ug/ml again with embodiment 2 It supports, the culture medium that the death rate is almost without exception, and replacement G418 concentration is 300ug/ml after 3 days is observed after 1 day, is further cultured for 3 days, G418 concentration is cell mortality 80% in the culture dish of 300ug/ml, continues to cultivate, and cell almost all is dead in culture dish, Can not the group's of being formed cell mass, cannot construct stablize expression AQP2 albumen humanization mouse podocytes strain.
The humanization mouse podocytes strain of expression AQP2 albumen is stablized in the building of comparative example 2
Step (4) and step (5) are trained in step (6) with the culture medium of G418 concentration 800ug/ml again with embodiment 2 Support, the death rate observed after 1 day and occurs large stretch of dead, it is dead to continue to cultivate cell almost all, can not the group's of being formed cell mass, cannot Construct the humanization mouse podocytes strain for stablizing expression AQP2 albumen.
As it can be seen that method of the invention can construct stable, high efficient expression AQP2 albuminous cell model.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, 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 are fallen within the scope of the claimed invention.
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Claims (8)

1. a kind of humanization mouse podocytes model of stable expression AQP2 albumen, which is characterized in that the cell model is to protect It is hidden in China General Microbiological culture presevation administrative center, and cell or its offspring that deposit number is CGMCC NO.:16201 Cell.
2. stablize the construction method of the humanization mouse podocytes model of expression AQP2 albumen described in a kind of claim 1, it is special Sign is that the method includes the steps as follows:
(1) using the cDNA of people as template, with primer amplification shown in SEQ ID No:1 and SEQ ID No:2, pass through PCR amplification Two restriction enzyme restriction enzyme site HindIII are introduced, the purpose AQP2 fragment amplification as shown in SEQ ID No:3 is obtained and produces The amplified production is recycled purpose AQP2 segment by object after agarose gel electrophoresis；
(2) the purpose AQP2 segment of recycling is connect with plasmid PUC19, after primer identification effectively, is extracted through alkaline lysis method Plasmid DNA obtains plasmid PUC19-hAQP2；
(3) by two restriction enzyme HindIII and Kpn1 digestions, by the purpose AQP2 of the plasmid PUC19-hAQP2 Segment is cloned into the corresponding restriction enzyme site of ANXA11-DYK carrier, after being identified with primer, obtains plasmid AQP2-DYK；
(4) mouse podocytes are subjected to cell culture；
(5) plasmid AQP2-DYK made from step (3) is transfected in the mouse podocytes cultivated to step (4)；
(6) Screening of Media for being again 400~700ug/ml with G418 concentration, individual cells continue culture and form group's cell mass, Positive colony cell screening is carried out to cell mass, obtains the humanization mouse podocytes strain for stablizing expression AQP2 albumen.
3. the construction method of the humanization mouse podocytes model of stable expression AQP2 albumen according to claim 2, It is characterized in that, is identified in the step (2) with primer shown in SEQ ID No:4 and SEQ ID No:5.
4. the construction method of the humanization mouse podocytes model of stable expression AQP2 albumen according to claim 3, It is characterized in that, is identified in the step (3) with primer shown in SEQ ID No:6 and SEQ ID No:7.
5. the humanization mouse podocytes model of stable expression AQP2 albumen according to claim 1, which is characterized in that institute Expression AQP2 albumen at least 40 generations can be stablized by stating cell model.
6. the humanization mouse podocytes model of stable expression AQP2 albumen according to claim 1, which is characterized in that institute Stating the specific production rate for remaining to express AQP2 after cell model 50 generations of passage is 15-22pg/cell/day.
7. claim 1, the humanization mouse podocytes model of 5,6 described in any item stable expression AQP2 albumen is in AQP2 egg Application in white research.
8. claim 1, the humanization mouse podocytes model of 5,6 described in any item stable expression AQP2 albumen is in antibody Application in terms of preclinical drug effect pharmacological property assessment or drug screening.
CN201810956096.7A 2018-08-21 2018-08-21 A kind of humanization mouse podocytes model of stable expression AQP2 albumen and its construction method and application Pending CN109266616A (en)

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