猫细小病毒分离鉴定及马抗猫瘟特异性IgG制备、检测和治疗效果研究
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摘要
猫泛白细胞减少症是一种由猫细小病毒(FPV)引起猫急性、高度接触性传染病,其致病性强,感染范围广泛,危害大。幼猫发病率很高,致死率可达86%以上。FPV对虎、豹、狮子等大型猫科动物以及体型较小的山猫、豹猫、野猫和鼬科动物中的水貂、雪貂等均为易感,目前,它是肉食兽细小病毒中感染范围最宽、致病性最强的一种病毒。针对目前猫泛白细胞减少症尚无有效可靠的特效药物治疗的实际,积极开展抗FPV抗体的研究,生产既具有治疗性又具有保护性且生产成本低廉的高效价抗体无论是在理论上,还是在生产实践上都具有非常重要的意义。
2008年3月,我们剖解了长春中日联谊动物医院疑似患猫细小病毒死亡猫的尸体,通过细胞敏感试验、理化试验、形态学观察、血凝及血凝抑制试验、动物感染、特征序列PCR扩增及测序分析,获得一株猫细小病毒强毒株。采猫肾、肠、肝制成悬浮,用韩国金标试纸检测呈强阳性,用F81细胞培养病毒,到第4代出现典型的CPE,用电子显微镜观察病毒的形态和结构,病毒粒子直径为20～24nm;通过理化性质检测,该病毒无囊膜,对热、乙醚、氯仿、胰蛋白酶有抗性,特征符合DNA病毒特征。该病毒在4℃对猪红细胞有凝集作用,HI试验显示其血凝效价为256倍,用FPV阳性血清作HA试验,表明其抑制效价在32倍以上。VP2结构蛋白是综合病毒产生抗体的主要部位,其氨基酸的改变诱发宿主范围的变化。用细小病毒通用引物进行PCR检测,结果表明该病毒是一株细小病毒。用2对特异引物进行PCR检测,结果与预期设计大小一致;扩增VP2全基因序列,得出一条约1755bp的条带,通过测序,与国内外或其他地区的11株猫细小病毒强毒株进行比较,同源性高达99%,仅有5～10个碱基发生变异,而决定其宿主范围、血凝性和致病性的几个关键碱基和氨基酸未发生改变。该病毒对实验猫较为易感,人工感染5天后,采取猫粪便,进行PCR试验均检测出阳性抗原,感染率达100%,对幼猫的致死率高达66.7%,即便是同居感染,也可致幼猫死亡;因此,本实验分离到的毒株为一株猫细小病毒强毒株。
本实验对病毒进行了4倍浓缩,用活病毒作免疫原,分别用白油-吐温和黄芪多糖作为佐剂,对两匹马进行基础免疫和高度免疫,用ELSIA方法测定其第1、7、14、40、60和90天的血液抗体,结果显示:马在免疫后第60天左右抗体滴度最高,随后逐渐下降,趋于稳定。两种免疫佐剂效价差异不明显。在免疫初期,马对白油-吐温的免疫反应较大,而对黄芪多糖无异常反应。马对猫细小病毒有较好的免疫原性,能有效抵制猫细小病毒活毒株而又无任何临床症状和不适反映,安全可靠。我们采取4℃自然沉降法分离血浆,对传统的IgG制备方法:硫酸铵沉淀法、正辛酸-碳酸铵沉淀法和低温乙醇法进行了比较,对IgG的酶切温度和酶量进行了探索。通过比较,表明37℃胃蛋白酶消化1小时或30℃消化2小时酶切效果较好,IgG片断纯度高。同时,对正辛酸-硫酸铵盐析沉淀法制备IgG进行了条件优化,设计了新的提纯IgG方法,通过对血清37℃,正辛酸去白蛋白、两次硫酸铵沉淀,利用蛋白A与免疫球蛋白的Fc区和大多数哺乳动物的IgG结合的特性,用Protein A柱过滤,收集和纯化F (ab)'2片断。通过分光光度计检测,每1 mL血清可获得12.59 mg IgG,其产品得率与硫酸铵沉淀法相当,比传统正辛酸-硫酸铵沉淀法提高了30%。通过SDS-PAGE电泳和薄层凝胶扫描,表明,用优化的正辛酸-硫酸铵盐析沉淀法制备的IgG,电泳条带清晰,杂蛋白少,获得的IgG片断纯度高。
据《中华人民共各国药典》(2005年版)的相关内容,本研究对抗FPV IgG生物制剂的物理性状、热原性、安全性、无菌性、保存期进行了检验,结果表明:该生物制剂呈乳白色,溶液稳定无分层、无可见颗粒,无需氧菌、厌氧菌、真菌和支原体等微生物污染,常温保存90天物理性状不发生显著变化,血凝抑制效价不降低;家兔、小白鼠、、豚鼠等试验动物注射该生物制品观察连续观察10天,其体温无显著升高、体重无减轻、饮食、排泄、精神均良好,无任何临床症状和异常变化,表明抗FPV IgG安全稳定、无毒副作用;对3只家兔进行热原性检测,注射生物制品后,体温升高均低于0.6℃,3只兔体温升高总和1.2℃,低于1.4℃。表明该制品所含热原的限度符合规定。用血凝抑制试验、双向琼脂扩散试验、细胞中和试验和动物治疗试验对该制品的效价进行了检验,结果表明该生物制剂具有较高的特异性,效价为128倍以上,对6只猫进行人工感染FPV,均按期发病,对其中5猫进行治疗,每次注射该制品2 mL,隔天治疗,连续3针,7天后病猫临床症状消失,治愈率达100%,表明该制品对猫泛白细胞减少症有很好的治疗效果。
本试验对猫细小病毒进行了分离鉴定,证明所分离的毒株为FPV强毒株;对传统抗体制备工艺进行改良后,对马抗FPV IgG进行了制备。每1 mL血清可获得12.59 mg IgG,通过对该产品的系列检查,表明该生物制剂具有较高的特异性,效价为128倍以上,对猫泛白细胞减少症安全有效、无毒副作用,符合生物制剂规定。
Feline panleukopenia is an acute, highly contagious infectious disease caused by FPV, with high pathogenicity and wide-ranged infection. The incidence of the diease is especially high in kettens with a mortality of 85% or more. Both of large felids such as tigers, leopards, lions and small felids such as smaller lynx, leopard cats, wild cats, and minks or ferrets are susceptible animals to FPV, the virus that is most widely distributed and with the strongest pathogenicity amongst carnivore parvoviruses. To date, there is still no specific remedy for this disease. It is of great significance to develop highly effective anti-FPV antibodies.
A strong strain of feline parvovirus was obtained from the isolations of a cat which was suspected to be died of FPV infetion in March, 2008, by cell sensitivity test, physical and chemical tests, morphological examination, HA and HT tests, animal infections, and sequencing of PCR produtcs for characteristic sequence.Suspensions were prepared from the tissues of kidney, intestine, and liver respectivesly and tested by FPV Golden sign indicator paper (made in Korea), which gave rise to a positive result. The F81 cells were used for propagation of FPV, and typical pathological changes appeared at the fourth generation. Electronic microscopy showed that thediameter of the virion was about 20-24nm. Physical and chemical features was coincide with characteristics of DNA viruses, such as free of envelope, high resistance to heat, chloroform ,ether and trypsin. The virus made blood clot easily to porcine blood cells under 4℃. The HI test showed a hemagglutinin titer of 256 folds, and HA test with FPV positive serum showed a inhibitory titer ofmore than 32 folds. As the primary structural protein, VP2 is the main protection antigen inducing the organism to produces the neutralizing antibody and determining the specificity of host. The result of PCR with universal primer was positive, indicating the virus was a kind of parvovirus. Two pairs of specific primers were used for further PCR testing, results showed consistency with the expected size of the design. The complete sequence of VP2 amplified by PCR was about 1755bp. Compared with the sequence of the 11 FPV strainws published previously , their homology was up to 99%. Only 5 to 10 base pairs were mutated but there is no change in the key bases and amino acids determining its host range,pathogenicity and hemagglutinin.The virus was highly susceptible to experimental cats,because infection rate was 100% of the kittens tested by PCR for feces samples 5 days after artificial infection, and mortility was 66.7%. Conclusively, the strain we isolated is a strong strain of parvovirus.
Basic and high degree immunizations against the live FPV were conducted in two horses with the 4-time-concentrated FPV culture medium as antigen, white oil - Twain and astragalus polysaccharide as adjuvants. Antibody titers were detected by ElISA on d 1,7,14,40, 60 and 90 and the results showed that the highest titer appeared on d 60 and decreased gradually thereafter and finally trended to stable.There was no singificant difference in antibody titers between two adjuvants. During the early stage of immunization, greater immune response was found in white oil–Twain treated horse while no response in astragalus polysaccharide treated horse. Experiments showed that horses have good immunogenicity for FPV, effectively resisting the strain of feline parvovirus withoutany clinical symptoms and uncomfortable signs.It suggests that immunization against FPV is safty and reliable.
Plasma was seperated by natural sedimentation at 4℃and cold ethanol method were compared with the traditional IgG preparation methods like the ammonium sulphate precipitation and bitter - ammonium carbonate precipitation. The optimal restriction tempreture and enzyme dosage was exploreed as well.The result showed that it was best to use pepsin at 37℃for 1h,or 30℃for 2h. We optimized a new method of purification of IgG, by digesting the serum at37℃with pepsase, removing albumin in caprylic acid, and the twice ammonium sulfate precipitation. According to the character that Protein A and Fc of immunoglobulin can connect with IgG of most mammals, the F (ab)'2 was collected and purified by Protein A column filter.The spectrophotometry showed that 12.59mg IgG was obtained from 1mL serum by the new method which increased by 30% than the traditional methods of caprylic acid and ammonium sulfate precipitation. SDS-PAGE gel electrophoresis and thin-layer scanning demonstrated that IgG prepared by optimized bitter - ammonium sulfate salting-out precipitation method showed clear electrophoretic bands and high purity.
According to the relavant items of "People's Republic of China States Pharmacopoeia" (2005 version), The physical properties, heat antigenicity, safety, aseptic, and shelf life of this biological product of horse anti- FPV IgG were examined.The results showed that: the product is a milk white solution that is non-layered, free of visible particles, bacteria, fungi, mycoplasma and other microbial contaminations. There is no changes in physical and chemical properties and no reducction of hemagglutination inhibition titer after store for 90 days at room temperature. Rabbits, mice, and guinea pigs injected with the product was observed for 10 successvie days and showed no significant increase in body temperature, no reduction in body weight, and good status of appetite, excrement, and spirit, suggesting the safty and stability
Thermogenic test was conducted by injected with the product in 3 rabbits. The results showed that the average increase in temperature of rabbits were lower than 0.6℃, the total increase was d 1.2℃, less than the standard limitation of 1.4℃. The potency was evaluated by HA and HI test, agarose double diffusion, cells neutralization test and animal treatment test. The results showed that the product is highly specific with the titer of 128-fold. Six artificial FPV- infected cats onsetted the disease on scheduled time. Five of the six cats were treated by 3 injections of 2ml of the product on every other day. The clinical symptoms of all the 5 cats disappeared 7 days later with the recovery rate of 100%, indicating the high effectiveness of the product in treatment of feline panleukopenia.
Conclusively, a strong FPV strain was isolated and identified and a safty, highly specific product of horse anti-FPV antibody was produced by improved techniques.
2008年3月,我们剖解了长春中日联谊动物医院疑似患猫细小病毒死亡猫的尸体,通过细胞敏感试验、理化试验、形态学观察、血凝及血凝抑制试验、动物感染、特征序列PCR扩增及测序分析,获得一株猫细小病毒强毒株。采猫肾、肠、肝制成悬浮,用韩国金标试纸检测呈强阳性,用F81细胞培养病毒,到第4代出现典型的CPE,用电子显微镜观察病毒的形态和结构,病毒粒子直径为20～24nm;通过理化性质检测,该病毒无囊膜,对热、乙醚、氯仿、胰蛋白酶有抗性,特征符合DNA病毒特征。该病毒在4℃对猪红细胞有凝集作用,HI试验显示其血凝效价为256倍,用FPV阳性血清作HA试验,表明其抑制效价在32倍以上。VP2结构蛋白是综合病毒产生抗体的主要部位,其氨基酸的改变诱发宿主范围的变化。用细小病毒通用引物进行PCR检测,结果表明该病毒是一株细小病毒。用2对特异引物进行PCR检测,结果与预期设计大小一致;扩增VP2全基因序列,得出一条约1755bp的条带,通过测序,与国内外或其他地区的11株猫细小病毒强毒株进行比较,同源性高达99%,仅有5～10个碱基发生变异,而决定其宿主范围、血凝性和致病性的几个关键碱基和氨基酸未发生改变。该病毒对实验猫较为易感,人工感染5天后,采取猫粪便,进行PCR试验均检测出阳性抗原,感染率达100%,对幼猫的致死率高达66.7%,即便是同居感染,也可致幼猫死亡;因此,本实验分离到的毒株为一株猫细小病毒强毒株。
本实验对病毒进行了4倍浓缩,用活病毒作免疫原,分别用白油-吐温和黄芪多糖作为佐剂,对两匹马进行基础免疫和高度免疫,用ELSIA方法测定其第1、7、14、40、60和90天的血液抗体,结果显示:马在免疫后第60天左右抗体滴度最高,随后逐渐下降,趋于稳定。两种免疫佐剂效价差异不明显。在免疫初期,马对白油-吐温的免疫反应较大,而对黄芪多糖无异常反应。马对猫细小病毒有较好的免疫原性,能有效抵制猫细小病毒活毒株而又无任何临床症状和不适反映,安全可靠。我们采取4℃自然沉降法分离血浆,对传统的IgG制备方法:硫酸铵沉淀法、正辛酸-碳酸铵沉淀法和低温乙醇法进行了比较,对IgG的酶切温度和酶量进行了探索。通过比较,表明37℃胃蛋白酶消化1小时或30℃消化2小时酶切效果较好,IgG片断纯度高。同时,对正辛酸-硫酸铵盐析沉淀法制备IgG进行了条件优化,设计了新的提纯IgG方法,通过对血清37℃,正辛酸去白蛋白、两次硫酸铵沉淀,利用蛋白A与免疫球蛋白的Fc区和大多数哺乳动物的IgG结合的特性,用Protein A柱过滤,收集和纯化F (ab)'2片断。通过分光光度计检测,每1 mL血清可获得12.59 mg IgG,其产品得率与硫酸铵沉淀法相当,比传统正辛酸-硫酸铵沉淀法提高了30%。通过SDS-PAGE电泳和薄层凝胶扫描,表明,用优化的正辛酸-硫酸铵盐析沉淀法制备的IgG,电泳条带清晰,杂蛋白少,获得的IgG片断纯度高。
据《中华人民共各国药典》(2005年版)的相关内容,本研究对抗FPV IgG生物制剂的物理性状、热原性、安全性、无菌性、保存期进行了检验,结果表明:该生物制剂呈乳白色,溶液稳定无分层、无可见颗粒,无需氧菌、厌氧菌、真菌和支原体等微生物污染,常温保存90天物理性状不发生显著变化,血凝抑制效价不降低;家兔、小白鼠、、豚鼠等试验动物注射该生物制品观察连续观察10天,其体温无显著升高、体重无减轻、饮食、排泄、精神均良好,无任何临床症状和异常变化,表明抗FPV IgG安全稳定、无毒副作用;对3只家兔进行热原性检测,注射生物制品后,体温升高均低于0.6℃,3只兔体温升高总和1.2℃,低于1.4℃。表明该制品所含热原的限度符合规定。用血凝抑制试验、双向琼脂扩散试验、细胞中和试验和动物治疗试验对该制品的效价进行了检验,结果表明该生物制剂具有较高的特异性,效价为128倍以上,对6只猫进行人工感染FPV,均按期发病,对其中5猫进行治疗,每次注射该制品2 mL,隔天治疗,连续3针,7天后病猫临床症状消失,治愈率达100%,表明该制品对猫泛白细胞减少症有很好的治疗效果。
本试验对猫细小病毒进行了分离鉴定,证明所分离的毒株为FPV强毒株;对传统抗体制备工艺进行改良后,对马抗FPV IgG进行了制备。每1 mL血清可获得12.59 mg IgG,通过对该产品的系列检查,表明该生物制剂具有较高的特异性,效价为128倍以上,对猫泛白细胞减少症安全有效、无毒副作用,符合生物制剂规定。
Feline panleukopenia is an acute, highly contagious infectious disease caused by FPV, with high pathogenicity and wide-ranged infection. The incidence of the diease is especially high in kettens with a mortality of 85% or more. Both of large felids such as tigers, leopards, lions and small felids such as smaller lynx, leopard cats, wild cats, and minks or ferrets are susceptible animals to FPV, the virus that is most widely distributed and with the strongest pathogenicity amongst carnivore parvoviruses. To date, there is still no specific remedy for this disease. It is of great significance to develop highly effective anti-FPV antibodies.
A strong strain of feline parvovirus was obtained from the isolations of a cat which was suspected to be died of FPV infetion in March, 2008, by cell sensitivity test, physical and chemical tests, morphological examination, HA and HT tests, animal infections, and sequencing of PCR produtcs for characteristic sequence.Suspensions were prepared from the tissues of kidney, intestine, and liver respectivesly and tested by FPV Golden sign indicator paper (made in Korea), which gave rise to a positive result. The F81 cells were used for propagation of FPV, and typical pathological changes appeared at the fourth generation. Electronic microscopy showed that thediameter of the virion was about 20-24nm. Physical and chemical features was coincide with characteristics of DNA viruses, such as free of envelope, high resistance to heat, chloroform ,ether and trypsin. The virus made blood clot easily to porcine blood cells under 4℃. The HI test showed a hemagglutinin titer of 256 folds, and HA test with FPV positive serum showed a inhibitory titer ofmore than 32 folds. As the primary structural protein, VP2 is the main protection antigen inducing the organism to produces the neutralizing antibody and determining the specificity of host. The result of PCR with universal primer was positive, indicating the virus was a kind of parvovirus. Two pairs of specific primers were used for further PCR testing, results showed consistency with the expected size of the design. The complete sequence of VP2 amplified by PCR was about 1755bp. Compared with the sequence of the 11 FPV strainws published previously , their homology was up to 99%. Only 5 to 10 base pairs were mutated but there is no change in the key bases and amino acids determining its host range,pathogenicity and hemagglutinin.The virus was highly susceptible to experimental cats,because infection rate was 100% of the kittens tested by PCR for feces samples 5 days after artificial infection, and mortility was 66.7%. Conclusively, the strain we isolated is a strong strain of parvovirus.
Basic and high degree immunizations against the live FPV were conducted in two horses with the 4-time-concentrated FPV culture medium as antigen, white oil - Twain and astragalus polysaccharide as adjuvants. Antibody titers were detected by ElISA on d 1,7,14,40, 60 and 90 and the results showed that the highest titer appeared on d 60 and decreased gradually thereafter and finally trended to stable.There was no singificant difference in antibody titers between two adjuvants. During the early stage of immunization, greater immune response was found in white oil–Twain treated horse while no response in astragalus polysaccharide treated horse. Experiments showed that horses have good immunogenicity for FPV, effectively resisting the strain of feline parvovirus withoutany clinical symptoms and uncomfortable signs.It suggests that immunization against FPV is safty and reliable.
Plasma was seperated by natural sedimentation at 4℃and cold ethanol method were compared with the traditional IgG preparation methods like the ammonium sulphate precipitation and bitter - ammonium carbonate precipitation. The optimal restriction tempreture and enzyme dosage was exploreed as well.The result showed that it was best to use pepsin at 37℃for 1h,or 30℃for 2h. We optimized a new method of purification of IgG, by digesting the serum at37℃with pepsase, removing albumin in caprylic acid, and the twice ammonium sulfate precipitation. According to the character that Protein A and Fc of immunoglobulin can connect with IgG of most mammals, the F (ab)'2 was collected and purified by Protein A column filter.The spectrophotometry showed that 12.59mg IgG was obtained from 1mL serum by the new method which increased by 30% than the traditional methods of caprylic acid and ammonium sulfate precipitation. SDS-PAGE gel electrophoresis and thin-layer scanning demonstrated that IgG prepared by optimized bitter - ammonium sulfate salting-out precipitation method showed clear electrophoretic bands and high purity.
According to the relavant items of "People's Republic of China States Pharmacopoeia" (2005 version), The physical properties, heat antigenicity, safety, aseptic, and shelf life of this biological product of horse anti- FPV IgG were examined.The results showed that: the product is a milk white solution that is non-layered, free of visible particles, bacteria, fungi, mycoplasma and other microbial contaminations. There is no changes in physical and chemical properties and no reducction of hemagglutination inhibition titer after store for 90 days at room temperature. Rabbits, mice, and guinea pigs injected with the product was observed for 10 successvie days and showed no significant increase in body temperature, no reduction in body weight, and good status of appetite, excrement, and spirit, suggesting the safty and stability
Thermogenic test was conducted by injected with the product in 3 rabbits. The results showed that the average increase in temperature of rabbits were lower than 0.6℃, the total increase was d 1.2℃, less than the standard limitation of 1.4℃. The potency was evaluated by HA and HI test, agarose double diffusion, cells neutralization test and animal treatment test. The results showed that the product is highly specific with the titer of 128-fold. Six artificial FPV- infected cats onsetted the disease on scheduled time. Five of the six cats were treated by 3 injections of 2ml of the product on every other day. The clinical symptoms of all the 5 cats disappeared 7 days later with the recovery rate of 100%, indicating the high effectiveness of the product in treatment of feline panleukopenia.
Conclusively, a strong FPV strain was isolated and identified and a safty, highly specific product of horse anti-FPV antibody was produced by improved techniques.
引文
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