Studies On The Proliferation Of Avian Reovirus In Different Cell Lines And The Expression Of ?B And ?C Gene In Baculovirus System

Avian reovirus(ARV)is an important member of the Orthoreovirus branch of Reoviridae.The infected host is usually chicken,Muscovy duck and some birds.Malabsorption syndrome and viral arthritis and immunosuppressive disease are common clinical disorder.ARV is a serious harm to the world poultry industry caused incalculable economic losses.It was found that ARV could proliferate in several cell lines,such as chicken liver cancer(LMH)cell line,green monkey kidney(Vero)cell line cell line,chicken embryo fibroblast(DF-1)cell line and so on.The study on the regulation of proliferation has not yet been reported.The baculovirus expression system is one of the four major expression systems of gene engineering.It has a high expression of foreign genes,can carry large gene fragments,and express the biological activity of the product with good.ARV encoded ?B and ?C protein which are the outer shell protein of its virus particles,and the ?B protein is carried in a group specific neutralizing antigen determinants,the aB protein enhances the ability of ARV infected cells through the action of ?C protein.The ?C protein carries a ARV specific neutralization reaction indicating antigen,which can stimulate the body to produce protective neutralization antibodies,and relevant to the adsorption and proliferation of the virus particles.Therefore,?B protein and ?C protein are the preferred proteins in the development of ARV vaccine.In this study,the proliferative characteristics of ARV in several cell lines were studied,then ARV ?B and ?C antigen proteins were expressed by baculovirus expression system and their biological activities were analyzed.In order to provide theoretical basis and technical support for the prevention and control of ARV.The results of this study are divided into four parts.1.Study on the proliferative characteristics of ARVThe ARV S1133 strain was inoculated with chicken liver cancer(LMH)cell line,African green monkey kidney(Vero)cell line and chicken embryo fibroblast(DF-1)cell line by gradient dilution.The titer of the virus was determined at 0 h,12 h,24 h,36 h,48 h,72 h,96 h,120 h total eight time points after ARV infection of three kinds of cell lines.The growth curve of ARV on three cell lines was plotted and its proliferation law was analyzed.The results showed that ARV in Vero cell line proliferated more rapidly,the titer of the virus was higher and the stable period was longer,so it was more suitable for the proliferation of ARV.Stable CPE was produced,and the virus titer reached a peak at 48 h after infection,which was 1×10-8.46/0.1 mL.2.Construction of expression Vector of ARV Gene ?B and ?CThis paper analyzed the sequence characteristics of NCBI gene and expression vector,designed primers to amplify the gene and construct its recombinant expression vector,and named ?B-pFast HTB and ?C-pFast Dual.Recombinant expression vector was confirmed by double enzyme digestion and sequencing.The results showed that the full-length gene was amplified successfully and the recombinant expression vector was constructed by transposing to the expression vector.3.Soluble expression of ?C protein in Sf9 cellsThe recombinant plasmid was transformed into DH10 competent cells,and the positive clones were screened.The recombinant baculovirus was obtained by extracting the recombinant bar granulocytes and transfected into the Sf9 cells respectively.The recombinant baculovirus was named BacSC-?B and BacSC-?C.The recombinant virus was obtained by cell morphological observation and mRNA transcriptional level verification,and the titer of the recombinant virus was determined.The results showed that the Sf9 cells were successfully infected with BacSC-?B and BacSC-?C,and the transfected Sf9 cells had large nuclei and typical CPEs,such as granular substances.The extractive of DNA and RNA of Sf9 cells transfected with recombinant baculovirus can detected Gene ?B and ?C.The TCID50 of BacSC-?B was 10-9 42/0.1 mL and the TCID50 of BacSC-?C was 10-8.31/0.1 mL.4.Preliminary validation of protein ?B and ?CThe recombinant protein was extracted,and named rBacSC-?B and rBacSC-?C,the recombinant protein were preliminary validated by IFA,SDS-PAGE and Western Blot.The results showed ?B and ?C protein was successfully expressed in Sf9 cells;IFA results showed a recombinant baculovirus that showed green fluorescein.The result of SDS-PAGE and Western Blot showed that rBacSC-?B has a protein band at 35 kDa and rBacSC-?C has a protein band at 35 kDa.It indicated that ?B and ?C recombinant protein was successfully expressed in Sf9 cells and could react with ARV positive serum antibody,showing a good reactivity.In this study,the data of ARV proliferation in several cell lines were obtained,and the expression and identification of ARV aC protein in baculovirus expression system were completed,which provided experimental basis and technical support for the further study of ARV vaccine.