發(fā)布時間：2018-05-09 20:24

  本文選題：禽白血病病毒 + 遺傳多樣性　； 參考：《山東農業(yè)大學》2017年博士論文

【摘要】：禽白血病病毒(Avian leukosis viruses,ALV)屬于反轉錄病毒科(Retroviridae)α反轉錄病毒屬(Alpharetrovirus)的一類病毒。ALV感染不僅誘發(fā)多種腫瘤性疾病,還可引起大多數(shù)感染雞的亞臨床癥狀,如產(chǎn)蛋下降、免疫抑制或生長遲緩。根據(jù)病毒中和試驗和gp85的特性,迄今為止將ALV分為A-J十個亞群,以及新鑒定的K亞群。自20世紀90年代以來,禽白血病特別是J亞群禽白血病病毒(ALV-J)給我國的養(yǎng)雞業(yè)造成了重大的經(jīng)濟損失,嚴重危害我國白羽肉雞、蛋用型雞、黃羽肉雞和固有的地方品種雞等各種類型雞的健康發(fā)展。先天垂直感染是ALV的重要傳播方式。由于宿主對ALV難以產(chǎn)生有效免疫應答反應,加之ALV基因組變異頻率較快,至今為止,尚未有商品化的疫苗可用。由于ALV亞群的多樣性,感染和傳播的復雜性,給我國的養(yǎng)雞業(yè)造成了重大的經(jīng)濟損失,本研究對我國不同雞群進行了ALV的流行病學調查,并對公雞精液在ALV傳播中作用進行了系統(tǒng)的研究;通過研究不同佐劑及免疫增強劑對ALV-J gp85重組蛋白誘導抗體的產(chǎn)生,從而加速和輔助我國ALV的凈化;對新分離到的ALV-K全基因組做了系統(tǒng)的分析,并研究了ALV-K的原型株JS11C1對SPF雞的致病性;本研究利用反向遺傳操作技術,構建JS11C1株的感染性克隆,并在此基礎上首次構建了env(J)-LTR(K)和env(K)-LTR(J)的嵌合感染性克隆,借以探究env基因在ALV-K的致病性中所發(fā)揮的作用。1.我國不同雞群中ALV的流行病學調查及其準種分析本研究對我國12個不同地方品系雞群中ALV進行了分離鑒定,共分離鑒定到14株ALV,對這些分離毒株gp85基因進行了克隆測序和亞型鑒定,結果顯示有2株ALV-A,1株ALV-B,8株ALV-J和3株ALV-K。其中,2012年從山東某地方品系雞群發(fā)生疑似為血管瘤的腫瘤病例中分離到5株J亞型ALV-J,定名為SDLY1201-SDLY1205。分別擴增五株ALV-J的gp85基因,并對五個毒株準種多樣性進行分析和比較。結果顯示:五個毒株其gp85核苷酸長度分別為921bp、921bp、924bp、918bp、912bp,其不同克隆之間的氨基酸同源性分比為99.3%-100%、99.3%-100%、99.4%-100%、98.4%-100%、99.0%-100%,其中序列完全相同的克隆比例分別為13/20、17/20、17/20、9/18、16/19,代表了五個毒株中優(yōu)勢準種的比例,對五個毒珠優(yōu)勢準種的gp85比較顯示其氨基酸同源性為89.2%-92.5%。本研究證實即使在同一時期同一雞群感染的不同個體中,ALV-J野毒株準種也存在巨大的差異,特別是其中優(yōu)勢準種發(fā)生了改變。2.公雞精液在ALV傳播中的作用長期以來,對ALV感染雞群過程中公雞的作用特別是對后代的傳播作用一直沒有得到明確的闡述。本研究通過雞胚靜脈接種J亞群禽白血病病毒(ALV-J),獲得持續(xù)病毒血癥的公雞,并將這些公雞的精液人工授精給產(chǎn)蛋期的母雞,1周后收集種蛋并孵化出雛雞。結果顯示,兩只受精的母雞在4-5w時產(chǎn)生ALV-J的抗體,在6只母雞的泄殖腔中檢測到p27抗原,母雞產(chǎn)的26個雞蛋中檢測到3個蛋清P27為陽性。此外,對12只母雞進行病毒分離時發(fā)現(xiàn),在授精后6w均沒有檢測到病毒血癥的存在。然而,1周齡時從34只后代的雛雞中的1只雛雞中分離到ALV-J,其與從公雞精液樣品中分離到的ALV-J的gp85基因的序列同源性為98.4%-99.2%。我們的研究表明,ALV-J可以通過公雞的精液的人工授精感染母雞并且垂直傳播給子代的雛雞。這一發(fā)現(xiàn)對于改進和完善種雞場ALV凈化程序有重要的指導作用。3.不同佐劑及免疫增強劑對ALV-J gp85重組蛋白誘導抗體產(chǎn)生的研究原核表達的ALV-J gp85囊膜蛋白能夠誘導SPF雞產(chǎn)生抗體,但抗體陽性率和抗體水平均較低,本研究觀察了免疫增強劑松花粉多糖(TPPPS)聯(lián)合不同佐劑(CPG、YF01)對增強ALV-J gp85囊膜蛋白免疫后抗體水平的影響。結果顯示,單一gp85重組蛋白只能誘導少數(shù)雞產(chǎn)生抗體,且產(chǎn)生的抗體效價較低、維持時間較短;而YF01或Cp G佐劑分別聯(lián)合重組蛋白能夠使大部分免疫雞產(chǎn)生抗體,且效價較高,當兩種佐劑再聯(lián)合使用免疫增強劑泰山松花粉多糖后能夠使產(chǎn)生的高效價抗體維持時間更長。對ALV-J抗體水平較高的4組收集種蛋進行孵化檢測母源抗體,人工攻毒后觀察母源抗體對雛雞的保護效果。結果發(fā)現(xiàn)發(fā)現(xiàn)添加免疫增強劑TPPPS組產(chǎn)生的母源抗體水平較沒有添加組高,且對雛雞具有較高的保護效力,與對母體的使用效果一致。通過本研究我們可以發(fā)現(xiàn)泰山松花粉多糖聯(lián)合使用YF01和CPG佐劑可以增強gp85重組蛋白的免疫原性,產(chǎn)生較好的免疫保護作用,這將為開發(fā)更加有效的ALV-J亞單位疫苗提供有力實驗依據(jù),高效亞單位疫苗的研制也將為J亞群禽白血病的凈化提供更大的幫助。4.K亞群ALV的全基因組分析及其致病性研究本研究從我國地方品系雞群中分離鑒定到了3株ALV-K,并對其做了全基因組分析,結果表明,這3株ALV-K與之前已發(fā)表的ALV-K毒株位于一個單獨的分支,與雞群中其他已知亞群的ALV均不在同一分支中。其中,與SDAUAK-11、SDAUAK-12和SDAUAK-13的全基因序列同源性最高的分別為中國廣東分離株GD14LZ,GDFX0601,江蘇分離株JS11C1,與其它ALV-K毒株的同源性分別為90.8%-97.7%,90.8%-97.7%,91.2%-97.3%。為研究ALV-K的致病性,將2011年分離自中國某地方品系雞的ALV-K原型株JS11C1株分別采用卵黃囊接種、雞胚靜脈接種和1日齡腹腔接種三種方式感染SPF雞,系統(tǒng)記錄分析了感染雞群的病毒血癥和抗體產(chǎn)生動態(tài)、對感染雞群體重增重的抑制以及感染雞群對疫苗免疫應答的影響等方面,以對JS11C1的致病性進行全面評估。結果表明,當通過卵黃囊接種和雞胚靜脈接種時,JS11C1可以誘發(fā)感染雞的持續(xù)病毒血癥,抑制感染雞群的體重增重和對疫苗的免疫應答,并可誘發(fā)一定比例的淋巴細胞腫瘤。相對于其他亞群ALV,JS11C1的傳播效率和致病性相對較弱。本研究系統(tǒng)研究了JS11C1株對SPF雞的致病性,為下一步研究其致病機制和科學防控奠定了基礎。5.env基因在ALV-K致病作用中的研究在所有亞群ALV中,ALV-J致病性最強,而ALV-K與ALV-A、B等類似。為了闡明env基因和LTR片段在ALV-J與ALV-K致病性差異的作用,本研究構建和比較了ALV-J野毒株SDAU1005和ALV-K野毒株JS11C1及其重組嵌合病毒renv(J)-LTR(K)和renv(K)-LTR(J)的感染性克隆的生物學特性。在這4個感染性克隆病毒中,r SDAU1005(ALV-J)在DF-1細胞上的復制能力最強,r JS11C1最弱,而2個重組病毒介于二者之間,其強度次序r SDAU1005renv(K)-LTR(J)renv(J)-LTR(K)r JS11C1。此外,在與致病性相關的對增重、對法氏囊和胸腺發(fā)育及疫苗免疫后抗體反應的抑制作用上,這一比較結果表明,env基因和LTR片段在影響ALV-J與ALV-K之間致病性差異中起著重要作用,其中env基因的作用似乎大于LTR片段。本研究發(fā)現(xiàn)ALV-K在細胞上的復制水平顯著低于ALV-J及其他亞群,這也為改進凈化程序中的檢測方法和判定標準提供了新的科學數(shù)據(jù)。
[Abstract]:Avian leukemic virus (Avian leukosis viruses, ALV) belongs to the retrovirus family (Retroviridae) alpha retrovirus genus (Alpharetrovirus), a class of viral.ALV infection not only induces a variety of tumor diseases, but also causes subclinical symptoms of most infected chickens, such as egg drop, immunosuppression or growth retardation. According to the virus neutralization test And the characteristics of gp85 have so far divided ALV into ten subgroups of A-J and a newly identified K subgroup. Since 1990s, avian leukaemia, especially the J subgroup of avian leukemic virus (ALV-J), has caused major economic losses to the poultry industry in our country, seriously endangering China's white feathered chicken, egg type chicken, Huang Yu broiler and inherent local breed chicken. The healthy development of various types of chickens. Congenital vertical infection is an important mode of transmission of ALV. Because the host is difficult to produce effective immune response to ALV, and the mutation frequency of the ALV genome is fast, so far, no commercialized vaccine is available. Because of the diversity of the ALV subgroup, the complexity of infection and transmission, the chicken industry is made in our country. It has been a major economic loss. This study conducted a ALV epidemiological survey on different chicken groups in China, and systematically studied the role of the cock semen in the transmission of ALV. Through the study of different adjuvants and immune enhancers, the production of antibodies induced by the recombinant protein of ALV-J gp85 was studied, thus accelerating and assisting the purification of ALV in China; The whole genome of ALV-K has been systematically analyzed, and the pathogenicity of ALV-K's prototype strain JS11C1 on SPF chicken is studied. In this study, the infective clones of the JS11C1 strain were constructed by using the reverse genetic manipulation technique, and on this basis, a chimeric infection clone of env (J) -LTR (K) and env (K) -LTR was constructed for the first time. The role of the disease in the epidemiological investigation of ALV in different chicken groups in China and its quasi species analysis, the ALV was isolated and identified in 12 different local chicken groups, 14 strains of ALV were isolated and identified, and the gp85 gene of these isolates was cloned, sequenced and identified. The results showed that 2 strains of ALV-A, 1 ALV-B, 8, 8, 8, 8. Strain ALV-J and 3 strains of ALV-K., 5 J subtypes ALV-J were isolated from a local strain of chicken group in Shandong in 2012. The gp85 gene of five strains of ALV-J was amplified by SDLY1201-SDLY1205., and the ratio of the five strains of quasi species diversity was analyzed and compared. The results showed that the gp85 nucleotide length of five strains. 921bp, 921bp, 924bp, 918bp, 912bp, the amino acid homology ratio between the different clones is 99.3%-100%, 99.3%-100%, 99.4%-100%, 98.4%-100%, 99.0%-100%, and the proportion of the identical sequences is 13/20,17/20,17/20,9/18,16/19, representing the proportion of the dominant species in the five strains, and the g of the five virulent bead predominant species. P85 showed that its amino acid homology was 89.2%-92.5%.. This study confirmed that even among the different individuals infected with the same chicken group at the same time, there were significant differences in the ALV-J wild strains, especially the dominant species changed the role of the.2. Rooster semen in the ALV transmission for a long time, for the rooster in the ALV infected chicken group. The effect of the effect, especially on the transmission of offspring, has not been clearly stated. This study was inoculated with the J subgroup of avian leukemic virus (ALV-J) from the chicken embryo vein to obtain the rooster of continuous viremia, and the semen of the cocks was artificially inseminated to the hens at the egg laying period. After 1 weeks, the eggs were collected and hatched out of the chicks. The results showed that two fertilized chickens were fertilized. The hens produced ALV-J antibodies at 4-5W, detected p27 antigen in the cloaca of 6 hens, and 3 egg white P27 were positive in 26 hen's eggs. Furthermore, when the virus was separated from 12 hens, none of the virus was detected in 6W after the insemination. However, 1 of the 34 chicks were 1 weeks old. ALV-J is isolated from the chicks, and the sequence homology of the gp85 gene of ALV-J isolated from the sample of the cock semen is 98.4%-99.2%.. Our study showed that ALV-J could infect hens through the seminal seminal insemination of the cock and propagate the chicks vertically to the progeny of the chicken. This discovery has a great effect on improving and improving the ALV purification procedure of the chicken farm. The effect of different adjuvant and immuno enhancers on the production of ALV-J gp85 recombinant protein induced antibodies by.3. and ALV-J gp85 capsule protein can induce antibody in SPF chicken, but the antibody positive rate and antibody level are low. This study observed the combination of immune enhancement agent pine pollen polysaccharide (TPPPS) combined with different adjuvant (CPG, YF01). The results showed that the antibody level of the ALV-J gp85 capsule protein was enhanced. The results showed that the single gp85 recombinant protein could only induce a few chickens to produce antibodies, and the antibody titer produced was lower and the maintenance time was shorter, while the combination of YF01 or Cp G adjuvant combined with recombinant protein could make most of the immunized chickens produce antibodies, and the titer was higher when two adjuvants were reproduced. The combined use of the immunosuppressive agent of Taishan pine pollen polysaccharide can keep the high effective antibody for longer. 4 groups of eggs collected from the high level of ALV-J antibody were incubated to detect the mother source antibody, and the protective effect of the mother antibody on the chicken was observed after the attack. The results showed that the parent source produced by the immune enhancement agent TPPPS group was found. In this study we can find that the combination of Taishan pine pollen polysaccharide and the use of YF01 and CPG adjuvant can enhance the immunogenicity of the gp85 recombinant protein and produce a better immune protective effect, which will be more effective for the development of A. The LV-J subunit vaccine provides a powerful experimental basis. The development of high efficiency subunit vaccine will also provide greater help for the purification of the J subgroup of avian leukosis. The whole genome analysis and pathogenicity of the.4.K subgroup ALV and its pathogenicity study, 3 strains of ALV-K were isolated and identified from the local chicken flock of our country, and the whole genome analysis was made. The 3 strains of ALV-K and the previously published ALV-K strains are located in a separate branch and are not in the same branch with the other known subgroups of the chicken group. Among them, the highest homology of the whole gene sequences of SDAUAK-11, SDAUAK-12 and SDAUAK-13 are GD14LZ, GDFX0601, Jiangsu isolates, and other ALV-K venom of Guangdong isolates in China. The homology of the plant was 90.8%-97.7%, 90.8%-97.7% and 91.2%-97.3%., respectively, to study the pathogenicity of ALV-K. In 2011, the JS11C1 strains of ALV-K prototype strains isolated from a local chicken in China were inoculated with yolk sac, chicken embryo vein inoculation and 1 day old abdominal inoculation were infected with three ways of SPF chickens. The virus infection of the infected chickens was recorded and analyzed systematically. The results showed that when the egg yolk sac was inoculated and the chicken embryo was inoculated, JS11C1 could induce the continuous viremia of the infected chicken and inhibit the weight of the infected chicken group. The results showed that JS11C1 was inoculated with the egg yolk sac and the chicken embryo vein. Weight gain and immune response to vaccines can induce a certain proportion of lymphocyte tumors. Relative to other subgroups ALV, the transmission efficiency and pathogenicity of JS11C1 is relatively weak. This study systematically studied the pathogenicity of JS11C1 strains to SPF chickens, and laid the foundation for the pathogenesis and scientific control of the.5.env gene for the pathogenesis of ALV-K in the next step. In all subgroup ALV, the pathogenicity of ALV-J is the strongest, while ALV-K is similar to ALV-A and B. In order to clarify the role of env gene and LTR fragment in the pathogenicity of ALV-J and ALV-K, this study constructs and compares the infectivity of SDAU1005 and ALV-K wild strains and their recombinant inlay viruses. In the 4 infectious clones, R SDAU1005 (ALV-J) has the strongest replicative ability on DF-1 cells and the weakest R JS11C1, while the 2 recombinant viruses are between two, and the intensity sequence R SDAU1005renv (K) -LTR (J) renv, in addition to the pathogenicity, and the development of the bursa and thymus. The results showed that the env gene and the LTR fragment played an important role in influencing the pathogenicity difference between ALV-J and ALV-K, in which the role of the env gene seemed to be greater than that of the LTR fragment. This study found that the level of ALV-K replication on the cell was significantly lower than that of ALV-J and other subgroups, which was also modified. The detection methods and criteria in the purification process provide new scientific data.

【學位授予單位】：山東農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S858.31

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