CN104099292A - Selective breeding method for EST sensitive cell line containing classical swine fever virus (CSFV) C strain - Google Patents
Selective breeding method for EST sensitive cell line containing classical swine fever virus (CSFV) C strain Download PDFInfo
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Abstract
The invention discloses a selective breeding method for an EST sensitive cell line containing a classical swine fever virus (CSFV) C strain. The method comprises the following steps: (1) monocloning EST cells, to be specific, the EST cells without exogenous microorganism pollution are infected by the CSFV C strain, are cultured for 48 hours under the condition of 37 DEG C and 5% CO2, and are digested with 0.25% trypsin to form decentralized single cells, the decentralized single cells are diluted in a continuous four times gradient manner, the diluted cells are planted in a 96-hole cell plate and are cultured under the condition of 37 DEG C and 5% CO2, after 8 hours, each hole is observed, culturing holes in which cell clusters exist are eliminated, then, the cells are continuously cultured for 1 week, the cell holes in which single cells are cloned for growth are selected, the cells in the holes are screened by subcloning, and after continuously screening the cells by subcloning by the method three times, a subcloning cell line is obtained. Through the adoption of the selective breeding method disclosed by the invention, a single virus inoculating procedure in the production process of traditional cell vaccines is simplified, the production cycle of classical swine fever cell vaccines is shortened, and the production cost is reduced.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of selection of Pestivirus suis C strain EST sensitive cell line.
Background technology
Swine fever (Classical swine fever, CSF) is a kind of highly pathogenic contagious disease being caused by Pestivirus suis (Classical swine fever virus, CSFV), and mortality ratio is up to more than 90%.This disease, taking hemorrhagic fever, aerodigestive tract symptom and neurological disorder as main clinical manifestation, is classified as one of the animal epidemic that must circulate a notice of by OIE (OIE), be classified as a class animal epidemic in China.China each province and city all have swine fever popular at present, annual because the pig of swine fever death accounts for the more than 1/3 of the pig that dies of illness, and therefore, swine fever is still one of great epidemic disease of harm China pig industry development so far.
At present the prevention of swine fever is mainly adopted to vaccine immunity, the hog cholera lapinised virus vaccine of being developed by China has good using value aspect prevention swine fever, and some American-European countries successfully put out the swine fever of this area by this vaccine.But the attenuated vaccine great majority for immunoprophylaxis make on living animal, and different animals individuality is larger to the reaction difference of CSFV, cause between vaccine different batches even same batch of amount that contains live virus inconsistent, directly affect the quality of vaccine.Cell cultures seedling was rapidly developed in recent years, and the production cost of swine Fever Vaccine is significantly reduced, and cell can reduce the stealthy chance infecting or overcome stealthy infection by checking in advance as the carrier of propagative viruses; The immunizing power having without animal individual, is beneficial to viral growth; Can produce in a large number, also can continue more for a long time to cultivate; Cost is low, and convenient sources be convenient to store, and effect is even, easy stdn etc.Therefore, select the CSFV low virulent strain carrier using the cell of high titre horizontal growth as production of vaccine, will effectively reduce the shortcoming of malicious seedling a little less than CSFV, improve security and the immune efficacy of vaccine.But the current cell for the production of CSFV vaccine; its virus titer is lower; the amount that causes containing live virus in the finished product is not high, adds the impact of transit link, and when immune animal, the amount of effective supply live virus has been not enough to stimulate body to produce solid immunoprotection.Therefore, screening Pestivirus suis C strain sensitive cell line, and make on this basis the high titre Pestivirus suis of its persistent infection, the production at swine fever cell vaccine and application aspect are had realistic meaning and economic worth by the foundation of this clone.
In prior art, pass through limiting dilution assay, to clone and subclone with the EST cell of CSFV C strain, and then by the method such as gene amplification, fluorescent dye, a few strain clone cells that obtain are carried out to seed selection and qualification, the EST clone (enhancement type pig testis clone) that final acquisition can high titre persistent infection Pestivirus suis.There is following defect: conventional EST clone does not contain Pestivirus suis C strain, each need of production Pigs Inoculated pestivirus C strain; The Pestivirus suis titre that conventional EST clone is produced is low, and vaccine effect is poor.
Summary of the invention
The object of the invention is to overcome the defect existing in prior art, a kind of selection of Pestivirus suis C strain EST sensitive cell line is provided, the method is screened the clonal cell line to Pestivirus suis sensitivity by EST cell monoclonalization, and to overcome now, cell cultures CSFV virus titer is lower causes the not high problem of live virus amount in vaccine finished product.Meanwhile, the single of simplifying in conventional cell seedling production process connects malicious program, for the production of swine fever cell vaccine is shortened the cycle and reduces costs.Its concrete technical scheme is:
A selection for Pestivirus suis C strain EST sensitive cell line, comprises the following steps:
(1) EST cell monoclonal
The EST cell polluting without inoculating microbe, infects 37 DEG C of 5%CO with CSFV C strain
2under condition, cultivate after 48h, after the individual cells that becomes to disperse through 0.25% tryptic digestion, carry out continuous 4 times of gradient dilutions, plant in 96 porocyte plates 37 DEG C of 5%CO
2under condition, cultivate, after 8 hours, observe every hole, get rid of the culture hole that has cell mass, then continue to cultivate 1 week, select the cell hole of being grown by single cell cloning, cell in hole is carried out to subclone screening, obtain subclonal cell line with after this method subclone screening 3 times continuously;
(2) can be high seed selection and the qualification of titre breeding Pestivirus suis C strain clone
The each cloned cell line of above-mentioned EST cell is inoculated in respectively in 24 porocyte culture plates to 37 DEG C of 5%CO
2under condition, cultivate 48h, discard nutrient solution, wash cell 2 times with PBS (phosphoric acid buffer), collecting cell after 0.25% tryptic digestion, utilize RNAiso Plus (TAKARA) reagent to extract total RNA (Yeast Nucleic Acid), after RT-PCR (inverse transcription polymerase chain reaction), preliminary evaluation goes out persistent infection the clone of Pestivirus suis C strain;
The Auele Specific Primer of RT-PCR qualification CSFV C strain:
Forward primer (upstream primer): TAACACGACTGTCAAGGTGC;
Reverse primer (downstream primer): AACCATGAGCAATGCCACT;
Preliminary evaluation is continued to lie in the culture dish of 35mm with malicious EST cell, after cell covers with individual layer, utilize indirect immunofluorescene assay to determine positive cell number, immunofluorescence detection display, almost 100% cell is all with CSFV antigen;
By determining with cultivations of going down to posterity of the EST clone of high titre Pestivirus suis C strain persistent infection, respectively 3,9, in 15 generations, carried out three freeze thawing repeatedly and receive malicious; After the centrifugal removal cell debris of virus stock solution used, by 10 times of gradient dilutions, viral dilution liquid is connect to poison in the PK15 cell that is paved with 96 orifice plates, 37 DEG C, 5%CO with DMEM (improvement Eagle substratum) liquid
2incubator is cultivated after 48h, detects virus infected cell with indirect immunofluorescence method; Calculate viral medium lethal dose TCID according to Reed-Muench method
50, taking the arithmetical av that repeats experimental results 3 times as cultivating viral infection titer; TCID
50measurement result demonstration, more than 15 generations of cell continuous passage, its viral minuent maintains 10
5.5tCID
50/ mL is higher 100 times than the virus titer after single inoculation EST parent cell.
Preferably, step utilizes indirect immunofluorescene assay to determine positive cell number described in (2), concrete steps are as follows: wash cell 2 times with PBS, through the fixing 20min of 4% paraformaldehyde, add permeable membrane agent 1%Tritonx-100 permeable membrane 20min after washing cell 2 times with PBS; Wash after cell 3 times with PBS, add the anti-CSFV serum of rabbit of dilution in 1: 300, with diluting containing 5% new-born calf serum PBS, 37 DEG C of wet boxes are hatched after 1 hour and are washed away unconjugated primary antibodie, 180r/min, 5min/ time with PBS, totally 3 times, then add the goat anti-rabbit igg-FITC of dilution in 1: 400, the same primary antibodie of dilution process, 37 DEG C of wet boxes are hatched 1 hour; Then wash away unconjugated two with PBS and resist, 180r/min, 5min/ time, totally 3 times, add containing 30% glycerine PBS solution, be placed in fluorescence microscopy Microscopic observation green fluorescence situation.
Compared with prior art, beneficial effect of the present invention is:
1. the EST sensitive cell line of the present invention's screening contains Pestivirus suis C strain for a long time, the cultivation of only need going down to posterity while producing vaccine;
2. the Pestivirus suis C strain titre of the EST sensitive cell line of the present invention's screening is high;
The present invention by set up can high titre persistent infection Pestivirus suis EST clone, be conducive to overcome the lower situation of current C SFV cell cultures titre, simplify the viral Single-infection process in cell production process second simultaneously, there is realistic meaning for reducing swine Fever Vaccine production cost.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in more detail.
A selection for Pestivirus suis C strain EST sensitive cell line, comprises the following steps:
(1) EST cell monoclonal
The EST cell polluting without inoculating microbe, infects 37 DEG C of 5%CO with CSFV C strain
2under condition, cultivate after 48h, after the individual cells that becomes to disperse through 0.25% tryptic digestion, carry out continuous 4 times of gradient dilutions, plant in 96 porocyte plates 37 DEG C of 5%CO
2under condition, cultivate, after 8 hours, observe every hole, get rid of the culture hole that has cell mass, then continue to cultivate 1 week, select the cell hole of being grown by single cell cloning, cell in hole is carried out to subclone screening, obtain subclonal cell line with after this method subclone screening 3 times continuously;
(2) can be high seed selection and the qualification of titre breeding Pestivirus suis C strain clone
The each cloned cell line of above-mentioned EST cell is inoculated in respectively in 24 porocyte culture plates to 37 DEG C of 5%CO
2under condition, cultivate 48h, discard nutrient solution, wash cell 2 times, collecting cell after 0.25% tryptic digestion with PBS, utilize RNAiso Plus (TAKARA) reagent to extract total RNA, after RT-PCR, preliminary evaluation goes out persistent infection the clone of Pestivirus suis C strain;
The Auele Specific Primer of RT-PCR qualification CSFV C strain:
Upstream primer: TAACACGACTGTCAAGGTGC;
Downstream primer: AACCATGAGCAATGCCACT;
Preliminary evaluation is continued to lie in the culture dish of 35mm with malicious EST cell, after cell covers with individual layer, utilize indirect immunofluorescene assay to determine positive cell number, immunofluorescence detection display, almost 100% cell is all with CSFV antigen;
By determining with cultivations of going down to posterity of the EST clone of high titre Pestivirus suis C strain persistent infection, respectively 3,9, in 15 generations, carried out three freeze thawing repeatedly and receive malicious; After the centrifugal removal cell debris of virus stock solution used, by 10 times of gradient dilutions, viral dilution liquid is connect to poison in the PK15 cell that is paved with 96 orifice plates, 37 DEG C, 5%CO with DMEM liquid
2incubator is cultivated after 48h, detects virus infected cell with indirect immunofluorescence method; Calculate viral medium lethal dose TCID50 according to Reed-Muench method, taking the arithmetical av that repeats experimental results 3 times as cultivating viral infection titer; The demonstration of TCID50 measurement result, more than 15 generations of cell continuous passage, its viral minuent maintains 10
5.5tCID
50/ mL is higher 100 times than the virus titer after single inoculation EST parent cell.
Step utilizes indirect immunofluorescene assay to determine positive cell number described in (2), concrete steps are as follows: wash cell 2 times with PBS, through the fixing 20min of 4% paraformaldehyde, add permeable membrane agent 1%Tritonx-100 permeable membrane 20min after washing cell 2 times with PBS; Wash after cell 3 times with PBS, add the anti-CSFV serum of rabbit of dilution in 1: 300, with diluting containing 5% new-born calf serum PBS, 37 DEG C of wet boxes are hatched after 1 hour and are washed away unconjugated primary antibodie, 180r/min, 5min/ time with PBS, totally 3 times, then add the goat anti-rabbit igg-FITC of dilution in 1: 400, the same primary antibodie of dilution process, 37 DEG C of wet boxes are hatched 1 hour; Then wash away unconjugated two with PBS and resist, 180r/min, 5min/ time, totally 3 times, add containing 30% glycerine PBS solution, be placed in fluorescence microscopy Microscopic observation green fluorescence situation.
The above; it is only preferably embodiment of the present invention; protection scope of the present invention is not limited to this; any be familiar with those skilled in the art the present invention disclose technical scope in, the simple change of the technical scheme that can obtain apparently or equivalence replace all fall within the scope of protection of the present invention.
Claims (2)
1. a selection for Pestivirus suis C strain EST sensitive cell line, is characterized in that, comprises the following steps:
(1) EST cell monoclonal
The EST cell polluting without inoculating microbe, infects 37 DEG C of 5%CO with CSFV C strain
2under condition, cultivate after 48h, after the individual cells that becomes to disperse through 0.25% tryptic digestion, carry out continuous 4 times of gradient dilutions, plant in 96 porocyte plates 37 DEG C of 5%CO
2under condition, cultivate, after 8 hours, observe every hole, get rid of the culture hole that has cell mass, then continue to cultivate 1 week, select the cell hole of being grown by single cell cloning, cell in hole is carried out to subclone screening, obtain subclonal cell line with after this method subclone screening 3 times continuously;
(2) can be high seed selection and the qualification of titre breeding Pestivirus suis C strain clone
The each cloned cell line of above-mentioned EST cell is inoculated in respectively in 24 porocyte culture plates to 37 DEG C of 5%CO
2under condition, cultivate 48h, discard nutrient solution, wash cell 2 times with PBS, collecting cell after 0.25% tryptic digestion, utilizes RNAisoPlus reagent to extract total RNA, and after RT-PCR, preliminary evaluation goes out persistent infection the clone of Pestivirus suis C strain;
The Auele Specific Primer of RT-PCR qualification CSFV C strain:
Upstream primer: TAACACGACTGTCAAGGTGC;
Downstream primer: AACCATGAGCAATGCCACT;
Preliminary evaluation is continued to lie in the culture dish of 35mm with malicious EST cell, after cell covers with individual layer, utilize indirect immunofluorescene assay to determine positive cell number, immunofluorescence detection display, almost 100% cell is all with CSFV antigen;
By determining with cultivations of going down to posterity of the EST clone of high titre Pestivirus suis C strain persistent infection, respectively 3,9, in 15 generations, carried out three freeze thawing repeatedly and receive malicious; After the centrifugal removal cell debris of virus stock solution used, by 10 times of gradient dilutions, viral dilution liquid is connect to poison in the PK15 cell that is paved with 96 orifice plates, 37 DEG C, 5%CO with DMEM liquid
2incubator is cultivated after 48h, detects virus infected cell with indirect immunofluorescence method; Calculate viral medium lethal dose TCID50 according to Reed-Muench method, taking the arithmetical av that repeats experimental results 3 times as cultivating viral infection titer; TCID
50measurement result demonstration, more than 15 generations of cell continuous passage, its viral minuent maintains 10
5.5tCID
50/ mL is higher 100 times than the virus titer after single inoculation EST parent cell.
2. the detection method of the relevant miRNA of chicken intestinal diorder Salmonella infection according to claim 1, is characterized in that,
Step utilizes indirect immunofluorescene assay to determine positive cell number described in (2), concrete steps are as follows: wash cell 2 times with PBS, through the fixing 20min of 4% paraformaldehyde, add permeable membrane agent 1%Tritonx-100 permeable membrane 20min after washing cell 2 times with PBS; Wash after cell 3 times with PBS, add the anti-CSFV serum of rabbit of dilution in 1: 300, with diluting containing 5% new-born calf serum PBS, 37 DEG C of wet boxes are hatched after 1 hour and are washed away unconjugated primary antibodie, 180rpm, 5min/ time with PBS, totally 3 times, then add the goat anti-rabbit igg-FITC of dilution in 1: 400, the same primary antibodie of dilution process, 37 DEG C of wet boxes are hatched 1 hour; Then wash away unconjugated two with PBS and resist, 180rpm, 5min/ time, totally 3 times, add containing 30% glycerine PBS solution, be placed in fluorescence microscopy Microscopic observation green fluorescence situation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100577204C (en) * | 2007-11-30 | 2010-01-06 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
| CN102965332B (en) * | 2011-11-30 | 2014-06-04 | 普莱柯生物工程股份有限公司 | Swine testicular clone cell line and production method of classical swine fever live vaccine |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100577204C (en) * | 2007-11-30 | 2010-01-06 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
| CN102965332B (en) * | 2011-11-30 | 2014-06-04 | 普莱柯生物工程股份有限公司 | Swine testicular clone cell line and production method of classical swine fever live vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 万婧: "猪瘟病毒疫苗C株体外适应性的分子基础研究", 《浙江大学硕士学位论文》, 8 June 2011 (2011-06-08) * |
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Inventor after: Jing Xiaoqi Inventor after: Wei Yanquan Inventor after: Huang Guangdong Inventor after: Zhang Qiong Inventor after: Chen Shengye Inventor before: Jing Xiaoqi Inventor before: Sun Shiqi Inventor before: Guo Huichen Inventor before: Wei Yanquan Inventor before: Huang Guangdong Inventor before: Zhang Qiong Inventor before: Chen Shengye |
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Free format text: CORRECT: INVENTOR; FROM: JING XIAOQI SUN SHIQI GUO HUICHEN WEI YANQUAN HUANG GUANGDONG ZHANG QIONG CHEN SHENGYE TO: JING XIAOQI WEI YANQUAN HUANG GUANGDONG ZHANG QIONG CHEN SHENGYE |
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