CN103789276B - A kind of respiratory syncytial virus attenuation strain and application thereof - Google Patents
A kind of respiratory syncytial virus attenuation strain and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of respiratory syncytial virus attenuation strain KM516 – AS, does is its deposit number in China typical culture collection center CCTCC? NO:V201344; And carrying out cell cultures in vitro with this attenuation strain, results attenuation strain, carries out purifying, is prepared into respiratory syncytial virus attenuated vaccine, after this vaccine immunity animal, have good immunogenicity, protectiveness and security to animal.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of respiratory syncytial virus attenuation strain, and use this strain to prepare the preparation method of attenuated vaccine and the immune protective effect etc. to animal.
Background technology
Respiratory syncytial virus (Respiratorysyncytialvirus, RSV) be the common virus of infant's acute respiratory infection, belong to paramyxovirus section, pneumonitis virus belongs to, virus particle diameter is 150 ~ 300nm, is the minus-stranded rna virus of non-segmental.In tissue culture, it can cause iuntercellular boundary to disappear, and merges formation synplasm, and by its called after respiratory syncytial virus.RSV is widely current in all over the world, is to cause infant, one of the elderly and the modal pathogenic micro-organism of immunocompromised crowd lower respiratory infection.The most effective approach of prevention rsv infection inoculates RSV vaccine exactly, one of Vaccine Project that RSV vaccine has developed as 21 century override by the World Health Organization, is therefore necessary to take different strategies to accelerate research and development preventative vaccine.
Go through the very long long road of decades, people differently successively develop formalin-inactivated vaccine, subunit vaccine and attenuated vaccine etc., but also not RSV vaccine appearance safely and effectively so far, because all there is problem in various degree in these vaccines.Formalin inactivation RSV vaccine (Formalin-InactivatedRSVVaccine, FI – RSV vaccine) just develop in the sixties in 20th century, when after human vaccination, natural infection RSV and laboratory animal inoculate FI – RSV vaccine at that time, find that there is the unforeseen circumstances such as granulation, aggravation, analyze reason and have 2 points: one is that FI – RSV vaccine can induce body to produce the serum antibody of high-titer, but the NAT produced is very low; Two is that immune FI – RSV vaccine can strengthen CD4 in inoculator's body
+t cell is active, and this also can increase the weight of inoculator's state of an illness.
Subunit vaccine, RSV film surface fusion protein F and adhesion protein G can produce protection antibody by excitating organism, are the main virus antigens of development RSV subunit vaccine, and body can be induced to produce the neutralizing antibody reaction of protection lower respiratory tract.RSV subunit vaccine has four principal characters: in seronegative orangutan and adult, the immunogenicity of subunit vaccine is very weak, and so in newborn infant, the immunizing dose of this vaccine cannot play effective level of protection; The Neutralization antibody of induction is very low, can not available protecting vaccine recipient; The same with FI – RSV vaccine, subunit vaccine can cause disease aggravation; This vaccine is parenteral route immunity, female immune response passing antibody and significantly can suppress this vaccine.Any one in these four features all can make subunit vaccine can not use in low age infant.
Attenuated live vaccine, it is the candidate vaccine of current most potentiality, it has following feature: RSV living vaccine can stimulate body generation to infect similar immune response to wild-type RSV, comprises neutralizing antibody, can protect the mucoantibody of lower respiratory tract and the cell immune response of balance; Even if there is female biography antibody, the RSV attenuated live vaccine still reproducible of Nasal immunization; After seronegativity infant immunization RSV attenuated live vaccine, in RSV natural infection afterwards, not there is aggravation.Therefore, attenuation RSV living vaccine is the available strategy of exploitation newborn infant vaccine, is one of outstanding candidate vaccine.And early stage RSV attenuated live vaccine exists the inadequate or attenuation of attenuation excessively or the problem such as virulence reversion, so be still necessary continual exploitation RSV attenuated live vaccine safely and effectively.
Summary of the invention
The object of this invention is to provide strain respiratory syncytial virus attenuation strain (humanrespiratorysyncytialvirusKM516-AS), this attenuation strain is the respiratory syncytial virus strain filtered out from First People's Hospital of Yunnan Province pneumonia children sputum sample, gone down to posterity by continuous low temperature and obtain, called after human respiratory syncytial virus KM516 – AS, this attenuation strain is preserved in China typical culture collection center on November 5th, 2013 and (is called for short CCTCC, address Wuhan, China, Wuhan University), preserving number is CCTCCNO:V201344; Respiratory syncytial virus attenuation strain KM516 – AS of the present invention proves to have good immunogenicity, protectiveness and security through animal experiment, can be used as the seed culture of viruses producing attenuated live vaccine.
Another object of the present invention respiratory syncytial virus attenuation strain is applied in preparation respiratory syncytial virus attenuated vaccine, and this preparation method comprises the steps:
Nutrient solution static gas wave refrigerator cell is used, until cell grows up to individual layer at (1) 37 DEG C; Described cell is the one in Vero cell, diploid cell MRC – 5 cell, KMB – 17 cell, 2BS cell, WI – 38 cell, these cells are purchased from ATCC or domestic relevant producer, and nutrient solution is contain the MEM of 10% new-born calf serum or be added with the DMEM nutrient solution of 2 – 10% new-born calf serum;
(2) be 0.01 ~ 0.5 be inoculated in above-mentioned cell culture fluid by respiratory syncytial virus attenuation strain KM516 – AS with infection multiplicity, cultivate until cytopathy reaches 75% ~ 90% at 25 DEG C;
(3) preparation of rough attenuated virus liquid adopts the ordinary method of this area, namely first discards the nutrient solution of sick cell, adds the phosphate buffered saline buffer that pH value is 7.2, after multigelation, sonicated cells, reaches more than 95% to cell crashing ratio, obtains rough attenuated virus liquid;
(4) purified virus prepares the method for vaccine is adopt sucrose density gradient centrifugation conventional in this area, the rough attenuated virus liquid that step (3) is obtained is centrifugal, extract supernatant liquor, by the supernatant liquor containing virus through saccharose gradient (concentration range: 2ml60% sucrose from bottom; 2ml45% sucrose; 2ml30% sucrose; 2ml15% sucrose), 40000rpm4 DEG C of centrifugal 6h, removes the impurity such as remaining sucrose ion, with phosphate solution as PBS suspension gained sample by Tape samples through the dialysis tubing that molecular retention amount is 50kd afterwards further, and survey virus titer, obtained respiratory syncytial virus attenuated vaccine thus.
The present invention adopts respiratory syncytial virus attenuation KM516 – AS strain, and Vero cell, diploid cell MRC – 5 cell, KMB – 17 cell, 2BS cell, WI – 38 cell matrix and corresponding method can obtain effective respiratory syncytial viral antigens; In the inventive method, the cell matrix of propagative viruses is Vero cell, diploid cell MRC – 5 cell or KMB – 17 cell or 2BS cell or WI – 38 cell, that the World Health Organization (WHO) is recommended as the desirable cell matrix producing vaccine for man, compared with other cell matrixs, these cell matrixs are safe and reliable as vaccine for man cell matrix.In addition, experimentation on animals has proved that KM516 – AS strain has good immunogenicity, protectiveness and security.
Compared to prior art, the present invention adopts respiratory syncytial virus attenuation strain KM516 – AS strain to cultivate in the cells such as Vero cell, diploid cell, High-efficient Production respiratory syncytial virus attenuated vaccine can be carried out, for significantly reducing production cost, preparing effective, cheap respiratory syncytial virus attenuated vaccine and providing guarantee; In addition, vaccine of the present invention is high purity attenuated vaccine, makes the security of respiratory syncytial virus vaccines, validity ensured.
Accompanying drawing explanation
Fig. 1 is the Electronic Speculum schematic diagram of the normal Vero cell used in the present invention;
Fig. 2 is that in the present invention, KM516-AS strain adapts to breed schematic diagram on Vero cell;
Fig. 3 is RSV strain Chao Shi PCR primer electrophoresis result schematic diagram in the present invention; Swimming lane 1:DNAMarkerDL2000 in figure; Lane2:RSV; Lane3:Negativecontrol;
Fig. 4 is the F gene comparison result schematic diagram in the present invention in RSV strain Chao Shi PCR primer and NCBI;
Fig. 5 is the BLAST result schematic diagram of RSV strain Chao Shi PCR primer sequence in NCBI in the present invention;
Fig. 6 is RSV strain virus titer result schematic diagram under differing temps in the present invention;
Fig. 7 is attenuation strain KM516-AS Cold tolerance and temperature sensitivity result schematic diagram in the present invention;
Fig. 8 is the 8th day ratio the 0th day body weight evolution result schematic diagram after small mouse of the present invention inoculation;
Fig. 9 is the photo (blank and cell controls) of small mouse pulmonary lesion of the present invention and lung pathologies section;
Figure 10 is the photo (KM516-AS and KM516-WT) of small mouse pulmonary lesion of the present invention and lung pathologies section;
Figure 11 is upper lower respiratory tract residual virus titres result schematic diagram after small mouse of the present invention inoculation;
Figure 12 is the organ coefficient result schematic diagram of mouse lungs after inoculation in the present invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail, but scope is not limited to described content; The main cell biology method adopting routine in embodiment, these methods are well known to those of ordinary skill in the art.According to following examples, be not difficult slightly modified and conversion and successful implementation the present invention as the case may be, and these amendments and conversion all drop in the scope of the application's claim.
Embodiment 1:Vero cell cultures
In liquid nitrogen, take out the hot water that Vero cell (buying in U.S. ATCC, numbering CCL – 81) puts into rapidly 37 DEG C, make cell suspension fast melt, then MEM or DMEM(added containing 10% new-born calf serum buys in GIBCOInvitrogenCorporation company) nutrient solution, pH to 7.2 is adjusted to cultivate about 4h in 37 DEG C, discard whole nutrient solution, add fresh nutrient solution to continue to cultivate, after growing up to fine and close individual layer, buy in GIBCOInvitrogenCorporation company with PBS() wash cell, Yi Mei – EDTA(buys in GIBCOInvitrogenCorporation company) digestion, add above-mentioned nutrient solution, kind of a rate passage cell is divided with 1:2 ~ 1:4, until obtain enough cells of planting needed for poison, be normal Vero cell as shown in Figure 1.
The separation andpreconcentration of embodiment 2: respiratory syncytial virus RSV
The 20 parts of pneumonia children sputum samples obtained from First People's Hospital of Yunnan Province, respectively get 1ml and be suspended into 20ml with PBS respectively, mixing, 0.22 μm of membrane filtration is degerming, respectively the filtrate 1ml obtained is inoculated Vero cell, 37 DEG C of cultivations, use maintenance medium (maintenance medium: add the new-born calf serum of 2%, the NaHCO of 3% in MEM or DMEM nutrient solution
3) cultivate, filter out synplasm phenomenon and breed, a strain RSV strain that pathology is the fastest, as shown in Figure 2.
The strain of acquisition is got 200 μ L, put into through DEPC(purchased from the great Bioisystech Co., Ltd in Shanghai) process is without the 1.5mLEP pipe of RNase, add 800 μ LTripure reagent (purchased from Tian Gen biochemical technology company limited (Beijing)) again, thermal agitation, leave standstill after add 100 μ L chloroforms (purchased from Chongqing Chuan Dong Chemical Group company limited) again, vibration, leave standstill, the centrifugal 10min of 12000rpm.Extract supernatant, add the Virahol of 1 times of volume and the 3M sodium-acetate (purchased from Shantou Xilong Chemical Factory Co., Ltd) of 1/10 volume, mixing is centrifugal after leaving standstill, abandon supernatant, precipitation washs 2 times with 70% ice ethanol (purchased from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd.), use 100% ice washing with alcohol 1 time again, add 20 μ L through the sterilized water of DEPC process without RNase, outstanding even precipitation, add 0.5 μ LRNaseinhibitor(purchased from precious biotechnology (Dalian) company limited), be the RNA template of RSV.Then prepare the reverse transcription system of RSV: RNA template 11 μ L, dNTP2 μ L, 5 × M-MLVbuffer(are purchased from precious biotechnology (Dalian) company limited) 4 μ L, primer EP21 μ L, adds sterilized water to 25 μ L.After mixing, 70 DEG C of water-bath 10min ,-20 DEG C of chilling 2min on ice, 70 DEG C of water-bath 2min, ice-water bath 2min, add 1 μ LRNaseinhibitor and 1 μ LM-MLV, reverse transcription in the PCR instrument of Ke Regai: 42 DEG C, 1h; 70 DEG C, 5min, results reverse transcription product cDNA, be the template of nest-type PRC qualification outer circulation amplification.
Use RSVF gene identification primer again: EP15 '-GTTTTTGTTAGGTGTTGGATCTGC-3 '; EP25 '-CATTTAGTTTTGCCATAGCATGAC-3; IP35 '-CATTTAGTTTTGCCATAGCATGAC-3 '; IP45 '-GAAGAAAGATACTGATCCTGC-3 ' carry out nest-type PRC (outer circulation increase: template cDNA3 μ L, 10 ×
taqthe each 1 μ L of buffer2.5 μ L, dNTP1.5 μ L, outer circulation primer EP1 and EP2,
taqdNApolymerase0.5 μ L, moisturizing to 25 μ L, PCR parameter: 94 DEG C, 3min; 94 DEG C, 30s, 50 DEG C, 30s, 72 DEG C, 30s, 30 circulations; 72 DEG C, 3min, results outer circulation amplified production; Internal recycle increases: template (outer circulation amplified production) 1 μ L, 10 ×
taqthe each 1 μ L of buffer2.5 μ L, dNTP1.5 μ L, internal recycle primer I P3 and IP4,
taqdNApolymerase0.5 μ L, moisturizing to 25 μ L, PCR parameter: 94 DEG C, 3min; 94 DEG C, 30s, 50 DEG C, 30s, 72 DEG C, 30s, 30 circulations; 72 DEG C, 3min, results internal recycle amplified production) (needed for nest-type PRC
taqbuffer, dNTP,
taqdNApolymerase etc. are all purchased from precious biotechnology (Dalian) company limited) qualification, as shown in Figure 3, can amplify the positive object band of expection 500bp size, Vero cell composition negative control does not amplify object band.After PCR primer order-checking (Sangon Biotech (Shanghai) Co., Ltd.), with DNAMAN(U.S. LynnonBiosoft company software) the F gene fragment recording RSVLong strain in sequence and NCBI is compared, as shown in Figure 4, consistence is 100%; Recording sequence BLAST(network address in NCBI: http://blast.ncbi.nlm.nih.gov/Blast.cgi) as shown in Figure 5, can find out that the strain of acquisition is A hypotype RSV virus strain.By the correct RSV strain of qualification Vero cell, 37 DEG C go down to posterity once, the virus of results is designated as KM516 – WT.
The adaptability attenuation culturing process of embodiment 3:RSV
By the RSV strain KM516 – WT of above-mentioned results cell (Vero cell), 37 DEG C of 5 generations of biography in embodiment 1, passed for 10 generations at 32 DEG C, passed for 15 generations at 28 DEG C, passed for 10 generations at 25 DEG C, finally obtain KM516 – AS strain; Again by Vero passage to 35cm
2in culturing bottle, when cell grows to 70 ~ 80% of individual layer, abandon supernatant substratum, add 0.5ml respiratory syncytial virus attenuation KM516 – AS strain and 0.5ml diluent (diluent: to not containing the NaHCO adding 1% in MEM or the DMEM nutrient solution of bovine serum
3), 25 DEG C of constant-temperature incubations, rock once every 30min, add 10ml maintenance medium in 25 DEG C of constant temperature culture after 2h.As shown in Figure 2, when cytopathy area is more than 80%, abandons supernatant, add 2mlPBS, multigelation makes cell come off completely, gets 0.5ml and again inoculates Vero cell, so repeat 10 times after results virus.
The attenuation characteristics of embodiment 4:RSV adapted strain KM516 – AS and virulence experiment
Select A549 cell (buying in U.S. ATCC, numbering CCL – 185) to measure the virus titer of RSV strain KM516 – AS and KM516 – WT in the present embodiment, particular content is as follows:
(A) A549 cell dissociation, counting, adjustment concentration to 5 × 10
4/ mL, 100 μ L/ holes are seeded to 96 orifice plates, when cell grows to 50% ~ 60% of individual layer, by RSV adapted strain KM516 – AS and street strain KM516 – WT respectively by 10
0, 10
-1, 10
-210
-9, 10
-10doubling dilution, 96 orifice plates are inoculated in 100 μ L/ holes, each extent of dilution establish 4 parallel, 37 DEG C of constant temperature culture, observation, add up the infection titer of RSV strain KM516 – AS and KM516 – WT when pathology no longer appears in cell.
(B) RSV adapted strain KM516 – AS is identical with measuring method during at 37 DEG C at the measuring method of 25 DEG C, 32 DEG C, 39 DEG C virus infection titer with street strain KM516 – WT, and the inoculation of RSV strain is complete is put in 25 DEG C, 32 DEG C, 39 DEG C mensuration virus infection titer respectively.
1) mensuration of KM516 – AS Cold tolerance
RSV adapted strain exceeds 100 times when virus titer is than 32 DEG C 25 DEG C time, namely thinks that the successful acclimatization to cold of RSV has been arrived on Vero cell; (A), (B) record the virus titer of RSV adapted strain KM516 – AS when 25 DEG C and 32 DEG C as stated above, evaluate RSV adapted strain whether have Cold tolerance according to criterion.KM516 – AS is less than 2 at the virus titer of 25 DEG C and the ratio of 32 DEG C as shown in Figure 6,7, illustrates that KM516 – AS does not have Cold tolerance.
2) mensuration of KM516 – AS temperature sensitivity
RSV adapted strain KM516 – AS exceeds 100 times at the virus titer of 32 DEG C than during at 39 DEG C, namely thinks that RSV strain has temperature sensitive properties; (A), (B) record the virus titer of RSV adapted strain when 32 DEG C and 39 DEG C as stated above, evaluate RSV adapted strain whether have temperature sensitivity according to criterion; As shown in Figure 6,7, KM516 – AS is greater than 2 at the virus titer of 32 DEG C and the ratio of 39 DEG C, illustrates that KM516 – AS has temperature sensitivity.
3) toxicity test in KM516 – AS body
Carry out experimentation on animals, adopt Kunming mouse (buying in Kunming Medical University's Experimental Animal Center), female, 6-8 week; KM516 – WT street strain and KM516 – AS adapted strain, each virus strain is set to one group, separately establishes cell controls group and blank group, totally 4 groups, often organizes 10 mouse.Immunity weighs Mouse Weight first three day every day, the 4th day collunarium inoculation strain, and attenuation group and open country poison group every mouse inoculation dosage 100 μ l, virus quantity is 10
5pFU.Continuous immunity three days, once a day, every day weighs in, and examines mouse drinking water diet, behavioral activity, the mental status, fur, ight soil etc. after immunity.8th day, often organize after mouse gets 5 etherizations and put to death, the left lung of mouse, for making pathological section, is often organized mouse and is got concha tissue survey upper respiratory tract residual virus titres, get right lung and survey lower respiratory tract virus titer.
As Fig. 8, body weight evolution relatively after mouse inoculation before ratio inoculation in the 8th day, inoculation KM516 – AS adapted strain can be seen, Mouse Weight increasing amount no significant difference (P>0.05) between cell controls group and blank group, but it is all high than KM516 – WT street strain, and adapted strain is compared with corresponding street strain, there were significant differences for body weight evolution (P<0.05), reason may be occur pathological change in Mice Body after inoculation street strain, cause appetite stimulator and weight loss, and the virulence of adapted strain is more weak, feed and the body weight that can not affect mouse after inoculation increase.Fig. 9, Figure 10, shown in table 1, few obviously than inoculation KM516 – WT street strain of lymphatic infiltration phenomenon in the mouse alveolar of inoculation KM516 – AS adapted strain and capillary vessel, illustrate that KM516 – AS has attenuating effects, shown in the upper and lower Respirovirus titre of Figure 11, in the upper respiratory tract that temperature is lower, adapted strain KM516 – AS is compared with corresponding street strain KM516 – WT, residual virus titres does not have significant difference (P>0.5), and in the lower respiratory tract that temperature is higher, there were significant differences (P<0.5), this illustrates that adapted strain has certain Virus reproductivity in vivo, but it is lower than street strain.Therefore, illustrate that strain KM516 – AS has attenuation.
Table 1: the dirty lesion degree of mouse lung
Note: "-" represents do not have pathology, and "+" indicates pathology, and "+" more multilist shows that lesion degree is more serious.
Embodiment 5: the preparation of attenuated vaccine
By the attenuation strain KM516 – AS suspension that obtains in embodiment 3 through ultrasonic grinding cell, 13000rpm4 DEG C of centrifugal 2h, collect supernatant liquor, then will containing virulent supernatant liquor through sucrose (buying in GIBCOInvitrogenCorporation company) gradient (concentration range: 2ml60% sucrose from bottom; 2ml45% sucrose; 2ml30% sucrose; 2ml15% sucrose), 40000rpm4 DEG C of centrifugal 6h, is respectively charged into centrifugal rear Tape samples the dialysis tubing that molecular retention amount is 50kd, puts in 300mlPBS, and 4 DEG C are stirred dialysis 4h, change a PBS, to remove sucrose, salt ion etc. completely every 1h.Gained sample is loaded to carry out dialysis in the dialysis tubing that molecular weight is 6000Da concentrated, room temperature places some hours, until sample volume is concentrated into minimum, adds 2mlPBS suspended sample, by packing viral after purifying, namely obtains respiratory syncytial virus attenuated vaccine.
The immunogenicity of embodiment 6:RSV attenuated strain KM516 – AS
Carry out experimentation on animals, adopt Kunming mouse, female, 6-8 week, KM516 – WT street strain and KM516 – AS attenuated strain, each virus strain is set to one group, separately establishes cell controls group and blank group, totally 4 groups, often organize 10 mouse, immunity weighs Mouse Weight first three day every day, the 4th day collunarium inoculation strain, attenuation group and open country poison group every mouse inoculation dosage 100 μ l, virus quantity is 10
5pFU.Continuous immunity three days, once a day, every day weighs in, and examines mouse drinking water diet, behavioral activity, the mental status, fur, ight soil etc. after immunity.8th day, often organize after mouse gets 5 etherizations and put to death, the left lung of mouse was for making pathological section.Get mouse concha and right lung homogenate respectively, by 1:3(1g concha or right lung weight: 3mlPBS) add PBS and make upper and lower respiratory tract homogenate respectively, measure remaining virus titer, often organize mouse and get concha tissue survey upper respiratory tract residual virus titres, get right lung and survey lower respiratory tract virus titer.13rd day, mouse tail venous blood sampling, surveyed the total antibody of serum and NAT.14th day, put to death after often organizing remaining mouse etherization, take out complete lungs, take lung weight, calculate the organ coefficient of mouse lungs, and survey the total antibody of lung homogenate liquid and NAT.The immunogenicity of KM516 – AS attenuated strain is evaluated with this.As Table 2,3, suitable with inoculation street strain of the serum of mouse inoculation KM516 – AS attenuated strain and total antibody titer of lung homogenate liquid and Neutralizing titer, illustrate that KM516 – AS attenuated strain has good immunogenicity, table 4 also illustrates, the immunogenicity of KM516 – AS attenuated strain is suitable with street strain.
Table 2: mice serum and the total antibody titer of lung homogenate liquid (geometrical mean)
Table 3: mice serum Neutralizing titer and lung homogenate liquid Neutralizing titer (geometrical mean)
The biological characteristics of table 4:RSV strain is summed up
Note: "-" represents do not have this biological characteristics, and "+" indicates this biological characteristics, and "+" more multilist shows that this biological characteristics is stronger.
The security of embodiment 7:RSV attenuated strain KM516 – AS and protectiveness
Carry out above-mentioned experimentation on animals, the lesion degree of cell controls group and the section of blank group mouse lungs is defined as " – "; inoculation street strain mouse lungs section lesion degree is defined as " +++ ++ "; how much weigh lesion degree with "+"; evaluate KM516 – AS strain attenuation degree, as Fig. 9,10, shown in table 1, compared with the mouse of inoculation street strain; the dirty lesion degree of mouse lung of inoculation KM516 – AS adapted strain is obviously low, proves that KM516 – AS has attenuating effects.Shown in Figure 11, mensuration mouse upper and lower Respirovirus titre can find out KM516 – AS strain, and levels of replication is lower than street strain in vivo, the security of further proof KM516 – AS attenuated strain, in addition, calculate the organ coefficient of mouse lungs, organ coefficient, also known as dirty body ratio, is the weight of laboratory animal internal organs and the ratio of its body weight.Time normal, the odds ratio of each internal organs and body weight is more constant.After animal contaminated, impaired organ weights can change, therefore organ coefficient also changes thereupon.Organ coefficient increases, and represents internal organs hyperemia, oedema or hypertrophy etc.; Organ coefficient reduces, and represents internal organs atrophy and other degenerative changes.The change of organ coefficient often can reflect the toxicity comprehensive condition of chemical toxicant to these internal organs preferably.As shown in figure 12, the mice organs coefficient of cell controls group, blank group and inoculation KM516 – AS attenuated strain is all 0.6 ~ 0.8%, and the mice organs coefficient of inoculation street strain is greater than 1%.Without significant difference (P>0.5) between cell controls group, blank group and the organ coefficient inoculating KM516 – AS attenuated strain mouse, and and between corresponding street strain, have significant difference (P<0.05).Prove that KM516 – AS has attenuating effects, there is security.
Carry out mouse challenge viral dosage, be divided into experimental group and blank group, often organize 10 mouse, experimental group first uses the immunity of KM516 – AS strain after one week, then attacks poison with KM516 – WT collunarium; Blank group directly attacks poison with KM516 – WT without immunity; within after attacking poison the 4th day, often organize anesthesia execution 5 mouse; get mouse right lung and upper and lower Respirovirus titre is surveyed in concha homogenate; as shown in table 5; the upper and lower Respirovirus titre of non-immune mouse is apparently higher than immune group; illustrate that KM516 – AS attenuated strain has protectiveness; simultaneously shown in table 2, table 3; inoculate the mice serum of KM516 – AS attenuated strain and total antibody titer and the Neutralizing titer of lung homogenate liquid and inoculate the suitable of street strain, also showing that KM516 – AS attenuated strain has protectiveness.Table 4 illustrates, KM516 – AS attenuated strain only has temperature sensitivity, and Cold tolerance is not obvious, but has certain attenuating effects, has immunogenicity, security and protectiveness.
Table 5 mouse is upper lower respiratory tract residual virus titres (geometrical mean) after attacking poison
The genetic stability test of embodiment 8:RSV attenuated strain KM516 – AS
By attenuated strain KM516 – AS in embodiment 1 cell (Vero cell), 25 DEG C cultivate and reached for the 50th generation, malicious mouse is attacked respectively with the KM516 – AS attenuated strain in the 10th generation, the 30th generation and the 50th generation and KM516 – WT street strain, separately establish cell controls group and blank group, totally 6 groups, often organize 10 mouse, attenuation group and open country poison group every mouse inoculation dosage purified virus liquid 100 μ l, virus quantity is 10
5pFU.Attack poison four days after, often organize mouse get 5 etherizations after put to death, the left lung of mouse for making pathological section, according to the method evaluation lesion degree of embodiment 4.As shown in table 6, the 30th generation and the KM516 – AS attenuated strain in the 50th generation are attacked poison and cause the lesion degree of mouse lungs identical with the KM516 – AS attenuated strain in the 10th generation, illustrate that KM516 – AS attenuated strain has the stable hereditary property of attenuation virulence.
The different generation KM516 – ASs of table 6 25 DEG C time attack the dirty lesion degree of malicious mouse lung
Note: "-" represents do not have pathology, and "+" indicates pathology, and "+" more multilist shows that lesion degree is more serious.
The embodiment of the present invention proves that KM516 – AS strain has the characteristic of efficient stable propagation on the cells such as Vero; Experimentation on animals has proved that KM516 – AS strain has good attenuation, security, immunogenicity and protectiveness.
Sequence table
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Claims (3)
1. a strain respiratory syncytial virus attenuation strain KM516 – AS, its deposit number in China typical culture collection center is CCTCCNO:V201344.
2. the application of respiratory syncytial virus attenuation strain described in claim 1 in preparation respiratory syncytial virus attenuated vaccine, is characterized in that carrying out as follows:
Nutrient solution static gas wave refrigerator cell is used, until cell grows up to individual layer at (1) 37 DEG C;
(2) be 0.01 ~ 0.5 be inoculated in above-mentioned cell culture fluid by respiratory syncytial virus attenuation strain KM516 – AS with infection multiplicity, cultivate until cytopathy reaches 75% ~ 90% at 25 DEG C;
(3) discard the nutrient solution of sick cell, add the phosphate buffered saline buffer that pH value is 7.2, after freeze thawing, sonicated cells, obtains rough attenuated virus liquid;
(4) rough attenuated virus liquid adopts conventional sucrose density gradient centrifugation to obtain respiratory syncytial virus attenuated vaccine;
Wherein said cell is the one in Vero cell, diploid cell MRC – 5 cell, KMB – 17 cell, 2BS cell, WI – 38 cell;
Described nutrient solution is be added with the MEM nutritive medium of 2 – 10% new-born calf serum or be added with the DMEM nutritive medium of 2 – 10% new-born calf serum, and pH value is 6.8 – 7.2.
3. respiratory syncytial virus attenuated vaccine obtained in application described in claim 2.
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| WO2004083397A2 (en) * | 2003-03-18 | 2004-09-30 | Wyeth Holdings Corporation | Process for increasing rsv surface glycoprotein yields using a mutant strain of rsv |
| CN101012454A (en) * | 1996-07-15 | 2007-08-08 | 美国政府健康及人类服务部 | Production of attenuated chimeric respiratory syncytial virus vaccines from cloned nucleotide sequences |
| CN102078606A (en) * | 2010-12-08 | 2011-06-01 | 中国人民解放军军事医学科学院微生物流行病研究所 | Respiratory syncytial virosome vaccine and preparation method thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012454A (en) * | 1996-07-15 | 2007-08-08 | 美国政府健康及人类服务部 | Production of attenuated chimeric respiratory syncytial virus vaccines from cloned nucleotide sequences |
| WO2004083397A2 (en) * | 2003-03-18 | 2004-09-30 | Wyeth Holdings Corporation | Process for increasing rsv surface glycoprotein yields using a mutant strain of rsv |
| CN102078606A (en) * | 2010-12-08 | 2011-06-01 | 中国人民解放军军事医学科学院微生物流行病研究所 | Respiratory syncytial virosome vaccine and preparation method thereof |
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| HRSV A2株细胞低温传代适应减毒指标的初步探索;谢忠平等;《现代预防医学》;20091231;第36卷(第20期);第3924-3927页 * |
| The Temperature-Sensitive (ts) Phenotype of a Cold-Passaged (cp) Live Attenuated Respiratory Syncytial Virus Vaccine Candidate, Designated cpts530, Results from a Single Amino Acid Substitution in the L Protein;KATALIN JUHASZ et al.;《JOURNAL OF VIROLOGY》;19970831;第71卷(第8期);第5814-5819页 * |
| 人呼吸道合胞病毒减毒活疫苗培养的初步探索;李志强等;《实用预防医学》;20080630;第15卷(第3期);第698-702页 * |
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