Preliminary Study On The Expression Purification And Crystallization Conditions Of ASFV Outer Envelope Protein EP402R And KP177R

African swine fever virus infects domestic pigs and wild suids,and then causes African swine fever.ASF is endemic all over the world.Current ASF control strategies largely rely on rapid detection and implementation of quarantine policies,and there is no effective vaccine yet.ASFV has a five-layer structure,proteins located on different positions have different functions.Both EP402 R and KP177 R proteins are structural proteins located on the ASFV envelope.Several studies reveal that EP402 R is related to the ASFV-mediated hemosorption process,but the specific mechanism is still unclear.However,few studies of KP177 R have been reported.By studying the structure of EP402 R and KP177 R,it is helpful to explore the substrate binding site,understand its function and study the ASFV-mediated hemosorption mechanism.In addition,it plays an important role in the development of ASFV vaccine.In this project,the prokaryotic and eukaryotic expression vectors of EP402 R and KP177 R truncations were constructed,and expressed in HEK293 F mammalian cells and E.coli.We used affinity chromatography technology to preliminarily purify the target protein,and used SDS-PAGE gel electrophoresis,molecular sieve and fluorescence molecular sieve to detect the stability and purity of the target proteins.The target protein that can be expressed stably and in large quantities is further purified by molecular sieve chromatography and then crystallized.We found that the five truncations of EP402 R couldn't be stably expressed in HEK293 F mammalian cells and E.coli.Later,we attempted to co-express EP402R/KP177R/O61 R in HEK293 F mammalian cells,but EP402 Rcould not be stably expressed with the help of KP177Rand/or O61 R.In this project,we found that the KP177 Rtruncation could be stably expressed in HEK293 F mammalian cells,but the expression level is low;and the KP177 R truncation could be stably expressed in E.coli in large quantities.We found two conditions suitable for crystallization,but failed in optimizing protein crystals.In addition,we found that the GFP-KP177 R fusion protein and MBP-N3S6N-KP177 R fusion protein could not be stably expressed in E.coli;MBP-N3S-KP177 R and MBPN3A-KP177 R fusion protein coud be expressed stably in E.coli in large quantities,but there were no suitable conditions for crystallization.