Site-specific Recombination And Expression Of Rabies Virus G And N Antigentic Genes In Chloroplast Of Chlamydomonas Reinhardtii

Rabies is a major lethal zoonosis of significant public health concern in many parts of the world,especially in China.It causes a fatal encephalomyelitis for which there is no treatment once the clinical symptoms have appeared.Vaccine inoculation is a useful way to control this disease.Although traditional vaccines have shown their efficiency in controlling the incidence of rabies,the security issues to be concerned. So developing novel vaccine that more efficient and cheaper has become a research hotspot.The scientists pay attention to plant bioreactor because they can produce cheaper high-efficient animal vaccines.Especially the use of plant chloroplasts expressed system has several practical advantages,such as high-lever expression of foreign genes,increasing environmental safrty and so on.It has been a researchable hotpot of plant bioreactors now.Besides,Single-cell eukaryotes- C.reinhardtii as a new bioreactor also has its own special advantages,which have ease of manipulation,cheap cost and large-scale production,and become more and more significant.The aim of this experiment is to transfer the main antigentic genes-glycoprotein gene(G) and nucleoprotein gene(N) into the chloroplast of C.reinhardtii,through the site-specific interagation and expression,finally gain the C.reinhardtii transfermants expressed the G,N antigens.The experiment makes a strong foundation for use C. reinhardtii chloroplast to produce rebies virus antigens.First,the gene G and N were cloned into C.reinhardtii chloroplast expression vector,and expressed vectors p64-G and p64-N containing the selective marker aadA gene and the homologous sequence of Chlamydomonas chloroplast,which is clpP-trnL-petB-chlL-rpl23-rpl2 were constructed.Then,the expressed vectors were transfered into the C.reinhardtii chloroplast by the biolistic method.In the C.reinhardtii chloroplast,the integration of foreign genes had occurred in the chloroplast genome at the specific site by homologous recombination,and by Spc selection we obtained resistant transformants. Finally,by homogeneity of the C.reinhardtii transformants,PCR analysis indicated that the G and the N were inserted into the C.reinhardtii chloroplast genome. Additionally,western blot and ELISA also indicate that the glycoprotein(G)gene and nucleoprotein(N) were expressed in C.reinhardtii chloroplast.