一株ALV-J全基因序列分析及其RNAi重组腺病毒与DNA疫苗的试验研究

发布时间：2018-06-22 11:51

本文选题：ALV-J + 全基因序列　；参考：《广西大学》2016年硕士论文

【摘要】：禽白血病(avian leukosis, AL)是一种能引起禽类免疫抑制的肿瘤性疾病,由禽白血病病毒(avian leukosis virus, ALV)引起,其中J亚型禽白血病病毒(subgroup J ALV, ALV-J)是目前养鸡业最为常见、危害最为严重的亚群。鸡群感染ALV后,可能会出现生产性能降低、免疫水平下降、出血、肿瘤和生长迟缓等病症。目前并没有很好的预防和治疗ALV感染的疫苗和药物,主要依赖种群的净化。为了辅助净化工作,课题组构建了一个DNA疫苗和一个RNAi的重组腺病毒,本课题将通过动物试验来验证它们的实际效果。另外从临床发病鸡分离鉴定了一株ALV-J并进行了全基因序列分析。本课题从广东某鸡场临床发病的病鸡体内采集病料,应用DF-1细胞培养分离病毒,通过对培养物的IFA、ELISA和PCR检测,确定获得一株ALV-J,命名为GDMMo通过特异性引物扩增获得并完成了对该分离株的全基因组序列测定,经与14个来自两广地区的分离株及国内其他地方的参考株进行比较分析,发现该分离株与广西分离株GX14ZS14、GX14HG04及广东参考株GDQY1201等之序列的相似性很高,尤其与GX14ZS14株的核苷酸相似性高达96.8%,且系统进化树上与GX14ZS14亲缘关系最近,同属一分支。为验证课题组制备的RNAi重组腺病毒pAd-gp85-shRNA2对鸡体内ALV-J感染的干扰效果,选择蛋清p27抗原阳性且血浆病毒分离呈阳性的种鸡群作为实验对象,其中实验组(28只产蛋母鸡)接种重组腺病毒100μL (1.6×109PFU)／只、对照组(28只产蛋母鸡)注谢PBS 100μL。分别检测试验前后母鸡的体重、其后代雏鸡胎粪的p27抗原阳性率；同时在试验后采集母鸡的蛋清进行p27抗原检测和抗凝血进行病毒分离培养；接种后每周采集实验组和对照组各5只母鸡的抗凝血和分泌物,对ALV-J和重组腺病毒分别进行定量检测。结果发现,实验组和对照组母鸡的体重无显著差异；实验组母鸡蛋清p27抗原阳转阴和血毒阳转阴的个体均为25%,后代雏鸡胎粪p27抗原阳转阴的个体有19%；在接种后2w,母鸡体内的ALV-J拷贝数最少、重组腺病毒对ALV-J的干扰效率也最高,可达68%；接种后4w,母鸡体内仍可检测到重组腺病毒,与接种后4w时实验组ALV-J拷贝数比对照组少的结果相一致,说明重组腺病毒在接种后4w仍能对母鸡体内的ALV-J发挥干扰作用。为验证课题组构建的DNA疫苗PVAX1-Ub1-gp85-p27-p10诱导接种母鸡产生抗体及其后代雏鸡产生母源抗体的效果,选择蛋清p27抗原检测阴性且ALV-J抗体也是阴性的产蛋母鸡作为实验对象。实验组(25只产蛋母鸡)接种DNA疫苗、对照组(25只产蛋母鸡)注射PBS,2w后以同样剂量重复注射一次。试验前后分别称量母鸡的体重、检测免疫母鸡血清中的抗体及其后代雏鸡血清中ALV-J的母源抗体。结果发现,实验组与对照组母鸡的体重无显著差异,有28%的免疫母鸡(7只)和8%(2只)母鸡之后代小鸡的抗体出现阴转阳。本课题研究的结果表明,从临床发病鸡成功分离到一株ALV-J并完成了其全基因序列的测定与分析；通过动物试验证明,课题组研发的重组腺病毒介导的RNAi能有效地干扰ALV-J在鸡体内的复制；研发的DNA疫苗可刺激产蛋母鸡产生抗体并诱导其后代雏鸡母源抗体的生成。
[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.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

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