鹅细小病毒的分离鉴定及其致病性和免疫性研究
发布时间：2018-08-04 10:15
【摘要】：鹅细小病毒病又称小鹅瘟,是由鹅细小病毒(Goose parvovirus,GPV)引起的雏鹅和雏番鸭的一种高度接触性传染病,使雏鹅及雏番鸭发生急性或亚急性败血症,具有传播速度快、死亡率高等特点。该病主要病理变化为渗出性肠炎,以空肠和回肠的急性卡他性-纤维素性坏死性肠炎为特征。发病后耐过的雏鹅及雏番鸭生长发育迟缓,形成“僵鸭”,给养鹅业造成了巨大的经济损失。自1961年方定一发现小鹅瘟病以来,国内外陆续也有本病的报道。由于该病对养鹅业危害严重,国内外很多研究学者对其开展了深入研究。小鹅瘟在吉林省局部地区也呈流行趋势,近几年吉林省的九台、德惠、辽源、白城、磐石、镇赉等多个地区发生小鹅瘟疫情,为了确诊该病及对分离到的病毒进行免疫分析,本研究从患病雏鹅病料中分离并鉴定了6株GPV,分别命名为JLJT、DH、LY、BCH、PSH、ZL株,并对各毒株VP3基因进行了克隆、测序分析,开展了各毒株致病性、免疫原性及交叉保护性的研究,为小鹅瘟疫苗的研制奠定基础。将来自不同地区发病死亡雏鹅的肝脏和栓塞段小肠及其内容物研磨制成匀浆,离心去除沉淀,双抗孵育除去细菌,接种12日龄鹅胚,收集尿囊液;通过PCR鉴定,以及将VP3基因片段纯化后克隆到p MD18-T载体中,进行重组质粒测序。由测序结果再次证明所分离的病毒均为GPV。同时,测定了包括本研究室已经分离鉴定的2株GPV在内的8株GPV VP3基因序列,并进行了基因及氨基酸序列分析。诱导表达了实验室保存的四平株(SP)VP3基因重组质粒pGEX-4T-VP3,经树脂纯化后得到VP3蛋白;提纯鹅卵黄IgY并免疫实验兔,获得抗血清;采用树脂亲和层析方法纯化兔IgG;通过改良的高碘酸钠法进行辣根过氧化物酶标记,获得兔抗鹅酶标抗体;通过各条件的优化建立能够检测鹅血清GPV抗体的间接ELISA方法。结果表明,GPV间接ELISA方法VP3蛋白最佳包被浓度为4μg/mL、血清稀释倍数为100倍、酶标抗体稀释倍数为1100倍。本研究通过攻毒实验,观察雏鹅发病及死亡情况;将死亡雏鹅肝脏及小肠制成石蜡切片,综合雏鹅发病死亡情况及病理切片观察结果,证明镇赉株GPV致病性较强。采取死亡雏鹅心血,获得血清,以本研究建立的间接ELISA方法测定并比较各血清中抗GPV抗体水平,结果发现镇赉株接种雏鹅血清抗体OD450nm值最高,为0.684。以镇赉株GPV与弗氏不完全佐剂制成油佐剂灭活苗免疫雏鹅,13d后以8株GPV进行攻毒实验。结果发现,攻毒后15d阴性对照组5只鹅全部死亡,死亡前表现明显的精神沉郁、食欲废绝、腹泻、排白色条状带泡沫的稀粪;剖检发现肝脏充血、出血,脾脏肿大、充血,肾脏肿大、充血出血,脑膜充血、出血;卵黄蒂附近小肠肿大,有长短不等的栓塞,剪开后栓塞呈黄白色、触摸坚实、呈明显的腊肠样栓塞。LY,CH,ZL,BCH,JLJT,DH株GPV攻毒组雏鹅未出现死亡,保护率达100%(5/5);PSH组有1只死亡,保护率为80%(4/5)。由此说明,镇赉株GPV致病性、免疫原性均较强且能够保护其它GPV毒株的感染。
[Abstract]:Goose parvovirus (goose parvovirus), also known as goose plague, is a highly contagious disease caused by goose parvovirus (Goose parvovirus, GPV) and young goose and Muscovy ducks. It has acute or subacute septicaemia of young goose and Muscovy ducks. It is characterized by rapid transmission and high mortality. The main pathological changes of this disease are exudative enteritis, jejunum and ileum. Acute catarrhal cellulosic necrotizing enteritis is characterized. The growth and development of geese and Muscovy ducks after the onset of disease, forming a "stiff duck", caused huge economic losses in the feeding goose industry. Since the discovery of goose plague in 1961, there have been reports of this disease at home and abroad. Because the disease has serious harm to the goose industry, it is serious at home and abroad. The goose plague in some areas of Jilin province is also prevalent. In recent years, nine stations in Jilin Province, Dehui, Liaoyuan, Baicheng, Panshi, Zhenlai and other regions have the situation of the goose plague. In order to diagnose the disease and the immunoassay of the isolated disease, this study is divided into the disease of the sick gosling. 6 strains of GPV, named JLJT, DH, LY, BCH, PSH, ZL, were isolated and identified, and the VP3 genes of each strain were cloned and sequenced, and the pathogenicity, immunogenicity and cross protection of the strains were studied to lay the foundation for the development of the goose plague vaccine. 12 day old goose embryos were inoculated with 12 days old goose embryos and collected urinic fluid. The recombinant plasmid was sequenced and the recombinant plasmid was sequenced by cloning the VP3 gene fragment into P MD18-T vector and sequencing. The GPV VP3 gene sequence of 2 strains of GPV, which was isolated and identified, was analyzed, and the gene and amino acid sequence was analyzed. The recombinant plasmid pGEX-4T-VP3 of the VP3 gene of the laboratory preserved Siping (SP) VP3 gene was induced and the VP3 protein was purified by the resin; the goose egg yolk IgY was purified and the rabbit was immunized to obtain the antiserum; the resin affinity chromatography was used. The rabbit IgG was purified by the modified sodium iodate method. The Rabbit anti goose peroxidase antibody was obtained. The indirect ELISA method for detecting GPV antibody in Goose Serum was established by optimizing the conditions. The results showed that the optimal concentration of VP3 protein in the indirect ELISA method of GPV was 4 u g/mL, the dilution multiple of the serum was 100 times, the enzyme labeled antibody was used. The dilution ratio was 1100 times. In this study, the incidence and death of gosling were observed by the attack. The liver and small intestine of the goose were made to make paraffin section, and the morbidity and mortality of the goose and the pathological sections were observed. It was proved that the pathogenicity of the Zhenlai strain GPV was stronger. The serum was obtained by the heart blood of the goose, and the indirect ELISA side established in this study was established. The anti GPV antibody level in each serum was measured and compared. The results showed that the serum antibody OD450nm of the Zhenlai goose was the highest, which was 0.684. with the Zhenlai strain GPV and the incomplete Freund adjuvant inactivated vaccine to inactivate the goose. After 13D, 8 strains of GPV were used to attack the virus. The results showed that all the 5 goose in the 15d negative control group died after the attack and all died and died. The former showed obvious depression, loss of appetite, diarrhoea, and white striped lath with dilute feces. The liver hyperemia, bleeding, splenomegaly, hyperemia, kidney enlargement, congestion and bleeding, meningeal congestion, bleeding, small intestinal swelling near yolhuang pedicle, embolization of different length and short length, yellow white, solid touch and obvious Dachshund samples were found. .LY, CH, ZL, BCH, JLJT, DH strain GPV in the GPV attack group did not appear to be dead, the protection rate was 100% (5/5); the PSH group had 1 deaths and the protection rate was 80% (4/5). Thus, it showed that the Zhenlai strain was pathogenic, the immunogenicity was both strong and could protect the infection of other GPV strains.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.33
本文编号：2163591
[Abstract]:Goose parvovirus (goose parvovirus), also known as goose plague, is a highly contagious disease caused by goose parvovirus (Goose parvovirus, GPV) and young goose and Muscovy ducks. It has acute or subacute septicaemia of young goose and Muscovy ducks. It is characterized by rapid transmission and high mortality. The main pathological changes of this disease are exudative enteritis, jejunum and ileum. Acute catarrhal cellulosic necrotizing enteritis is characterized. The growth and development of geese and Muscovy ducks after the onset of disease, forming a "stiff duck", caused huge economic losses in the feeding goose industry. Since the discovery of goose plague in 1961, there have been reports of this disease at home and abroad. Because the disease has serious harm to the goose industry, it is serious at home and abroad. The goose plague in some areas of Jilin province is also prevalent. In recent years, nine stations in Jilin Province, Dehui, Liaoyuan, Baicheng, Panshi, Zhenlai and other regions have the situation of the goose plague. In order to diagnose the disease and the immunoassay of the isolated disease, this study is divided into the disease of the sick gosling. 6 strains of GPV, named JLJT, DH, LY, BCH, PSH, ZL, were isolated and identified, and the VP3 genes of each strain were cloned and sequenced, and the pathogenicity, immunogenicity and cross protection of the strains were studied to lay the foundation for the development of the goose plague vaccine. 12 day old goose embryos were inoculated with 12 days old goose embryos and collected urinic fluid. The recombinant plasmid was sequenced and the recombinant plasmid was sequenced by cloning the VP3 gene fragment into P MD18-T vector and sequencing. The GPV VP3 gene sequence of 2 strains of GPV, which was isolated and identified, was analyzed, and the gene and amino acid sequence was analyzed. The recombinant plasmid pGEX-4T-VP3 of the VP3 gene of the laboratory preserved Siping (SP) VP3 gene was induced and the VP3 protein was purified by the resin; the goose egg yolk IgY was purified and the rabbit was immunized to obtain the antiserum; the resin affinity chromatography was used. The rabbit IgG was purified by the modified sodium iodate method. The Rabbit anti goose peroxidase antibody was obtained. The indirect ELISA method for detecting GPV antibody in Goose Serum was established by optimizing the conditions. The results showed that the optimal concentration of VP3 protein in the indirect ELISA method of GPV was 4 u g/mL, the dilution multiple of the serum was 100 times, the enzyme labeled antibody was used. The dilution ratio was 1100 times. In this study, the incidence and death of gosling were observed by the attack. The liver and small intestine of the goose were made to make paraffin section, and the morbidity and mortality of the goose and the pathological sections were observed. It was proved that the pathogenicity of the Zhenlai strain GPV was stronger. The serum was obtained by the heart blood of the goose, and the indirect ELISA side established in this study was established. The anti GPV antibody level in each serum was measured and compared. The results showed that the serum antibody OD450nm of the Zhenlai goose was the highest, which was 0.684. with the Zhenlai strain GPV and the incomplete Freund adjuvant inactivated vaccine to inactivate the goose. After 13D, 8 strains of GPV were used to attack the virus. The results showed that all the 5 goose in the 15d negative control group died after the attack and all died and died. The former showed obvious depression, loss of appetite, diarrhoea, and white striped lath with dilute feces. The liver hyperemia, bleeding, splenomegaly, hyperemia, kidney enlargement, congestion and bleeding, meningeal congestion, bleeding, small intestinal swelling near yolhuang pedicle, embolization of different length and short length, yellow white, solid touch and obvious Dachshund samples were found. .LY, CH, ZL, BCH, JLJT, DH strain GPV in the GPV attack group did not appear to be dead, the protection rate was 100% (5/5); the PSH group had 1 deaths and the protection rate was 80% (4/5). Thus, it showed that the Zhenlai strain was pathogenic, the immunogenicity was both strong and could protect the infection of other GPV strains.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.33
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