Characterization Of The Binding Patterns Between The VP8* Protein Of Rotavirus And The Histo-blood Group Antigens

Objective: This research was designed to express the VP8* protein of six different RVs(Rotateq-P[8]、Rotateq-P[5]、Rotarix、RRV、LLR、P[14])by genetic engineering technology,and to study the characterization of the binding patterns between the VP8* protein of rotavirus with different P gene type and the HBGAs by saliva- and synthetic oligosaccharides-based binding tests which were based on EIA,witch may be helpful for the further research of the features of the VP8* protein of rotavirus and the role of HBGAs in rotavirus infection and the discovery of new type vaccines and drugs for prevention and treatment of rotavirus infection.Methods:(1)The VP8* gene of all the vaccine virus strain involved in Rota Teq 、 Rotarix and Rotashield were retrived from Genbank and synthesized;(2)The LLR gene was obtained by virus RNA extraction from the rotavirus vaccine rotorway, and was amplified by RT-PCR;(3)The P[14] gene was obtained by virus RNA extraction from the fecal samples,and was amplified by RT-PCR;(4)the VP8* gene of all the six virus was ampified by PCR and cloned into the expression vector p ET-30a;(5)The recombinant plasmid p ET-30a-VP8* were identified by double enzyme digestion and sequencing identification and then transformed to the E.coli BL21(DE3),and then the His-VP8* protein were expressed and purified by the His Trap TMFF affinity chromatograph and validated by SDS-PAGE and Western-blot;(6)The binding patterns between the recombinant VP8* protein and saliva were analyzed by saliva-binding assays;(7)The binding patterns between the recombinant VP8* protein and the synthetic oligosaccharide were analyzed by oligosaccharide-binding assays.Results:(1)The six VP8* genes were successfully cloned into the expression vector p ET-30 a, respectively;(2)The recombinant VP8*protein of all the six virus strains were successfully expressed and purified,and the VP8* protein was about 26 KD;(3)Both of the recombinant protein His-Rotateq-P[8]-VP8* and His-Rotarix-VP8* could bind to saliva witch can express A or B or both A and B or O antigens,namely, saliva of sectors;the recombinant protein His-P[14]-VP8* could bind to saliva witch can express A or both A and B antigens;However,the recombinant protein His-Rotateq-P[5]-VP8* and His-RRV-VP8* and His-LLR-VP8* could bind to none of A, B, AB, O+ and Osaliva;(4)Both of the recombinant protein His-Rotateq-P[8]-VP8* and His-Rotarix-VP8* could bind to H type 1 HBGAs;the recombinant protein His-P[14]-VP8* could specially bind to type A HBGAs;the recombinant protein His-Rotateq-P[5]-VP8* and His-RRV-VP8* and His-LLR-VP8* could bind to none of all the synthetic oligosaccharides.Conclusions:(1)The recombinant VP8* proteins of all the six rotavirus strains with different P genetype were successfully expressed using E.coli prokaryotic expression system and purified by the His Trap TMFF affinity chromatograph;(2)The saliva- and synthetic oligosaccharides-based binding tests were successfully established for the study of the binding patterns between the VP8* protein of rotavirus and the HBGAs;(3)the VP8* protein of rotavirus recognizes HBGAs in a type-specific manner,Strains of P[8] genotype could bind with H type 1 antigen and saliva of sectors but nonsector type;Strains of P[14] could bind with type A antigen specially;Strains of P[5]、P[3] and P[15] couldn’t bind to any type of saliva or oligosaccharides.