Study On The Crucial Amino Acid Sites In Multimerization Of The Rep Protein Of Porcine Circovirus Type 2

Porcine circovirus(PCV) belongs to the genus Circovirus of the family Circoviridae, which is the known smallest animal virus. In the 1970 s, it was found in the PK-15 cells. Followed by series of outbreaks in Canada, PCV was isolated from post-weaning multisystemic wasting syndrome(PMWS) pigs. The pathogenic PCV was named PCV2, while the non-virulence PCV was named PCV1. PCV2 can induce immuno-suppression, and then cause co-infection with other pathogens to increase mortality. Currently PCV2 was spread all over the world, and has brought mass economic losses to the pig industry.In present, the mechanism about PCV2 Rep protein involved in viral rolling circle replication is still unclear, such as what kind of multimeric forms presents in rolling circle replication, the critical sites involved in the interaction, etc. Therefore, it is meaningful to explore the critical sites that are between Rep proteins and to draw the spatial structure of Rep protein. All these studies may provide new ideas to explore new functional domain of Rep protein and explain the mechanism of rolling circle replication. Besides, all the results may lay foundations for the development of effective vaccines and drugs.According to previous studies of our laboratory, we found that the cysteine at position 107, the leucine at position 35 and the isoleucine at position 37 within the Rep protein considered as key sites of the Rep protein dimer formation. In this study, three potential crucial sites mentioned above which are mutanted are explored to detect whether they have any influnece on the multimerization of Rep protein. The influence of mutants on the replication rate by PCV2 replicon and enzyme activity were detected. Besides, the crystal of the full-length Rep protein was preliminary screened. Research contents and results are as follows:1. The expression and purification of the PCV2 Rep proteinThe PCV2 Rep gene was amplified by PCR and then cloned into the p ET28a-SUMO prokaryotic expression vector. The plasmid was transformed into BL21(DE3) competent cells and then induced by IPTG. The SUMO-Rep fusion protein was purified by Ni2+-affinity chromatography. The purified SUMO-Rep fusion protein were cleavaged at 4℃ for 2 h combine with SUMO protease to completely removed SUMO-tag.2. The multimerization detection of the Rep full-length protein and its mutantsThe pc DNA3.1-Rep, pc DNA3.1-Rep-C107 A and pc DNA3.1-Rep-L35A-I37AC107 A eukaryotic expression plasmids were constructed and the protein from transfected 293 T cells were then detected by non-reducing SDS-PAGE. The results showed that under the non-reducing and denaturing conditions, the wild-type and mutant proteins didn’t show the dimerization or multimerization in eukaryotic cells. Then the multimerization of the purified wild-type and C107 A, L35A-I37A-C107 A mutant protein after the cleavage of the SUMO-tag were detected. The results showed that the multimerization of the full-length Rep protein C107 A mutant was the same as the wild-type protein, but the multimerization of the L35A-I37A-C107 A mutant protein was affected. So the cysteine at position 107 was not the key site of the multimerization of full-length Rep protein, while the leucine at position 35, the isoleucine at position 37 were involved in Rep full-length protein-protein interactions.3. The construction of PCV2 replicon mutants and the detection of the replication rate and Renilla luciferase activity of mutant repliconsRep gene C107 A and L35A-I37A-C107 A mutant replicons were successfully constructed. The replication rates of replicons with Realtime-PCR and Renilla luciferase activity by using Renilla Luciferase Assay System kit after transfected mutant PCV2 replicons in PK-15 cells were detected. The results showed that the replication rate and Renilla luciferase activity of C107 A mutant replicon were significantly higher than the wild-type replicon, while the replicon replication rate and Renilla luciferase activity of the L35A-I37A-C107 A mutant replicon were significantly lower than the wild-type replicon and given non-significant as the non-replication replicon.4. The detection of the helicase activity of mutant Rep full-length proteinsThe helicase activity of the full length protein and its mutant were detected by NativePAGE. The results showed that helicase activity of the both C107 A and L35A-I37A-C107 A mutant proteins were no significant differences between and the wild type Rep protein.5. The preliminary screening of the PCV2 Rep full-length protein crystalAfter the cleavage of SUMO-tag and purified by gel filtration chromatography, the Rep protein was further concentrated. The crystal of PCV2 Rep full-length protein was preliminary screened by the sessile drop method based on a total of 5 kits and 480 crystal screening conditions. Unfortunately, we were not succeed in getting any crystal results.