Study On The ATPase And Helicase Activity Of PCV2Rep

Porcine circoviruses belong to the genus Circovirus of the family Circoviridae, which are the smallest mamalian virus yet known. PCVs contain a circular ssDNA molecule with a size of1.76kb, encoding only two major open reading frames.There are two types of PCV:Porcine circoviruses type1(PCV1) and Porcine circoviruses type2(PCV2). PCV1dose not induce a disease in swine while PCV2is the etiological agent of Postweaning Multisystemic Wasting Syndrome(PMWS),it is also the cause of porcine dermatitis and nephropathy syndrome (PDNS)> congenital tremors (CT)-. Reproductive barriers. PCV has spreaded all over the world and brought serious damage to the pig industry.At present, there has not been effective treatment method for the Porcine circoviruses associated disease which only based on the vaccine prevention with a poor effect. To find a better control method, a deep detection of Replication mechanism and protein function is needed.Rep is a protein with multiple functions, which play an important role in virus replication. Homologous analysis shows that there exists a helicase domain on the C-term portion of the Rep protein. Up to now, helicase activity of PCV2Rep has not been demonstrated. This research is mainly on the helicase related functions of PCV2Rep to to provide some reference on the research of protein function, viral replication and pathogenesis. The detail work is as follows:1. Expression and purification of the SUMO-RepThe rep gene was amplified by polymerase chain reaction, and then cloned into the expression vector pET-30a, pET-28a SUMO, etc. Then different expression conditions were explored, and finally we chose the pET-28a SUMO as the express vector. The protein SUMO-Rep was purified by Ni2+affinity column chromatography, and identified by SDS-PAGE and western blot assay.2. ATPase activity assay of SUMO-RepTo detect the ATPase activity of SUMO-Rep, The Kinase-Glo Plus Luminescent Kinase Assay kit was used for detecting the amount ATP remaining in the reaction. The enzymatic kinetics parameter was obtained. The optimal divalent ion was the Mg2+And the addition of oligomeric nucleotides did not bring a boost on ATPase activity.3. The detection of binding activity with nucleic acid of SUMO-RepA nonspecific binding activity with ssDNA and dsDNA with a single-stranded oligo(dT) tail at the3’or5’end proved by EMSA, and this activity dose not rely on divalent ions and ATP.4. The detection of DNA duplex-unwinding activity of SUMO-RepBased on the FRET, method for unwinding activity detection was established. Then the DNA duplex-unwinding activity was detected and the enzymatic kinetics parameter was obtained. The impacts of NTP, capture strand concentration, divalent ions concentration, and reaction temperature on unwinding activity were evaluated. And the optimal helicase reaction condition has been established which is15mM、2mM ATP,37℃.Under the optimized condition, the Km of SUMO-Rep was determined to be104.32nM.5. The construction of SUMO-Rep K180A mutant and the detection of its ATPase and unwinding activity.In our experiments, we constructed the mutant K180A protein of SUMO-Rep. It showed that the ATPase activity of K180A mutant protein was abolished, while the DNA duplex-unwinding activity unwinding activity was reduced to a large extent, which demonstrated that180K is the key site of ATPase activity.