Expression And Identification Of Rabies Virus G Protein In Rice Endosperm

Rabies is an acute contact zoonotic infection caused by rabies virus,also known as water scare,commonly known as rabies.Clinical features:disturbance of consciousness and nervous excitement,followed by local or general paralysis and death.Main features of pathological changes:non-suppurative encephalitis and the appearance of internal basal bodies in the cytoplasm of nerve cells.Rabies virus(RABV)belongs to the Rhabdoviridae genus Lyssavirus.The RABV is typically bullet-shaped or test-tube shaped with a diameter of 75 nm and a length of 200-300 nm.The size is around 12 kb.The viral genome is a non-staged single-stranded RNA virus consisting of 11928?11932 nucleotides encoding a total of five different structural proteins,from the 3' end to the 5' end:nuclear protein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G)and transcriptase large protein(L).G protein is the only structural protein that can stimulate the body to produce cellular immunity and induce the body to produce neutralizing antibodies.It plays a decisive role in the pathogenesis of rabies,and is also involved in immune mechanisms such as rabies virus-mediated cell fusion and antibody binding.In order to obtain G protein with high immunological activity,high expression,easy storage and low cost in vitro,this study introduced rabies G protein into the endosperm of rice.Firstly,the rabies G gene sequence was selected from NCBI,and the G gene sequence was optimized according to the rice preference codon,then sent to Nanjing Jinsrui Company for synthesis,and pUC57 was used as the vector to link the optimized G gene sequence to pUC57.The pUC57-G recombinant plasmid was inserted into the intermediate vector pMP3 by double digestion of Mlyl and Xhol,and the intermediate vector pMP3-G was successfully constructed,and then the intermediate vector pMP3-G and the plant vector pCAMBIA1300 were digested with EcoRI and Hind?.The recombinant plasmid pCAMBIA1300-G was transformed into Agrobacterium tumefaciens EHA105 by electroporation.The recombinant plasmid pCAMBIA1300-G was successfully constructed by restriction endonuclease digestion and sequencing.After PCR identification,the recombinant plasmid has been successfully transformed into Agrobacterium.The rice seeds were selected for disinfection and induced to induce callus on the induction medium.The recombinant plasmid pCAMBIA1300-G was inoculated with Agrobacterium EHA105 and introduced into rice callus.The cells were cultured at 27? for 3 days and washed with cephalosporin.The callus was then placed on a cephalosporin-containing and hygromycin-containing screening medium to grow a new callus,transformed into a differentiation medium,and then rooted on a rooting medium to obtain 313 plants and 313 plant leaves.The leaf genomic DNA was extracted by CTAB method,and 79 strains were positive by PCR.50 transgenic plants were obtained through refining and planting.The obtained 50 plants were tested,firstly ground,followed by extract(25 mM Tris pH 8.0),extracted at room temperature for 1 h,centrifuged,and detected for G protein by antigen test strip and Western blotting.Finally,the T1 generation seeds of four lines with high expression and good reactivity were selected,and the T1 generation seeds of the selected four lines were rooted and planted,then the rice leaves were taken,and the total DNA of the leaf genome was extracted by CTAB method.The rice-specific single-copy gene REB4 was used as an internal reference.The homozygous was screened by real-time fluorescent quantitative PCR(qPCR)and the copy number was calculated.The results showed that 35 plants were selected from 132 plants.The copy number of the homozygous plants was 1,2,3,5,7,11,and 16,respectively.The obtained T2 homozygous seeds were tested for expression and activity again,and finally 7 plants with high expression levels were selected and planted again to obtain plants with stable genetic traits.At the same time,different buffers were screened to dissolve G protein,and then different fillers were screened to enrich G protein.Finally,PB buffer and enriched protein cationic filler of solubilized protein were screened,which laid a foundation for further intermediate purification and fine purification.This study is dedicated to the expression of rabies virus G protein in rice,the identification of immune activity and the preliminary enrichment and purification of the target protein,laying the foundation for the preparation of new rabies subunit vaccine in the future.