鵝細(xì)小病毒的分離鑒定及其致病性和免疫性研究
發(fā)布時(shí)間：2018-08-04 10:15
【摘要】：鵝細(xì)小病毒病又稱小鵝瘟,是由鵝細(xì)小病毒(Goose parvovirus,GPV)引起的雛鵝和雛番鴨的一種高度接觸性傳染病,使雛鵝及雛番鴨發(fā)生急性或亞急性敗血癥,具有傳播速度快、死亡率高等特點(diǎn)。該病主要病理變化為滲出性腸炎,以空腸和回腸的急性卡他性-纖維素性壞死性腸炎為特征。發(fā)病后耐過(guò)的雛鵝及雛番鴨生長(zhǎng)發(fā)育遲緩,形成“僵鴨”,給養(yǎng)鵝業(yè)造成了巨大的經(jīng)濟(jì)損失。自1961年方定一發(fā)現(xiàn)小鵝瘟病以來(lái),國(guó)內(nèi)外陸續(xù)也有本病的報(bào)道。由于該病對(duì)養(yǎng)鵝業(yè)危害嚴(yán)重,國(guó)內(nèi)外很多研究學(xué)者對(duì)其開(kāi)展了深入研究。小鵝瘟在吉林省局部地區(qū)也呈流行趨勢(shì),近幾年吉林省的九臺(tái)、德惠、遼源、白城、磐石、鎮(zhèn)賚等多個(gè)地區(qū)發(fā)生小鵝瘟疫情,為了確診該病及對(duì)分離到的病毒進(jìn)行免疫分析,本研究從患病雛鵝病料中分離并鑒定了6株GPV,分別命名為JLJT、DH、LY、BCH、PSH、ZL株,并對(duì)各毒株VP3基因進(jìn)行了克隆、測(cè)序分析,開(kāi)展了各毒株致病性、免疫原性及交叉保護(hù)性的研究,為小鵝瘟疫苗的研制奠定基礎(chǔ)。將來(lái)自不同地區(qū)發(fā)病死亡雛鵝的肝臟和栓塞段小腸及其內(nèi)容物研磨制成勻漿,離心去除沉淀,雙抗孵育除去細(xì)菌,接種12日齡鵝胚,收集尿囊液;通過(guò)PCR鑒定,以及將VP3基因片段純化后克隆到p MD18-T載體中,進(jìn)行重組質(zhì)粒測(cè)序。由測(cè)序結(jié)果再次證明所分離的病毒均為GPV。同時(shí),測(cè)定了包括本研究室已經(jīng)分離鑒定的2株GPV在內(nèi)的8株GPV VP3基因序列,并進(jìn)行了基因及氨基酸序列分析。誘導(dǎo)表達(dá)了實(shí)驗(yàn)室保存的四平株(SP)VP3基因重組質(zhì)粒pGEX-4T-VP3,經(jīng)樹(shù)脂純化后得到VP3蛋白;提純鵝卵黃IgY并免疫實(shí)驗(yàn)兔,獲得抗血清;采用樹(shù)脂親和層析方法純化兔IgG;通過(guò)改良的高碘酸鈉法進(jìn)行辣根過(guò)氧化物酶標(biāo)記,獲得兔抗鵝酶標(biāo)抗體;通過(guò)各條件的優(yōu)化建立能夠檢測(cè)鵝血清GPV抗體的間接ELISA方法。結(jié)果表明,GPV間接ELISA方法VP3蛋白最佳包被濃度為4μg/mL、血清稀釋倍數(shù)為100倍、酶標(biāo)抗體稀釋倍數(shù)為1100倍。本研究通過(guò)攻毒實(shí)驗(yàn),觀察雛鵝發(fā)病及死亡情況;將死亡雛鵝肝臟及小腸制成石蠟切片,綜合雛鵝發(fā)病死亡情況及病理切片觀察結(jié)果,證明鎮(zhèn)賚株GPV致病性較強(qiáng)。采取死亡雛鵝心血,獲得血清,以本研究建立的間接ELISA方法測(cè)定并比較各血清中抗GPV抗體水平,結(jié)果發(fā)現(xiàn)鎮(zhèn)賚株接種雛鵝血清抗體OD450nm值最高,為0.684。以鎮(zhèn)賚株GPV與弗氏不完全佐劑制成油佐劑滅活苗免疫雛鵝,13d后以8株GPV進(jìn)行攻毒實(shí)驗(yàn)。結(jié)果發(fā)現(xiàn),攻毒后15d陰性對(duì)照組5只鵝全部死亡,死亡前表現(xiàn)明顯的精神沉郁、食欲廢絕、腹瀉、排白色條狀帶泡沫的稀糞;剖檢發(fā)現(xiàn)肝臟充血、出血,脾臟腫大、充血,腎臟腫大、充血出血,腦膜充血、出血;卵黃蒂附近小腸腫大,有長(zhǎng)短不等的栓塞,剪開(kāi)后栓塞呈黃白色、觸摸堅(jiān)實(shí)、呈明顯的臘腸樣栓塞。LY,CH,ZL,BCH,JLJT,DH株GPV攻毒組雛鵝未出現(xiàn)死亡,保護(hù)率達(dá)100%(5/5);PSH組有1只死亡,保護(hù)率為80%(4/5)。由此說(shuō)明,鎮(zhèn)賚株GPV致病性、免疫原性均較強(qiáng)且能夠保護(hù)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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.33
本文編號(hào)：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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.33
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