CN102329780A - Anti-mycoplasma ST (swine testis) cell, and methods for culturing hog cholera virus low virulent strain and preparing hog cholera attenuated vaccine using same - Google Patents

Abstract

The invention provides a new anti-mycoplasma ST (swine testis) cell line, a preparation method of the cell line and methods for preparing hog cholera virus attenuated vaccine and the hog cholera virus low virulent strain using the cell line. The cell provided by the invention is sensitive to the hog cholera virus low virulent strain, can be used for preparing the hog cholera virus attenuated vaccine and can effectively prevent mycoplasma pollution in the culture process.

Description

The ST cell of mycoplasma and with this cell cultures CSFV low virulent strain and prepare the method for swine fever less toxic vaccine

Field under the invention

The invention belongs to the genetically engineered field, specifically, the present invention relates to the mycoplasma cell, prepare the method for this cell and with this cell cultures CSFV low virulent strain and prepare the method for swine fever less toxic vaccine.

Background technology

With ST cell cultures CSFV low virulent strain is one of common technology in the CSFV research work; With this technology serves as that the basis produces the swine fever attenuated live vaccines, is becoming the new trend that present domestic swine Fever Vaccine is produced.But in cell and virus culture link, mycoplasma contamination is a ubiquity and extremely scabrous problem.Mycoplasma contamination in the cell will have a strong impact on result of study, and to virus vaccines such as swine fever less toxic vaccine, mycoplasma contamination will mean that quality product is defective, have great Biosafety hidden danger.(appoint outstanding " Chinese animal and veterinary " 2008 the 35th volumes the 10th phase 39-42; The detection of mycoplasma contamination in the animal cell cultures such as Zhao Jun " Anhui agricultural sciences ".2010,38 (3): 1151-1153; Mycoplasma contamination and countermeasure in the cultured cell in vitro work such as Liu Yuqin " Chinese pathology journal the 33rd volume the 6th phase Chin J Pathol December in 2004, December 2004, Vol 33 No.6)

The method that is usually used in controlling and remove the culturing process mycoplasma contamination mainly contains following two kinds: 1, strict control culture environment and operating process prevent that mycoplasma from sneaking into culture system; 2, with tetracyclines and in cyclic ester class antibiotic etc make an addition in the nutrient solution by certain concentration and program, it killed or suppress to have polluted in the culture mycoplasma that maybe possibly pollute.These 2 kinds of methods all have its major defect: the former is extremely strict to the requirement of culture environment; Existence or introducing that the culture device condition that present China generally uses can't be stopped mycoplasma in the environment; This also makes operator be in technological high pressure conditions for a long time, and the careless slightly culture that just possibly cause by the gross is all discarded.According to statistics; Produce the technology of swine fever less toxic vaccine at present with the bull testis primary cell; Have 30% culture discarded by all because of mycoplasma contamination before harvest product approximately, with conventional ST clone production swine Fever Vaccine, its culturing process is suitable basically therewith by the probability of mycoplasma contamination.And the latter is because employed medicine pair cell when killing or suppressing mycoplasma can produce stronger toxicity; Particularly cause existing in the vaccine antibiotic remains; When therefore using this type medicine control mycoplasma contamination, can only take lower concentration, progressively screening for a long time, this method not only efficient is low; Length consuming time, and the possibility of mycoplasma contamination still exists.(2008 24 the 8th phases of volume of several kinds of treatment scheme curative effects comparative studies " modern medicine health " of mycoplasma contamination in the Liu Lan attached cell culturing process; The product of 200,610,048,802 1 kinds of prevention and control of pollution of cell culture of the number of applying for a patent).

Alexin is one type of natural intravital polypeptides matter of animal that is present in; Has the anti-infective function of wide spectrum; It is one of defensive barrier in the animal body; Many researchs prove: the gene transfection that will express such material is in the genome of multiple vitro culture thing, and these transgenic cultures can be expressed such material or increased such expression of Substance amount (reorganization and the amalgamation and expression " heredity " 25 (2) of Luo Gang porcine defensin gene PBD-I in intestinal bacteria: 146～150,200).Present existing research work obtains the PBD-1 gene transfection to have the mycoplasma pollution and can cultivate some viral cells in clones such as Marc145, BHK21; But these cells are insensitive to the CSFV low virulent strain, can't be applied to a large amount of amplifications and the preparation swine fever less toxic vaccine of CSFV low virulent strain.

The invention technology contents

An object of the present invention is to provide the ST cell that a kind of new mycoplasma pollutes; This cell is responsive to the CSFV low virulent strain; Can be used for CSFV low virulent strain vaccine production, ability excreting beta alexin can effectively be prevented mycoplasma contamination in cell cultures and the virus amplification.

Another object of the present invention provides a kind of method for preparing the ST clone of mycoplasma pollution.

A further object of the present invention provides the application of ST cell in cultured swine pestivirus low virulent strain and preparation swine fever less toxic vaccine that new mycoplasma pollutes.

A further object of the present invention provides the method for the weak poison of ST clone cultured swine pestivirus that utilizes the mycoplasma pollution that obtains.

A further object of the present invention provides ST clone that the mycoplasma that utilize to obtain pollutes and prepares the swine fever less toxic vaccine.

The invention discloses the ST clone that a new mycoplasma pollutes; It is characterized in that; This cell manual work has changed pig PBD-1 gene over to, and the pig PBD-1 gene that changes over to has the nucleotide sequence shown in the SEQ ID NO:1, this cell called after " PBD-1/ST cell "; Be preserved in Chinese typical culture collection center, deposit number is CCTCC C201196.The PBD-1/ST cell is that pig PBD-1 gene is imported pig testis clone ST and alternately screens acquisition through G418 and individual cells clone culture method.This cell also has following characteristic: the PBD-1/ST cell is responsive to the CSFV low virulent strain, can high titre cultured swine pestivirus low virulent strain, can produce pig beta-alexin-1, and can effectively prevent the mycoplasma contamination in the culturing process.

The invention also discloses the method for the ST cell of preparation mycoplasma pollution, this method comprises the steps:

1) gene amplification: with the pig liver total tissue RNA is template, with RT-PCR technology amplification pig PBD-1 gene;

2) plasmid construction: the PBD-1 gene fragment of amplification is cut through enzyme, be connected on the pIRES2-EGFP, and transformed into escherichia coli DH5 α, screening obtains to contain the plasmid of PBD-1 gene;

3) transfection and screening: contain the plasmid and the pig testis clone ST cotransfection of PBD-1 gene, and obtain to stablize the cell of high expression level pig PBD-1 gene through the G418 screening.

The invention also discloses a kind of method of optimizing the ST clone of preparation mycoplasma pollution, this method comprises the steps:

1) gene amplification: according to GenBank gene pool PBD-1 complete genome sequence (gi:47523177), design a pair of primer: the upper reaches and downstream primer are respectively (SEQ ID NO:2): 5'-CGGAATTCATGAGACTCCACCGCCTC-3'; (SEQ ID NO:3): 5'-CGGGATCCCTTCTGAGCCATATCTGTG-3'; And introduce EcoRI and two restriction enzyme sites of BamHI respectively and protect base at 5 ' end of upstream and downstream primer; With the pig liver total tissue RNA is template, with RT-PCR technology amplification PBD-1 gene;

2) plasmid construction: the PBD-1 fragment and the pIRES2-EGFP that will reclaim purifying cut through EcoRI and BamHI enzyme respectively; The purpose fragment of band sticky end reclaims; Under the effect of T4 ligase enzyme, carrying out cohesive end connects; To connect product and transform, choose through kalamycin resistance screening that the positive colony bacterium colony is cultivated and extract plasmid in a small amount in bacillus coli DH 5 alpha;

3) transfection and screening: with logarithmic phase ST cell inoculation in petridish; In gnotobasis, cultivated 24 hours under 37 ℃, 5%CO2 condition; Treat cytogamy to 70%～80% o'clock; Carry out the cell transfecting operation according to Lipofectamine 2000 process specificationss, replacing has serum, unparalleled anti-high sugared DMEM substratum to continue to cultivate after 6 hours; After the transfection 48 hours, begin the screening with G418, then the concentration with 2 000 μ g/ml continued screening with 1000 μ g/ml in 1-3 days, until most necrocytosiss, changed G418 concentration into 1000 μ g/ml and kept 6 weeks of screening.To obtain cell and adopt the Method of Limited Dilution method to reach 96 porocyte culture plates, about 1 cell in every hole is used alternatingly conventional substratum and 1000 μ g/ml G418 culture medium culturing individual cells, grows single clone to it.Under fluorescent microscope, pick out optimum cell clone and continue to cultivate, and constantly expand numerously, finally build up the transgenic ST clone of stable transfection PBD-1.

This cell called after " PBD-1/ST cell " contain the pig PBD-1 gene that manual work changes over to, and pig PBD-1 gene has the nucleotide sequence shown in the SEQ ID NO:1; The PBD-1/ST cell is preserved in Chinese typical culture collection center on September 27th, 2011, and deposit number is CCTCC C201196.This cell has following characteristic: the PBD-1/ST cell is responsive to the CSFV low virulent strain, can high titre cultured swine pestivirus low virulent strain, can produce beta-defensin, efficiency prevention mycoplasma contamination.

Plasmid vector pIRES2-EGFP among the present invention can obtain through commercially available commercial biotech firm; Like Clontech Laboratories; Inc. A Takara Bio Company company; Bacillus coli DH 5 alpha and ST cell can obtain through commercially available commercial biotech firm or culture presevation mechanism, like China's typical culture collection center preservation this bacterial classification are arranged, and the investigator can ask for to this center.

The ST clone that the invention also discloses new mycoplasma pollution is used with preparing in the CSFV low virulent strain vaccine at cultured swine pestivirus low virulent strain.

The invention also discloses the method for utilizing the PBD-1/ST cell cultures CSFV low virulent strain that obtains, this method comprises the steps:

1) cell is prepared: cultivate the PBD-1/ST cell to needed cell concentration with ordinary method; Observe at fluorescence (wavelength is about 490nm) microscopically; Counting is wherein dispersed the ratio of cell of fluorescence, and its ratio is qualified cell 95% when above, can be used for virus culture;

2) virus infection liquid preparation: the ID50 that learns from else's experience measures the rabbit spleen of tiring at 50,000 times-80,000 times CSFV low virulent strain and drenches poison, mixes according to the ratio of/1000 milliliters of 2 grams and cell maintenance medium and processes;

3) virus inoculation: the culture supernatant liquid in the sucking-off cell culture container, inject and the isopyknic viral liquid of supernatant then;

4) virus amplification: the PBD-1/ST cell of virus inoculation is put into 37 ℃ of culture environment continue to cultivate;

5) collection virus: virus culture 4-5 days, detect CSFV with fluorescent quantitative RT-PCR method, receive poison during greater than 10ng/ul at viral RNA content, whenever later on received poison once at a distance from 3 days, overcharge poison most 5 times, acquisition CSFV low virulent strain.

The invention also discloses the method for utilizing the PBD-1/ST cell preparation CSFV low virulent strain vaccine that obtains, this method comprises the steps:

1) cell is prepared: cultivate the PBD-1/ST cell to needed cell concentration with ordinary method; Under fluorescent microscope, observe; Counting is wherein dispersed the ratio of cell of fluorescence, and its ratio is qualified cell 95% when above, can be used for virus culture or vaccine production;

2) virus infection liquid preparation: the ID50 that learns from else's experience measures the rabbit spleen of tiring at 50,000 times-80,000 times CSFV low virulent strain and drenches poison, mixes according to the ratio of/1000 milliliters of 2 grams and cell maintenance medium and processes;

3) virus inoculation: the culture supernatant liquid in the sucking-off cell culture container, inject and the isopyknic virus infection liquid of supernatant then;

4) virus amplification: the PBD-1/ST cell of virus inoculation is put into 37 ℃ of culture environment continue to cultivate;

5) collection virus: virus culture 4-5 days, detect CSFV with fluorescent quantitative RT-PCR method, receive poison during greater than 10ng/ul at viral RNA content, whenever later on received poison once at a distance from 3 days, overcharge most malicious 5 times;

6) vaccine production: with the virus-culturing fluid that is up to the standards and the 5% sucrose skimmed milk stablizer mixed by 1:1, freeze-drying is carried out in packing, promptly becomes malicious freeze dried vaccine a little less than the swine fever.

The swine fever less toxic vaccine of the present invention's preparation can be used for preventing the sick or treatment swine fever disease of swine fever.

Advantage of the present invention

Compare in prior art, the present invention has following advantage:

1) PBD-1/ST cell provided by the invention can high be tired and produced the pig beta-alexin-1 of higher concentration: the rolling bottle culture condition is issued to 225.6 ± 10.6 ng/ul; So the pig beta-alexin of high density can be resisted the mycoplasma contamination in the culturing process, and this is the not available ability of common ST cell.

2) PBD-1/ST cell provided by the invention is also comparatively responsive to the CSFV low virulent strain simultaneously; Can high-efficient culture the CSFV low virulent strain: with the PBD-1/ST cell cultures pestivirus low virulent strain of raising pigs that increases; Its cultivation of ID50 method mensuration is tired and can be reached 500,000 times, and this is other not available abilities of cell of changeing the PBD-1 gene.The CSFV low virulent strain that height like this is tired can be used for research of CSFV low virulent strain and preparation swine fever less toxic vaccine.

3) Using P BD-1/ST clone cultured swine pestivirus low virulent strain provided by the invention and the method for preparing the swine fever less toxic vaccine; It is the novel method that above-mentioned two characteristics of PBD-1/ST clone are combined and form; This method is when cultured swine pestivirus low virulent strain and preparation swine fever less toxic vaccine; Need not to take special specific aim measure to take precautions against mycoplasma contamination, mycoplasma contamination can not take place in the vaccine that the ability of leaning on the unique mycoplasma of this cell to pollute just can make the viral product of being cultivated obtain preparation.This is that the method for present any cultured swine pestivirus is beyond one's reach.

Description of drawings

Fig. 1 is the recombinant plasmid organigram

Fig. 2 is that the PBD-1 gene enzyme is cut gel electrophoresis figure

M representes to contrast Mark molecule band 1 expression PBD-1 plasmid molecule band

Fig. 3 is the pcr amplification pig PBD-1The sequencing result figure of gene

Fig. 4 is that the enzyme of pig PBD-1 recombinant plasmid is cut evaluation figure

M1 representes to contrast DL 2000 marker; 1 expression PBD-1 recombinant plasmid is through EcoRI and BamHI double digestion; 2 expression PBD-1 recombinant plasmids are cut through the BamHI enzyme; 3 expression PBD-1 recombinant plasmids are cut through the EcoRI enzyme; M2 representes to contrast 1kb Ladder DNA Marker

Fig. 5 is in the recombinant plasmid PBD-1The sequencing result figure of gene

Fig. 6 is a PBD-1/ ST cell fluorescence microgram

Fig. 7 is the PCR detection figure of mycoplasma in the cell cultures

Detection of mycoplasma after 1 expression PBD-1/ ST cell and the infection mycoplasma ST co-culture of cells

2 expression PBD-1/ ST cells are handled the detection of mycoplasma of back and infection mycoplasma ST co-culture of cells through MTC

Detection of mycoplasma behind the 3 expression PBD-1/ ST cell conditioned medium liquid cultivation infection mycoplasma ST cells.

Embodiment

Come further to set forth the present invention below in conjunction with concrete embodiment.Should be appreciated that these embodiment only are used to explain the present invention, and can not limit protection scope of the present invention.

Embodiment 1 sets up the ST clone of stable transfection PBD-1 gene

According to Fig. 1 signal, follow these steps to operation

1.1 design of primers is with synthetic: according to GenBank gene pool PBD-1 complete genome sequence (gi:47523177), design a pair of primer: the upper reaches and downstream primer are respectively: (SEQ ID NO:2) 5'-CGGAATTCATGAGACTCCACCGCCTC-3'; (SEQ ID NO:3) 5'-CGGGATCCCTTCTGAGCCATATCTGTG-3', and introduce EcoRI and two restriction enzyme sites of BamHI respectively and protect base at 5 ' end of upstream and downstream primer.After the design of primers, seek professional institution's synthetic primer that qualification is arranged.

1.2 gene amplification: with the pig liver total tissue RNA is template, with RT-PCR technology amplification PBD-1 gene.The PCR reaction system is 25 μ L, and wherein template liver organization cDNA is 50 ng, and dNTPs concentration is 200 μ mo1/L, and (upper reaches and downstream primer are respectively (SEQ ID NO:2) 5'-CGGAATTCATGAGACTCCACCGCCTC-3' to every primer; (SEQ ID NO:3) 5'-CGGGATCCCTTCTGAGCCATATCTGTG-3') concentration is 0.4 μ mol/ L, and 1U Taq archaeal dna polymerase adds deionized water to TV 25 μ L.Reaction conditions: 94 ℃ of preparatory sex change 4min, 94 ℃ of sex change 40s, 59 ℃ of annealing 30s, 72 ℃ are extended 30s, 35 circulations.Last is taken turns 72 ℃ and extends 10min.Again reaction product is carried out gel electrophoresis, collect the PBD-1 gene fragment that the electrophoresis product is purifying at the 252bp place.Gene molecule gel electrophoresis figure according to this schedule of operation amplification obtains is seen Fig. 2, obtain reclaiming base sequence such as Fig. 3 of gene fragment through gene sequencing

1.3 Construction of eukaryotic: the PBD-1 fragment and the pIRES2-EGFP that will reclaim purifying cut through EcoRI and BamHI enzyme respectively; The purpose fragment of band sticky end is carried out purifying and recovering by the operation instruction that glue reclaims test kit; Under the effect of T4 ligase enzyme, carrying out cohesive end connects; To connect product and transform α, choose through kalamycin resistance screening that the positive colony bacterium colony is cultivated and extract plasmid in a small amount in DH5.Plasmid carried out PCR identifies and order-checking behind EcoRI and the BamHI double digestion.

According to the method described above, PCR evaluation figure saw Fig. 4 after plasmid carried out EcoRI and BamHI double digestion, and sequencer map is seen Fig. 5

1.4 transfection and screening: 3 times the ST cell of going down to posterity after will recovering is with tryptic digestion, with the high sugared DMEM substratum furnishing 5 * 10 that contains 10% foetal calf serum ⁵The density of individual/ml; Be inoculated in the petridish that several diameters are 35mm; In gnotobasis, cultivated 24 hours under 37 ℃, 5%CO2 condition; Treat cytogamy to 70%～80% o'clock, carry out the cell transfecting operation according to Lipofectamine 2000 process specificationss, change after 6 hours serum is arranged, unparalleled anti-high sugared DMEM substratum continues to cultivate.After the transfection 2 days, begin to screen with G418.Process is: first three days 1000 μ g/ml, then the concentration with 2 000 μ g/ml continues screening, until most necrocytosiss, changes G418 concentration into 1000 μ g/ml and keeps 6 weeks of screening.To obtain cell and adopt the Method of Limited Dilution method to reach 96 porocyte culture plates, about 1 cell in every hole is used alternatingly conventional substratum and G418 (1000 μ g/ml) culture medium culturing individual cells, grows single clone to it.Under fluorescent microscope, pick out optimum cell clone and continue to cultivate, and constantly expand numerously, finally build up the transgenic ST clone of stable transfection PBD-1.The method of building cell bank according to general clone, with the frozen step by step transgenic cell storehouse of setting up of cell of cultured commentaries on classics PBD-1 gene with subsequent use.This cell called after " PBD-1/ST cell " is preserved in Chinese typical culture collection center on September 27th, 2011, and deposit number is CCTCC C201196.

The ST cell fluorescence microscopically observations of the commentaries on classics PBD-1 gene of setting up is according to the method described above seen Fig. 6, detects through mycoplasma PCR, does not detect mycoplasma all the time and exists.

1.5 the proteic observation of PBD-1/ST cell expressing PBD-1: get the culture supernatant liquid that rolling bottle is cultivated, Kolle flask is cultivated about 24 hours PBD-1/ST cell respectively; The E-lisa method detects wherein PBD-1 protein content, and the MV of every group of 10 sample determinations is respectively 87.2 ± 2.2 ng/ul, 225.6 ± 10.6 ng/ul.

The cultivation of embodiment 2 PBD-1/ST cells

In full accord with the cultivation operation of common ST cell; Just in the passage process; Every biography 3-5 is for taking a sample once; At the fluorescence of fluorescence (wavelength is about 490nm) microscopically observation of cell, as cell observation more than 5% occurs, show that then this batch cell the parts of fine dysuria with lower abdominal colic occurs and goes into gene and lose in culturing process less than fluorescence; Such cell just can not continue to cultivate and uses as the cell that changes the PBD-1 gene, has only batch cell just not occur the cell that transgene loses more than 95% and can continue to cultivate as target cell.

According to the method described above, the ST cell cultures of the commentaries on classics PBD-1 gene that will from cell bank, take out after 10 generations fluorescence (wavelength is about 490nm) microscopically observations see Fig. 7, detect through mycoplasma PCR, do not detect mycoplasma all the time and exist.

The ST cell cultures CSFV low virulent strain of embodiment 3, applying transgene PBD-1 gene

3.1 keep the liquid preparation: MEM substratum degerming stoste or conventional sterile milk Chinese liquid add 2% NBCS, promptly become the liquid of keeping of cultured swine pestivirus.

3.2 cell is prepared: the ST cell cultures that will change the PBD-1 gene with the conventional ST passage cultured method of cultivation arrives needed cell concentration; Aseptic condition sampling is down observed; Observe at fluorescence (wavelength is about 490nm) microscopically; Counting is wherein dispersed the ratio of cell of fluorescence, and its ratio is qualified cell 95% when above, can be used for virus culture.

3.3 virus is prepared: get the rabbit spleen of tiring and drench poison at the CSFV low virulent strain of (ID50 mensurations) more than 50,000 times-80,000 times, according to 2 restrain/1000 milliliters ratio and cell maintenance medium mix and promptly become virus infection liquid.

3.4 inoculation: the culture supernatant liquid under the aseptic condition in the sucking-off cell culture container, inject and the isopyknic virus infection liquid of supernatant then.

3.5 cultivate: the cell of virus inoculation is put into 37 ℃ of culture environment continue to cultivate.

3.6 receive poison: virus culture is after 5 days, and the supernatant that takes a morsel detects with quantifying PCR method, carries out receiving first time poison during like viral RNA content wherein＞10ng/ul, every later at a distance from 3 days receipts poison once, according to the definite malicious number of times of the highest receipts of different purposes.

3.7 according to the method described above, respectively on January 5th, 1 2010 on November 7th, 2009; On May 5th, 1 2010 on March 4th, 2010; Carry out three batches of virus culture tests on July 3rd, 1 2010 on May 20th, 2010, through detecting: the viral liquid of results does not have mycoplasma contamination, and ID50 method mensuration virus is on average tired and is respectively: 500,000 times, and 500,000 times, 200,000 times.

Embodiment 4, the ST cell preparation swine fever less toxic vaccine of applying transgene PBD-1 gene

Get the weak viral disease poison of swine fever of in March, 2010-2010 batch cultivation in year April in the instance 3, the virus of preceding 5 collections mixes common 5000ml, and the virus titer that records its hybrid virus liquid is 500,000 times; Lyophilized vaccine thorough mixing with equivalent; In the Freeze Drying Equipment freeze-drying, become the test group freeze dried vaccine, get simultaneously the common ST cell cultures of another batch usefulness same strain, with tire, with receiving time CSFV; Be prepared into freeze dried vaccine as the control group freeze dried vaccine with same method; Test group vaccine and control group vaccine be 5 piglets of immunity respectively, and immunity is the precaval vein blood sampling after 14 days, separation of serum; The forward indirect hemagglutination method records on average tiring of two groups of serum antibodies and is respectively: 1:152.4 and 1:164.2, all minimal protection antibody horizontal (1:32) head and shoulders above.Prove that it possesses the vaccine immunity function fully.

Embodiment 5, and PBD-1/ST cell mycoplasma pollutes functional verification

5.1 infect the PBD-1/ST cell with the positive substratum of mycoplasma: what in the substratum of cultivating the PBD-1/ST cell, add 10% ratio is determined as mycoplasma male nutrient solution composition mycoplasma infection liquid through PCR; Infect liquid with this and cultivated the PBD-1/ST cell 48 hours; Use no mycoplasma culture medium instead and continue to cultivate this PBD-1/ST cell, changed substratum 1 time in per 2 days, after the 5th day; The one-time detection of taking a sample every day mycoplasma; As a result, detect in the culture supernatant liquid less than the mycoplasma existence after 5 days, fail to detect mycoplasma later on always and exist.

5.2 the positive ST co-culture of cells of transgenic cell and mycoplasma: respectively with well-grown transgenic ST cell provided by the present invention, well-grown and through special-purpose PCR test kit detect for the common ST cell of mycoplasma male with 0.25% trypsinase-0.02%EDTA mixed solution digestion, 3 times of substratum piping and druming diffusing after; Again will and this mixes; Pass 6 ratio average mark in 2 and pack into and continue to cultivate in 6 new culturing bottles, go down to posterity later on and undertaken by routine.Get supernatant every day once, special-purpose PCR test kit detects and makes the mycoplasma qualitative detection, and the result sees Fig. 7 .1

5.3 the transgenic cell and mycoplasma positive ST co-culture of cells handled by MTC: get one bottle of well-grown transgenic ST cell provided by the present invention; In super-clean environment sucking-off supernatant; Adding the DMEM high glucose medium that contains the 20ug/ml MTC continues to cultivate 3 hours; Contain the MTC substratum in the super-clean environment sucking-off, more than 5 times, loose with 0.25% trypsinase-0.02%EDTA mixed solution digestion, 3 times of substratum piping and druming with the PBS washing; Getting one bottle of well-grown simultaneously and detecting through special-purpose PCR test kit is that the common ST cell of mycoplasma male looses with 0.25% trypsinase-0.02%EDTA mixed solution digestion, 3 times of substratum piping and druming; Then these two cell suspensions are mixed; Pass 6 ratio average mark in 2 and pack into and continue to cultivate in 6 new culturing bottles, go down to posterity later on and undertaken by routine.Get supernatant every day once, special-purpose PCR test kit detects and makes the mycoplasma qualitative detection, and the result sees Fig. 7 .2

5.4 the supernatant that transgenic cell is cultivated is cultivated the positive ST cell of mycoplasma by 50% ratio preparing culture medium: collect the supernatant in the cultivation well-grown transgenic ST cell provided by the present invention 24-48 hour; 0.22um filtration sterilization is subsequent use, the preparation serum content is 15% DMEM or newborn Chinese liquid (select which kind of substratum consistent with the used substratum of supernatant).The substratum of supernatant and new preparation respectively mixed by 50% ratio row becomes new substratum; Use this culture medium culturing to detect and be the common ST cell of mycoplasma male through special-purpose PCR test kit; Other operations are undertaken by routine; Get supernatant every day once, special-purpose PCR test kit detects and makes the mycoplasma qualitative detection, and the result sees Fig. 7 .3.

SEQUENCE?LISTING

 

< 110>the ST cell of mycoplasma and with this cell cultures CSFV low virulent strain and preparation swine fever less toxic vaccine

Method

< 120>the ST cell of mycoplasma and with this cell cultures CSFV low virulent strain and preparation swine fever less toxic vaccine

Method

 

< 130>the ST cell of mycoplasma and with this cell cultures CSFV low virulent strain and preparation swine fever less toxic vaccine

Method

 

<160> 3

 

<170> PatentIn?version?3.3

 

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ggcaagtgtg?ctccaaagat?gaaacagatc?ggcacctgtg?gcatgcccca?agtcaaatgc 180

 

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Claims (6)

1. ST cell that new mycoplasma pollutes; It is characterized in that; This cell contains the pig PBD-1 gene that manual work changes over to, and pig PBD-1 gene has the nucleotide sequence shown in the SEQ ID NO:1, and this cell is the PBD-1/ST cell; Be preserved in Chinese typical culture collection center, deposit number is CCTCC C201196.

2. prepare the method for the ST cell of the new mycoplasma pollution described in the claim 1, this method comprises the steps:

1) gene amplification: with the pig liver total tissue RNA is template, with RT-PCR technology amplification pig PBD-1 gene;

2) plasmid construction: the PBD-1 gene fragment of amplification is cut through enzyme, be connected on the pIRES2-EGFP, and transformed into escherichia coli DH5 α, screening obtains to contain the plasmid of PBD-1 gene;

3) transfection and screening: contain the plasmid and the pig testis clone ST cotransfection of PBD-1 gene, and alternately screen the cell that obtains stably express pig PBD-1 gene through G418 and single cell clone culture method.

3. the application of ST cell in cultured swine pestivirus low virulent strain that new mycoplasma pollutes described in the claim 1.

4. the application of the ST cell that new mycoplasma pollutes described in the claim 1 in preparation swine fever less toxic vaccine.

5. the method for the ST cell cultures CSFV low virulent strain that new mycoplasma pollutes described in the claim 1, this method comprises the steps:

1) cell is prepared: cultivate the PBD-1/ST cell to needed cell concentration with ordinary method; Under fluorescent microscope, observe; Counting is wherein dispersed the ratio of cell of fluorescence, and its ratio is qualified cell 95% when above, can be used for virus culture or vaccine production;

2) virus infection liquid preparation: the ID50 that learns from else's experience measures the rabbit spleen of tiring at 50,000 times-80,000 times CSFV low virulent strain and drenches poison, mixes according to the ratio of/1000 milliliters of 2 grams and cell maintenance medium and processes;

3) virus inoculation: the culture supernatant liquid in the sucking-off cell culture container, inject and the isopyknic virus infection liquid of supernatant then;

4) virus amplification: the PBD-1/ST cell of virus inoculation is put into 37 ℃ of culture environment continue to cultivate;

5) collection virus: virus culture 4-5 days, detect CSFV with fluorescent quantitative RT-PCR method, receive poison during greater than 10ng/ul at viral RNA content, whenever later on received poison once at a distance from 3 days, acquisition CSFV low virulent strain.

6. the method for the ST cell preparation CSFV low virulent strain vaccine that new mycoplasma pollutes described in the claim 1, this method comprises the steps:

1) cell is prepared: cultivate the PBD-1/ST cell to needed cell concentration with ordinary method; Under fluorescent microscope, observe; Counting is wherein dispersed the ratio of cell of fluorescence, and its ratio is qualified cell 95% when above, can be used for virus culture or vaccine production;

2) virus infection liquid preparation: the ID50 that learns from else's experience measures the rabbit spleen of tiring at 50,000 times-80,000 times CSFV low virulent strain and drenches poison, mixes according to the ratio of/1000 milliliters of 2 grams and cell maintenance medium and processes;

3) virus inoculation: the culture supernatant liquid in the sucking-off cell culture container, inject and the isopyknic virus infection liquid of supernatant then;

4) virus amplification: the PBD-1/ST cell of virus inoculation is put into 37 ℃ of culture environment continue to cultivate;

5) collection virus: virus culture 4-5 days, detect CSFV with fluorescent quantitative RT-PCR method, receive poison during greater than 10ng/ul at viral RNA content, whenever later on received poison once at a distance from 3 days, overcharge most malicious 5 times;

6) vaccine production: with the virus-culturing fluid that is up to the standards and the 5% sucrose skimmed milk stablizer mixed by 1:1, freeze-drying is carried out in packing, promptly becomes malicious freeze dried vaccine a little less than the swine fever.

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