CN102533660B - Preparation method of permanent cell line for multiplying orf virus - Google Patents
Preparation method of permanent cell line for multiplying orf virus Download PDFInfo
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- CN102533660B CN102533660B CN201110452708.7A CN201110452708A CN102533660B CN 102533660 B CN102533660 B CN 102533660B CN 201110452708 A CN201110452708 A CN 201110452708A CN 102533660 B CN102533660 B CN 102533660B
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Abstract
The invention relates to a preparation method of a permanent cell line for multiplying an orf virus. The aim that the virus can be stably multiplied in a large amount is achieved by inoculating a calf testicular cell line to the orf virus and a stable cell environment and a standardized culture system for developing and producing an attenuated vaccine of the orf virus are provided. The preparation method comprises the following steps of: firstly, acquiring, separating and culturing a calf testicular cell; secondly, permanently establishing the calf testicular cell; and thirdly, carrying out multiplication culture on the orf virus in the cell line.
Description
One, technical field:
The present invention relates to that a kind of it utilizes bovine testicle cell for one and is prepared into clone for breeding the permanent cell line preparation method of sheep of virus, sheep of virus utilizes the method for this cell line proliferation.
Two, background technology:
In background technology, sheep infective warts is commonly called as sheep aphtha (Orf virus), the a kind of by the Zoonosis transmissible disease due to blue tongue virus of sheep and goat, OIE (OIE) classifies this disease need declare class Animal diseases as, and China is classified as a class animal epidemic.This disease is almost distributed in all sheep raisings country of the world, main infringement sheep and goat in natural situation, and goat is comparatively multiple.It is popular that this disease is often mass-sending property, the easy infection of 3 ~ 6 monthly age lamb, and the sheep morbidity of growing up is less, sick sheep and be this sick contagium with malicious sheep.Virus can be discharged with the saliva of sick sheep, warts and blister secretory product and the crust coming off, and its route of transmission is mainly to infect through the skin of damage or mucous membrane.Healthy Sheep directly contacts with sick sheep, or feed trough, feed, drinking-water, apparatus, pad grass, pasture and the mew etc. that are polluted by sick sheep of contact and infecting.
Along with the developing rapidly of China mutton sheep, wool sheep and milk goat aquaculture, China has become the first in the world sheep raising big country in recent years, and sheep raising had become one of the pillar in the many places of China and specialty industries already.On the other hand, sheep epidemic disease, especially sheep aphtha have also become to threaten one of main epidemic disease of sheep husbandry.High-quality and efficient cheap sheep aphtha vaccine is badly in need of at sheep raising family.
But do not have in the market the sheep aphtha vaccine can be for user, cause the reason of this problem to be that production of vaccine enterprise is unwilling to produce sheep aphtha attenuated vaccine, because the cell that this production of vaccine needs is the primary testicular cell of calf, these cell preparation procedure complexity, production cost is too high, expend time in and financial resources, and be difficult to realize quality control effectively, enterprise is difficult to obtain compared with rich profit.Therefore the clone that, research and development are suitable for sheep of virus (vaccine strain) propagation has important using value.
Three, summary of the invention:
The present invention is in order to solve the weak point in above-mentioned background technology, provide a kind of for breeding the permanent cell line preparation method of sheep of virus, it utilizes this clone inoculation sheep of virus (vaccine strain), virus reaches stable propagation object, for sheep of virus attenuated vaccine research and production provides stable cellular environment and standardized culture condition.
For achieving the above object, the technical solution used in the present invention is:
For breeding a permanent cell line preparation method for sheep of virus, it is characterized in that comprising the following steps: the obtaining, separate and cultivate of (1) bovine testicle cell; (2) foundation of bovine testicle cell permanence; (3) the stable propagation of sheep of virus.
In step (1), bovine testicle cell be retrieved as after calf birth the testis that gathers calf before feed, low-temperature transport, then separates testicular cell.
Bovine testicle cell is separated into mechanical process or tryptic digestion method in step (1).
In step (1), the nutrient solution of bovine testicle cell is the high sugar of M199, DMEM or RPMI-1640 substratum.
In step (2), the foundation of bovine testicle cell permanence realizes by importing Telomerase reverse transcription genetic method.
In step (2), in the process of establishing of bovine testicle cell permanence, utilize liposome 2000 to carry out carrier for expression of eukaryon cell transfecting, and utilize RT-PCR method to detect the expression of hTERT in the bovine testicle cell of stable transfection.
Compared with prior art, the present invention has advantage and effect are as follows:
Separation and Culture bovine testicle cell of the present invention, reaches after stable culture condition, and transfected with human telomerase reverse transcriptase gene (hTERT) makes bovine testicle cell immortalization, sets up bovine testicle cell system.Utilize this bovine testicle cell system inoculation sheep of virus (vaccine strain), virus (vaccine strain) can be stable propagation in cell culture environment at this, for sheep of virus attenuated vaccine research and production provides stable cellular environment and standardized culture systems, can be used for a large amount of productions of sheep aphtha vaccine.
Four, attached caption:
Fig. 1 is cultivation (normally) result of bovine testicle cell system;
Fig. 2 is the expression of results that RT-PCR detects hTERT in transfectional cell series;
Fig. 3 is the cytopathy result after the inoculation sheep of virus Local Isolates 96h of bovine testicle cell system;
Fig. 4 is the cytopathy result after the inoculation sore mouth toxic vaccine strain 96h of bovine testicle cell system;
Five, embodiment:
Referring to Fig. 1, Fig. 1 is cultivation (normally) result that bovine testicle cell of the present invention is; Referring to Fig. 2, utilizing the amplification of RT-PCR method. the cell of the negative contrast untransfected of N hTERT, 20 and 60 bovine testicle cells that are respectively transfection hTERT pass 20 generations, 60 generation hTERT expressions, the positive contrast of P.Referring to Fig. 3, the cytopathy after inoculation sheep of virus Local Isolates 96h, there is cavity in cell; Referring to Fig. 4, the cytopathy after inoculation sore mouth toxic vaccine strain 96h, cytopathy is slower; Referring to table 1, table 1 is the titer determination result of 96h after sheep aphtha vaccine strain inoculation bovine testicle cell system.
Embodiment:
Following examples are used for illustrating the present invention, but do not limit use range of the present invention.
1, the separation of bovine testicle cell and cultivation
Under aseptic condition, Hank ' s liquid (dual anti-) rinses testis 3 times, and sterile scissors cuts off epididymis and tunica albuginea, leaves parenchyma of testis.Hank ' s liquid rinses parenchyma of testis, until without blood, then moved in aseptic triangular flask.
Testis is shredded with eye scissors, approximately 1 ~ 3mm size, Hank ' s liquid rinses, and leaves standstill 2-3min.Supernatant discarded, repetitive scrubbing 3 times, adds PH7.6,0.25% trypsinase, consumption is tissue volume 2 ~ 4 times, 4 DEG C of hold over night.Every other day, suck trypsinase, add M199 nutrient solution, piping and druming, dispels cell gently, and 50mL centrifuge tube is centrifugal, 1000rpm, and centrifugal 10min, supernatant discarded, adds nutrient solution re-suspended cell, adjusts cell density, and 37 DEG C, 5% CO
2cultivate.
2, the foundation of bull testis epithelial cell permanence
Cultivate bovine testicle cell confluent culture bottle 60%～70% time, utilize liposome 2000 to carry out carrier for expression of eukaryon pCI-neo-hTERT(and be purchased from Addgen) cell transfecting, carry out Liu Suanyan NEOMYCIN SULPHATE (neo) screening and culturing, carry out cell expand go down to posterity.Extract the 20th, 60 generation transfectional cell and total RNA of the cell of untransfected, reverse transcription reaction synthesizes cDNA, utilizes primer amplified hTERT fragment and internal reference GAPDH, detects the expression of hTERT.HTERT primer: hup:5 ' GCTGCTCAGGTCTTTCTTTTATG 3 '; Hdown:5 ' CGACGTAGTCCATGTTCACAA 3 ', 55 DEG C of annealing temperatures; GAPDH primer: Gup:5 ' GAAGGTGAAGGTCGGAGT 3 '; Gdown:5 ' GAAGATGGTGATGGGATTTC 3 ', 56 DEG C of annealing temperatures.Result demonstration, after the bovine testicle cell after screening transfection was passaged to for 60 generations, hTERT is positive, and Growth of Cells is stone road shape.
3. the stable propagation of sheep of virus
Get after the recovery of clone freeze-stored cell, with the 1640 substratum suspension cells containing 10% calf serum, 37 DEG C, 5%CO
2cultivate 48h.Get the sheep aphtha vaccine strain virus of 300 L～500 L, inoculating cell system, continues to cultivate 96h, observes pathology.In the time there is plaque in culturing cell, freezing whole culture.Then after multigelation three times, centrifugal, collect supernatant, measure virus titer.
Claims (1)
1. for a permanent cell line preparation method for sheep of virus propagation, it is characterized in that comprising the following steps: the obtaining, separate and cultivate of (1) bovine testicle cell; (2) foundation of bovine testicle cell permanence; (3) sheep of virus is at the multiplication culture of clone;
In step (1), bovine testicle cell be retrieved as after calf birth the testis that gathers calf before feed, low-temperature transport, then separates testicular cell;
Bovine testicle cell is separated into mechanical process or tryptic digestion method in step (1);
In step (1), the nutrient solution of bovine testicle cell is the high sugar of M199, DMEM or RPMI-1640 substratum;
In step (2), the foundation of bovine testicle cell permanence realizes by importing Telomerase reverse transcription genetic method;
In step (2), in the process of establishing of bovine testicle cell permanence, utilize liposome 2000 to carry out carrier for expression of eukaryon cell transfecting, and utilize RT-PCR method to detect the expression of hTERT in the bovine testicle cell of stable transfection;
The obtaining, separate and cultivate of described step (1) bovine testicle cell;
Under aseptic condition, Hank ' s liquid rinses testis 3 times, and sterile scissors cuts off epididymis and tunica albuginea, leaves parenchyma of testis, and Hank ' s liquid rinses parenchyma of testis, until without blood, then moved in aseptic triangular flask;
Testis is shredded with eye scissors, approximately 1 ~ 3mm size, Hank ' s liquid rinses, and leaves standstill 2-3min; Supernatant discarded, repetitive scrubbing 3 times, adds PH7.6,0.25% trypsinase, consumption is tissue volume 2 ~ 4 times, 4 DEG C of hold over night; Every other day, suck trypsinase, add M199 nutrient solution, piping and druming, dispels cell gently, and 50mL centrifuge tube is centrifugal, 1000rpm, and centrifugal 10min, supernatant discarded, adds nutrient solution re-suspended cell, adjusts cell density, and 37 DEG C, 5% CO
2cultivate;
The foundation of described step (2) bovine testicle cell permanence:
Cultivate bovine testicle cell confluent culture bottle 60%～70% time, utilize liposome 2000 to carry out carrier for expression of eukaryon pCI-neo-hTERT cell transfecting, carry out Liu Suanyan NEOMYCIN SULPHATE screening and culturing, carry out cell expand go down to posterity; Extract the 20th, 60 generation transfectional cell and total RNA of the cell of untransfected, reverse transcription reaction synthesizes cDNA, utilize primer amplified hTERT fragment and internal reference GAPDH, detect the expression of hTERT, hTERT primer: hup:5 ' GCTGCTCAGGTCTTTCTTTTATG 3 '; Hdown:5 ' CGACGTAGTCCATGTTCACAA 3 ', 55 DEG C of annealing temperatures; GAPDH primer: Gup:5 ' GAAGGTGAAGGTCGGAGT 3 '; Gdown:5 ' GAAGATGGTGATGGGATTTC 3 ', 56 DEG C of annealing temperatures;
Described step (3) sheep of virus is at the multiplication culture of clone:
Get after the recovery of clone freeze-stored cell, with the 1640 substratum suspension cells containing 10% calf serum, 37 DEG C, 5%CO
2cultivate 48h, the sheep aphtha vaccine strain virus of getting 300 L～500 L, inoculating cell system, continues to cultivate 96h, observes pathology; In the time there is plaque in culturing cell, freezing whole culture; Then after multigelation three times, centrifugal, collect supernatant, measure virus titer.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103849602B (en) * | 2013-07-08 | 2016-02-10 | 山东省滨州畜牧兽医研究院 | A kind of bull testis clone and establishment method thereof and application |
| CN104694484B (en) * | 2015-02-13 | 2018-04-06 | 西北农林科技大学 | A kind of fast separating process of sheep of virus |
| CN104745539B (en) * | 2015-04-02 | 2018-02-06 | 西北农林科技大学 | The virus isolation procedure of sheep of virus low content sample |
| CN107058212B (en) * | 2017-03-31 | 2021-03-19 | 金宇保灵生物药品有限公司 | Subculture sheep testicular subculture cell, subculture domestication culture method, special culture system and application thereof |
| CN107287149B (en) * | 2017-05-09 | 2020-12-29 | 杨凌博德越生物科技有限公司 | Permanent cell line for orf virus proliferation and establishment method thereof |
| CN111019904A (en) * | 2018-10-10 | 2020-04-17 | 安徽东方帝维生物制品股份有限公司 | Construction method of immortalized sheep testis cell line adapting to sheep aphtha virus proliferation |
| CN109652451A (en) * | 2018-12-05 | 2019-04-19 | 安徽农业大学 | A kind of construction method of sheep Testis Caprae seu Ovis cell immortality cell line and its application |
| CN111139226A (en) * | 2019-11-08 | 2020-05-12 | 内蒙古农业大学 | Sore throat virus attenuated strain and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613674A (en) * | 2008-11-13 | 2009-12-30 | 西北农林科技大学 | Porcine vein endothelial cell line and establishment method thereof |
| CN101921729A (en) * | 2009-04-08 | 2010-12-22 | 柯明哲 | Telomerase immortalized skin fibroblast line and construction process thereof |
| CN101974488A (en) * | 2010-06-21 | 2011-02-16 | 西北农林科技大学 | Immortalized porcine pancreatic stem cell line and construction and differentiation methods thereof |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613674A (en) * | 2008-11-13 | 2009-12-30 | 西北农林科技大学 | Porcine vein endothelial cell line and establishment method thereof |
| CN101921729A (en) * | 2009-04-08 | 2010-12-22 | 柯明哲 | Telomerase immortalized skin fibroblast line and construction process thereof |
| CN101974488A (en) * | 2010-06-21 | 2011-02-16 | 西北农林科技大学 | Immortalized porcine pancreatic stem cell line and construction and differentiation methods thereof |
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