Nanochitin-CBM Conjugated Protein For Oral FMDV Vaccine Formulation

Foot and mouth disease(Foot-and-Mouth Disease,FMD)caused by foot-and-mouth disease virus(Foot-and-Mouth Disease Virus,FMDV)mainly infects cloven hoofed animals.It is an acute onset,febricity and highly contagious infectious animal disease and also brings a great harm to livestockes.This virus is the first and smallest virus found in animals.Foot and mouth disease virus has 7 serum types,which are O,A,C,Asia1,SAT 1,SAT 2,SAT 3,each of which contains multiple subtypes,there is no cross reaction between the various types.The main popular serum type in China is O type,A type and Asia 1 type.But the O type is most important.The outbreak of the disease brings a huge impact on animal husbandry economy.`In order to control and prevent the outbreak of this disease,this research was to develop a highly efficient,simple and efficient oral vaccine using bionanomaterial-nanochitin,which formulated with recombinant of chitin-binding domain–FMDV protein and expressed in bacterial expression host E.coli BL 21(DE3).Nanochitin was synthesized by acidic hydrolysis and/or TEMPO oxidization.The results showed thatby acidic hydrolysis with 3M HCl at reaction ratio of chitin to HCl 1:300(wt/v),the length of resulted a positively charged nanochitin particles were in the range of 150-350nm in length and 15-50nm in width,while those of nanochitin produced in conditions of Tempo:NaBr=0.020:0.15,chitin:H2O=1:100,pH=10.5 were in the range of 100-300nm in length and 10-40nm in width,which were carrying on net negative charges.The results also showed that recombinant DNA containing CBM-FMDV could be expressed in the expression vector of E.coli BL21(DE3).We found that the cells carrying on insertion of CBM-FMDV gene grew well in LB/Aml medium and reached logarithmic phase OD600 value to 0.6-0.8 after 3hours incubation at 37~oC,.It is the best phase for protein expression under IPTG induction.SDS-PAGE,Western-Blot analysis showed that the recombinant protein molecular weight was about 95kD and it could be recognized by type O FMDV monoclonal antibodies,it meant the protein correctly expressed and had immunological response to O type FMDV antibody.After ultrasonic crushing,the SDS-PAGE shows that most of recombinant protein was in the form of inclusion bodies.Then we used the urea gradient dialysis renaturation by two different pH value inclusion body dissolving liquid,the Western-blot and test strip show that the refolding protein in two different pH buffer have biological activity.We tried to use the refolding protein in two different pH buffer binding with two nanochitin with different charge,the protein concentration and Dot-Blot analysis showed that they could bind to each other..And the nanochitin with positive charge hads a higher affinity to bindwith recombinant protein in pH4.3 buffer,the ratio 2:1 is the most suitable condition.The significance of this study is that nanochitin carrying with different charges,a novel bionanomaterials have many advantages;Construction,expression,refolding of the recombinant protein can specifically bind to nanochitin,providing evidence that bionanomaterials could be used in novel oral vaccine formulation for FMDV disease control.