Establishment Of Real-time PCR Assay For Detecting Aleutian Mink Disease Virus , Analysis For Its Entire Gene And Prokaryotic Expression And Identification For The Main Regions Of VP2 Gene

Aleutian Disease(AD),caused by Aleutian mink disease virus, belongs to Parvovirinae Amdovirus.It leads to a mainly chronic and progressive infectious disease for mink and causes pathology disharmony of the immunologic system.AD is widely spreaded all over the world in the forms of horizontal and vertical transmission.The disease decreases mink's productivity and fur quality.Therefore,it results in enormous economic loss for mink cultivation.Over the past few yesrs,the epidemic conditions about AD ,which have been reported in the mink cultivation regions in our countries,block servely the development of Chinese mink cultivation.Thus, the study of isolation and identification for the domestic Aleutian mink disease virus and the establishment of the serodiagnosis which is suitable for the domestic strains will provide more scientific information for the future clinical diagnosis,effective prevention and treatment and further research.In this study,one pair of specific primers based on the conservative region of whole gene sequences from ADV in GenBank was designed.With SYBR GreenⅠrandom conjugated incorporation method,a real-time PCR method was established to detect ADV's standard DNA templates.A good linearity relationship was showed between cycle threshold and DNA templates within 0.78×101～0.78×108 copies/ul,and the correlation coefficient was 0.9967.The method was of high specificity and could be used to detect ADV rapidly,which has 100 times higher than PCR in sensitivity.Second,according to the DNA sequence from the standard strain ADV-G and strong pathogenic strain of ADV-Utah1, five pairs of primers were designed and synthesized.The whole genome of two Aleutian Mink Disease Virus (ADV-LN1, ADV-LN2) Chinese strains was amplified by PCR technology, cloned to the vector pMD18-T, sequenced and compared phylogenetically along with the west isolation.The result showed that the highest homology of ADV-LN1 to those of viruses of ADV-G,ADV-Utah1and ADV-SL3 types reached 94.0%(ADV-Utah1). Phylogenetic tree analysis showed that strain ADV-LN1, ADV-LN2 and strain ADV-G,ADV-Utah1,ADV-SL3 belonged to different branches which had a distant relationship.So we concluded that the Chinese strain we isolated before may be a new strain far from Euramerican or the Europe and the United States ADV strain long-term evolution in China.According to analyze inheritance and evolution between the strain tested and strains from other Parvovirus category,we could conclude that ADV as the new genus of the parvovirus subfamily,had the different evolutionary branches with the other genus viruses.The result validated that the rationality of the new classify system reported by ICTV8.The last, according to the full-length gene sequences of chinese ADV-LN1,ADV-LN2 virus strains, we designed a pair of specific primers to go on PCR amplification of ADV VP2 gene. The PCR products were cloned into the vector pMD-18T and sequenced. The fragment of the sequenced recombinant plasmid pMD-18T-M double-digested was obtained and was connected to prokaryotic expression vector pET-28a(+),recombinant plasmid pET-28a(+)-M was constructed, then converted to the host bacteria BL21,the conditions(induction temperature,concentrations of IPTG, induction time,) were optimized on the induced expression. As a result ,in 37℃, 1.0 mmol/L IPTG induces 5 h to realize effective expression in the recombinant M protein highly. Through Ni-NTA affinity chromatography recombinant M protein was purified.Western-blotting analysis showed that expression recombinant M protein specifically reacted with ADV-positive sera. So recombinant M protein prepared in this study had a good immunological reaction activity, it provided the foundation for ADV's serodiagnosis.In a word,the initial establishment of the ADV SYBR GreenⅠquantitative PCRdetection mode would offer effective detection technique for ADV. At the same time,the study provided lots of foundational information including the separating completed two full-length gene sequencing of the ADV separated strains in China. And the results would supply foundational data for the evolutionary characters and regularities of the ADV virus strains.VP2 functional domain was highly expressed by the prokaryotic expression vector pET-28a(+),it provided the foundation for ADV's serodiagnosis.