A Preliminary Stury On Production And Imminological Function Of Oral Rotavirus Ferritin Nanovaccine

VP6 protein can be used as candidate vaccine for rotavirus to induce immune protection in mice,and ferritin nanoparticle structure can be used as antigen delivery system to improve the immunogenicity of antigens.The advantage of using mammary bioreactor to produce recombinant protein lies in the safety,efficiency,low production cost,high expression quantity and biological activity of the protein produced.Rice is one of the most important food crops in human society,feeding the world's largest population.For a long time,most studies on rice have focused on the cultivation of high-yield rice and disease-resistant rice,while few studies focused on the application of expressing pharmaceutical recombinant proteins.The aim of this study is to use the transgenic biotechnology combine with nanobiotechnology to produce the mammal gland-based bioreactor mouse model and transgenic rice of rotavirus Nano-vaccine.The results of this study are showed as below:1.Expression,purification and function analysis of the rotavius inner protein VP6 and Helicobacter pylori non-haem iron-containing ferritin.The gene encoding VP6 protein of human rotavirus group A?GenBank accession No:AB022768.1?and Helicobacter p.ylori non-haem ironcontaining ferritin?GenBank NP₂23316?was synthesized based on the E.coli preferred codons and constructed into the pET-32a vector and transferred into the E.coli The protein VP6,ferritin and VP6-ferritin were expressed and the expression factor was optimized.Western blot analysis showed that the molecular weight was about 38 kDa,61 kDa and 80 kDa respectively.They were then purified by Ni-affinity FPLC and the His-tag was removed by enterokinase digestion.The rVP6-ferritin protein self-assembled to spherical particles??20 nm?as visualized by transmission electron microscopy?TEM?,which is similar to the structure of rFerritin.Oral administration of rVP6 and rVP6-ferritin induced efficiency anti-VP6 IgG and IgA titers in mice and rVP6-ferritin protein induced higher antibody titers than those of rVP6 peptides with statistical significance.Though mice gavage rVP6 or rVP6-ferritin together with CTB adjuvant demonstrate higher anti-VP6 IgG or IgA titers than those of rVP6-ferritin,no statistical significant difference was found between them.The group immunized with PBS did not produce any anti-VP6 antibodies.2.Construction and function analysis of the rVP6-ferritin mammal gland specific expression vector.The gene encoding VP6 protein of human rotavirus and Helicobacter pylori non-haem ironcontaining ferritin was synthesized based on the mammals preferred codons and constructed into the pBC1 vector.In order to facilitate cell screening and identification in the following studies,the cmv-EGFP-IRES-Neo fragment was cloned into the downstream of the vector.The mammal gland specific expression vector was identified by enzyme digestion and sequencing and was transferred into the Bcap-37 cell line using the nuclear transfection instrument.The expression of green fluorescent protein was observed by fluorescence microscopy,the mRNA of GFP and rVP6-ferritin was identified by RT-PCR and the rVP6-ferritin protein was identified by Western blot.Results showed that the rVP6-ferritin protein can be expressed in the mammal gland cells and with immunogenicity specific to VP6 protein.3.Production and function analysis of the mammal bland-based bioreactor mice model of rotavirus VP6 Nano-vaccine.The pBC 1 vector was linearized and the prokaryotic sequences are removed from the DNA fragment to be microinjected.The fragments containing rVP6-ferritin were recovered and microinjected into the zygotes of mice.the transgenic mice and its offspring were screened and identified by PCR,Southern blot,Western blot and ELISA.5 founder transgenic mice integrate rVP6-ferritin in the genome were obtained and the rVP6-ferritin was expressed specifically in the mammal gland.Western blot analysis showed that a 65 kDa specific hybridization bands were detected by anti-rotavirus VP6 antibody and ELISA analysis showed that the quantification is in the range of 52.04 to 125.25 mg/L.The titers of anti-VP6 specific IgG and IgA antibody in pup mice breast feeding by transgenic mice or wild type mice were analyzed by ELISA respectively.Specific anti-VP6 IgG and IgA antibodies were detected both in transgenic and wild type pup mice breastfed by transgenic mice,while none of the pup mice breastfed by wild type mice generated any anti-VP6 IgG or IgA antibodies.5-days-old pup mice fed by transgenic mother or wild type mother mice were challenged with 200-fold TCID50 simian rotavirus SA-11 to evaluate the protection against rotavirus induced diarrhea.Pups fed by wild type mother mice suffered watery diarrhea,and the feces pollute their body and legs,while pups fed by VP6-ferritin transgenic mice appeared mild diarrhea relatively and pups without rotavirus challenge did not show any diarrhea.Analysis of the change of body weight showed that rotavirus-challenged pups fed by wild type mice suffered from significant growth retardation,while the pups fed by transgenic mice did not.In brief,compared to pups fed on milk lacking rVP6-ferritin,the proportion of pups with diarrhea symptoms,the duration and intensity of diarrhea,and the deleterious effects on overall growth resulting from the diarrhea were all significantly reduced in the pups fed on milk containing rVP6-ferritin4.Preliminary study on the production of rotavirus nano-vaccine using transgenic rice.To investigate the feasibility of the expression of rVP6-ferritin nano-vaccine by transgenic rice,the rVP6-ferritin gene sequence was optimized according to the preferential codon of rice,and a rice over-expressed rvp6-ferritin gene vector and two rice endosperm-specific expressing vectors were constructed.We successfully obtained three transgenic rice lines and detected the transcription of rVP6-ferritin mRNA and protein expression using RT-PCR and Western blot Although we observed abundant expression of exogenous protein in the primary rice lines,decreased exogenous protein expression and exogenous gene silencing appeared in the subsequent expanded planting test,which seriously affected the further study on the function of rvp6-ferritin protein.This study explored the use of rice as a bioreactor to produce rotavirus nano-vaccines and provided a reference for the application of rice to produce pharmaceutical proteinsConclusion:Our study thus illustrates two important proof-of-principle demonstrations.First,we show that our designed self-assembling rotavirus VP6 nanoparticle vaccine is an effective antigenic system that confers immunogenicity and protective effects against rotavirus challenge in mice.This successful demonstration of a new alternative to the presently available live-attenuated vaccines against rotavirus adds Reoviridae family viruses to the list of viral taxa for which a ferritin-viral peptide strategy can be used effectively for antigen design.Second,our study successfully establishes that edible vaccines can be produced in mammary glands and that both immunogenicity and protective effects against a pathogen can result from feeding on the vaccine-containing milk.The use of genetically modified rice to produce edible vaccines has great potential,but its specific application requires further research.