| This study established a TaqMan probe fluorescent quantitative PCR method of detection of chicken infectious anemia virus. First of all,according to the specificity of the test requirements,we select a highly conserved fragment of chicken anemia virus genome which across the VP1 and VP2 genes for the target to detect in order to achieve a wide range of testing to the majority of strains.Then we amplified the fragment by PCR,and insert it into the pMD18-T vector,ectracted positive plasmid,quantitied by a spectrophotometer as a standard sample. Then we diluted it in 10-fold dilution and carry out the real time PCR to achieve a standard curve,and evaluate the indicators of the standard curve. The results show that this method has a high specificity,high sensitivity,good reproducibility. Its detection range is 101-1010copy/uL.In order to research the function of VP3 gene,we according to theCux-1 strain gene sequence on GenBank,using molecular biology software Oligo6.0 designed primers to gradually delete the VP3 gene.The research is taken on the basis of the pBluemCAV recombinant plasmid. And then we extact the recombinant plasmid,incomplete digested it,and achieved a vector fragment with CAV and the CAV genome fragment,connected the two fragments,achieved a plasmid of double CAV genome forword in the vector. Then we transfected the plasmid into MDCC-MSB1 cells,and detecet whether the virus are rescued by PCR,sequencing,IFA. Then detect where the virus which loss the nuclear localization signals locate in cells By laser scanning confocal microscope and the change of the ability of the mutant virus rCAV-VP3N88,rCAV-VP3N80, rCAV-VP3N65 inducing cell apoptosis.The results showed that: all the mutant virus we constructed can replicate in the cells. Using real time PCR to detect the replication dynamics of the virus at different stages in the cells,we find that rCAV-VP3N88 and rCAV-VP3N80'replication capacity are slightly reduced compared with the wild strain,rCAV-VP3N65'replication capacity is significantly lower than the wild strain,rCAV-VP3N45 and rCAV-VP3N25'replication is slightly.Through the IFA,confocal laser scanning microscope revealed that loss of the nuclear localization signal,virus locate in the cytoplasm which is contrast to the wild strain. illustrate the nuclear localization signal is important to virus to locate in nuclear. Apoptosis was detected by flow cytometry,results showed that 96h after transfection,the positive control group caused apoptosis is 85.39±2.18%,rCAV-VP3N88 and rCAV-VP3N80 caused apoptosis is 63.08±4.78% and 62.56±7.35%,rCAV-VP3N65 withered cells death is only about 31.46±2.55%.The mutant virus (rCAV-VP3N88,rCAV-VP3N80,rCAV-VP3N65) and wild strain inject the leg and neck of 1-day-old CAV-negative SPF chicks. Autopsy chicks after 5,9,11,14,21,28 days,pathology analysis showed that the positive control group appeared reduce the size of thymic lobules,a significant reduction in lymphocytes,hemorrhage,congestion,atrophy medulla; rCAV-VP3N88,rCAV-VP3N80 have similar change as positive control; rCAV-VP3N65'thymic cortex and medulla are mild bleeding and congestion; negative control group had no significant pathological changes in thymus.Real time PCR test revealed that the virus replicate kinetics curve in vivo was identity with in vitro. |
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