本文選題：ALV-J + 全基因序列��； 參考：《廣西大學》2016年碩士論文

【摘要】：禽白血病(avian leukosis, AL)是一種能引起禽類免疫抑制的腫瘤性疾病,由禽白血病病毒(avian leukosis virus, ALV)引起,其中J亞型禽白血病病毒(subgroup J ALV, ALV-J)是目前養(yǎng)雞業(yè)最為常見、危害最為嚴重的亞群。雞群感染ALV后,可能會出現(xiàn)生產性能降低、免疫水平下降、出血、腫瘤和生長遲緩等病癥。目前并沒有很好的預防和治療ALV感染的疫苗和藥物,主要依賴種群的凈化。為了輔助凈化工作,課題組構建了一個DNA疫苗和一個RNAi的重組腺病毒,本課題將通過動物試驗來驗證它們的實際效果。另外從臨床發(fā)病雞分離鑒定了一株ALV-J并進行了全基因序列分析。本課題從廣東某雞場臨床發(fā)病的病雞體內采集病料,應用DF-1細胞培養(yǎng)分離病毒,通過對培養(yǎng)物的IFA、ELISA和PCR檢測,確定獲得一株ALV-J,命名為GDMMo通過特異性引物擴增獲得并完成了對該分離株的全基因組序列測定,經與14個來自兩廣地區(qū)的分離株及國內其他地方的參考株進行比較分析,發(fā)現(xiàn)該分離株與廣西分離株GX14ZS14、GX14HG04及廣東參考株GDQY1201等之序列的相似性很高,尤其與GX14ZS14株的核苷酸相似性高達96.8%,且系統(tǒng)進化樹上與GX14ZS14親緣關系最近,同屬一分支。為驗證課題組制備的RNAi重組腺病毒pAd-gp85-shRNA2對雞體內ALV-J感染的干擾效果,選擇蛋清p27抗原陽性且血漿病毒分離呈陽性的種雞群作為實驗對象,其中實驗組(28只產蛋母雞)接種重組腺病毒100μL (1.6×109PFU)／只、對照組(28只產蛋母雞)注謝PBS 100μL。分別檢測試驗前后母雞的體重、其后代雛雞胎糞的p27抗原陽性率；同時在試驗后采集母雞的蛋清進行p27抗原檢測和抗凝血進行病毒分離培養(yǎng)；接種后每周采集實驗組和對照組各5只母雞的抗凝血和分泌物,對ALV-J和重組腺病毒分別進行定量檢測。結果發(fā)現(xiàn),實驗組和對照組母雞的體重無顯著差異；實驗組母雞蛋清p27抗原陽轉陰和血毒陽轉陰的個體均為25%,后代雛雞胎糞p27抗原陽轉陰的個體有19%；在接種后2w,母雞體內的ALV-J拷貝數(shù)最少、重組腺病毒對ALV-J的干擾效率也最高,可達68%；接種后4w,母雞體內仍可檢測到重組腺病毒,與接種后4w時實驗組ALV-J拷貝數(shù)比對照組少的結果相一致,說明重組腺病毒在接種后4w仍能對母雞體內的ALV-J發(fā)揮干擾作用。為驗證課題組構建的DNA疫苗PVAX1-Ub1-gp85-p27-p10誘導接種母雞產生抗體及其后代雛雞產生母源抗體的效果,選擇蛋清p27抗原檢測陰性且ALV-J抗體也是陰性的產蛋母雞作為實驗對象。實驗組(25只產蛋母雞)接種DNA疫苗、對照組(25只產蛋母雞)注射PBS,2w后以同樣劑量重復注射一次。試驗前后分別稱量母雞的體重、檢測免疫母雞血清中的抗體及其后代雛雞血清中ALV-J的母源抗體。結果發(fā)現(xiàn),實驗組與對照組母雞的體重無顯著差異,有28%的免疫母雞(7只)和8%(2只)母雞之后代小雞的抗體出現(xiàn)陰轉陽。本課題研究的結果表明,從臨床發(fā)病雞成功分離到一株ALV-J并完成了其全基因序列的測定與分析；通過動物試驗證明,課題組研發(fā)的重組腺病毒介導的RNAi能有效地干擾ALV-J在雞體內的復制；研發(fā)的DNA疫苗可刺激產蛋母雞產生抗體并誘導其后代雛雞母源抗體的生成。
[Abstract]:Avian leukosis (AL) is a cancer disease that can cause immune suppression in poultry. It is caused by the avian leukemic virus (avian leukosis virus, ALV), and J subtype avian leukosis virus (subgroup J ALV, ALV-J) is the most common and most serious subgroup in the chicken breeding industry. A DNA vaccine and a RNAi recombinant adenovirus have been constructed to help the purification of ALV. The subject will verify them through animal tests. In addition, a strain of ALV-J was isolated from the clinical chicken and the whole gene sequence analysis was carried out. The subject collected the disease from the sick chicken in a chicken farm in Guangdong, used DF-1 cells to isolate the virus, and determined to obtain a ALV-J by the IFA, ELISA and PCR detection of the culture, named GDMMo through the specificity. The whole genome sequencing of the isolate was obtained by primer amplification. After comparison and analysis with 14 isolated strains from two Guangxi regions and other domestic reference strains, it was found that the isolates were highly similar to the Guangxi isolate GX14ZS14, GX14HG04 and the Guangdong reference strain GDQY1201, especially with the GX14ZS14 strain. Nucleotide similarity is as high as 96.8%, and the phylogenetic tree is closely related to GX14ZS14 in the same branch. In order to verify the interference effect of RNAi recombinant adenovirus pAd-gp85-shRNA2 on ALV-J infection in chicken, we selected the chicken group with positive p27 antigen and positive plasma virus isolation as the experimental object, in which the experiment was carried out. The group (28 egg laying hens) was inoculated with recombinant adenovirus 100 L (1.6 x 109PFU) / only, and the control group (28 egg laying hens) was injected with PBS 100 u L. to detect the body weight of the hens before and after the test, and the positive rate of p27 antigen of the meconium in the offspring of the offspring, and the p27 antigen detection and anticoagulant virus isolation and culture of the hen's egg white were collected after the test. After inoculation, the anticoagulant and secretions of 5 hens in the experimental group and the control group were collected every week, and the ALV-J and the recombinant adenovirus were measured. The results showed that there was no significant difference in weight between the experimental group and the control group; the p27 antigen of the mother egg white and the blood toxic yang to the Yin were all in the experimental group, and the meconium P27 of the offspring was resistant to p27. After inoculation, the number of ALV-J copies in 2W was 19%, the number of ALV-J copies in the hen's body was least, the interference efficiency of recombinant adenovirus to ALV-J was also the highest, up to 68%. After inoculation, the recombinant adenovirus could still be detected in the hen's body, and the result of the ALV-J copy number in the experimental group was less consistent than that of the control group at 4W after inoculation, indicating that the recombinant adenovirus was in connection with the inoculation. 4W can still exert interference to the ALV-J in the hens. In order to verify the effect of the DNA vaccine PVAX1-Ub1-gp85-p27-p10 induced by the group to produce the antibody of the hen and the offspring of the offspring to produce the mother source antibody, the egg laying hen with negative p27 antigen and negative ALV-J antibody is selected as the experimental object. (25 The egg laying hens were inoculated with DNA vaccine, the control group (25 egg laying hens) was injected with PBS, and the same dose was repeated once after 2W. The weight of the hen was weighed before and after the test, and the antibodies in the serum of the immune hen and the maternal antibody of the ALV-J in the serum of the offspring were detected. The results showed that there was no significant difference between the experimental group and the control group, and there was a 2 difference between the hen and the control group. The antibody of 8% immune hens (7) and 8% (2) hens appeared to be Yin Yang. The results of this study showed that a strain of ALV-J was successfully isolated from the clinical chicken and the whole gene sequence was determined and analyzed. Through animal experiments, the recombinant adenovirus mediated RNAi could effectively interfere with ALV- J replication in chickens; the DNA vaccine developed can stimulate egg laying hens to produce antibodies and induce the generation of maternal antibodies in their offspring.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.31

【參考文獻】

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1 王新宇;張繼明;尹有寬;謝怡;黃玉仙;鄔祥惠;翁心華;;RNA干擾對乙型肝炎病毒復制的體外影響[J];世界華人消化雜志;2007年15期

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