Expression And Assembly Of Chimeric Rotavirus And Enterovirus 71 Virus-like Particles In Baculovirus Expression System

Infective diarrhea and hand-foot-mouth disease are common diseases of infants and young children.These two diseases have always occupied the top two positions in the number of class c infectious diseases in China.They seriously threaten the health and life quality of infants and young children,consume a large number of medical and health resources every year,brought huge economic burden to families and society.According to previous research,60% of infective diarrhea in infants under 5 years old is caused by group A Rotavirus(RV)infection,while hand-foot-mouth disease is mostly caused by enterovirus71(EV71)infection.Currently,there is no specific drug for rotavirus and EV71 infection,and vaccination is still the most effective way to prevent the infection.Therefore,it is necessary to develop bivalent vaccines that can prevent both virus infections.Virus like particles(VLPs)vaccine is a new generation of candidate vaccine as its high efficiency and safety.In previous research,our laboratory has successfully obtained rotavirus-like particles by simultaneously expressing rotavirus capsid proteins VP2,VP6 and VP7 in baculovirus expression system.The stability of rotavirus-like particles is similar to real virions,but the level of immune response induced by rotavirus-like particles still needs to be further improved.Studies have found that VP4 plays a key role in rotavirus invasion into small intestinal epithelium,and virus-like particles expressing VP2,VP4,VP6 and VP7 can enter small intestinal epithelial cells more effectively and enhance mucosal and systemic immune.In addition,previous work also showed that fusion proteins replacing 92 amino acids at the N-terminal of RV-VP2 with full-length EV71 VP1 could still be packaged into virus-like particles with VP6 and VP7.This research first obtained EV71 VP1,RV VP2,VP4,VP6,VP7 gene and ER1,2gene with PCR technology.Transfer vector pYBDM-IM and pUCDM-XIGP was selected to clone genes and Multibac baculovirus expression system was then used to build recombinant baculovirus which carrying EV71 VP1 and RV VP2,VP4,VP6,VP7 gene at the same time.Sf9 insect cells were transfected and observed.The protein expression wasidentified by Western Blot.Sucrose density gradient centrifugation was used to purify recombinant protein and the structure of VLPs was observed under transmission electron microscope.The results showed that ER1,2,VP2,VP4,VP6,and VP7 proteins were 127 KD,104 KD,88 KD,46 KD,and 38 KD,respectively.The protein bands were relatively single and the protein size was consistent with the expectation,indicating that the related proteins of virus-like particles were correctly expressed.The VLPs obtained in this study is not to prepare and purify enterovirus and rotavirus particles respectively,and then mixed,but to prepare chimeric bivalent virus particles,giving full play to the advantages of baculovirus multigene expression system,reducing production cost and improving production efficiency.Based on the nanometer characteristics of rotavirus particles and the characteristics that VP2 gene can carry large foreign gene,this study fused EV71 VP1 gene and truncated capsid protein VP2 of rotavirus to obtain ER1,2,and expressed RV VP6 gene in the middle tier,RV VP4 and VP7 gene in the outer layer,finally obtained chimeric type bivalent virus-like particles.This research can further expand the design idea of virus-like particle vaccine,which is of significance for the construction of other multi-valent virus-like particle vaccine,and lays a foundation for obtaining bivalent virus-like particle vaccine that can simultaneously prevent hand-foot-mouth disease and rotavirus infectious diarrhea.