Immunogenicity Of Outer Capsid Proteins And Screening Of Potential Host Proteins Interacting With These Proteins In Type â…¡ GCRV

Grass carp reovirus(GCRV), the type strain of Aquareovirus C, is the most important pathogen of grass carp hemorrhagic disease. Among the three genotypes of GCRV, Type Ⅱ GCRV accounts for the major pandemic of grass carp hemorrhagic disease at present in China. The viral genome is composed of 11 segments of double stranded(ds)RNA, and bioinformatic analysis predicted that VP4,VP35 and VP56 of Type Ⅱ GCRV might function as the outer capsid proteins of reoviruses. In this study, the outer capsid proteins of Type Ⅱ GCRV, genes cloning of these proteins and expression, purification and antibodies preparation, and analysis of the immunogenicity of these proteins, screening of potential host proteins interacting with these proteins by Yeast two-hybrid system, The main contents are as follows: 1. Expression, antibody preparation, immunogenicity of outer capsid protein gene S6,and screening of host proteins interacting with VP4 in Type Ⅱ GCRVAccording to the complete segment 6 sequence of Type Ⅱ GCRV HZ08 published on Gene Bank, two pairs of primers for cloning recombinant plasmids p GEX-4t-3-S6 and p GBKT7-VP4 were designed respectively. Total RNAs, which were extracted from CIK infected with GCRV JX02, were used as the template for amplifying the outer capsid protein gene S6 by RT-PCR, the amplified product was about 1954 bp. Gene S6 were cloned into p GEX-4t-3 expression vector, and transformed into Escherichia coli BL21 for protein expression. After induction with IPTG and SDS-PAGE analysis, recombinant VP4 protein(r VP4) of about 98 k Da in molecule weight was produced in E. coli and mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified r VP4, the titer of which was up to 1:4×105. The results of Western blot showed that antibody was able to identify both r VP4 and VP4 in virus particles. Furthermore, serum antibodies of grass carp infected by JX02 were able to recognize r VP4 specifically. Gene S6 was used to generate the bait plasmid p GBKT7-VP4 which was proved to have no self-activation in yeast, subsequently, c DNA library plasmid of CIK cells was screened in yeast AH109 transformed with p GBKT7-VP4, 4 positive clones were obtained and analyzed by plasmid sequencing and nucleotide sequence blasting. 4 potential CIK proteins were suggested to hold the potential to bind VP4. In this paper, the polyclonal antibody against VP4 was successfully prepared, this study also screened potential host proteins interacting with VP4, which laid a foundation of developing a serological diagnostic method for major epidemic strains of GCRV and functional studies of VP4. 2. Expression, antibody preparation, immunogenicity of outer capsid protein geneS11, and screening of potential host proteins interacting with VP35 in Type ⅡGCRVAccording to the complete segment 11 sequence of HZ08 strain, two pairs of primers for cloning recombinant plasmids p GEX-4t-3-S11 and p GBKT7-VP35 were designed respectively, the amplified product was was about 933 bp. p GEX-4t-3-S11 plasmid was transformed into Escherichia coli BL21 for protein expression. After induction with IPTG and SDS-PAGE analysis, r VP35 was about 61 k Da in molecule weight and mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified r VP35, The titer of which was up to 1:106. The results of Western blot showed that antibody was able to identify both r VP35 and VP35 in virus particles. Furthermore, serum antibodies of grass carp infected by JX02 were able to recognize r VP35 specifically. The bait plasmid p GBKT7-VP35 was proved to have no self-activation in yeast, subsequently, c DNA library plasmid of CIK cells was screened in yeast AH109 transformed with p GBKT7-VP35, 16 potential CIK proteins were suggested to hold the potential to bind VP35. In this paper, the polyclonal antibody against VP35 was successfully prepared, This study also screened potential host proteins interacting with VP35, which laid a foundation of developing a serological diagnostic method for major epidemic strains of GCRV and functional studies of VP35. 3. Expression, antibody preparation of outer capsid protein gene S7, and screening ofpotential host proteins interacting with VP56 in Type Ⅱ GCRVAccording to the complete segment 7 sequence of HZ08 strain, two pairs of primers for cloning recombinant plasmids p GEX-4t-3-S7 and p GBKT7-VP56 were designed respectively. Total RNAs, which were extracted from tissues in grass carp carrying GCRV JX02 W, were used as the template for amplifying gene S7 by RT-PCR, the amplified product was about 1539 bp. The p GEX-4t-3-S7 plasmid was transformed into Escherichia coli BL21 for protein expression. After induction with IPTG and SDS-PAGE analysis, r VP56 was about 83 k Da in molecule weight and mainly existed in the form of inclusion bodies. Polyclonal antibody was generated by immunization of mices with the purified r VP56, the titer of which was up to 1:106. The results of Western blot showed that antibody was able to identify r VP56. The bait plasmid p GBKT7-VP56 was proved to have no self-activation in yeast, p GADT7-Gc JAM-A vector was constructed for the sake of further validation of its interaction with VP56. Subsequently, c DNA library plasmid of CIK cells was screened in yeast AH109 transformed with p GBKT7-VP56, 8 potential CIK proteins were suggested to hold the potential to bind VP56, no interaction between VP56 and Gc JAM-A protein was detected in yeast cell. In this paper, the polyclonal antibody against VP56 was successfully prepared, this study also screened potential host proteins interacting with VP56. These results should pave the way for further functional analysis on VP56 protein for major epidemic strains of GCRV. 4. Identification and analysis of VP56 deficient strains in Type Ⅱ GCRVSpecific primer pairs were designed to target each JX02 ORF based on the HZ08 genome sequence. All genomic fragments were amplified and sequenced successfully, the sequences were deposited in the Gen Bank database. The deletion in the S7 genomic fragment was identified in JX02 by using polyclonal antibody against VP56 and PCR. Unlike the well-characterized HZ08 strain, GCRV JX02 has a 431 bp deletion in the S7 genomic fragment, the other 10 genomic segments shared between GCRV JX02 and GCRV HZ08 exhibit over 99% sequence identity, while the deletion was not exsited in JX02 W strain. The results of Western blot showed that VP56 protein was not exsited in JX02 virus particles. This study reveals the presence of Type Ⅱ GCRV VP56 deficient strains, the deletion in GCRV JX02 indicates that VP56 may not be essential for viral replication, which laid the foundation for further study of Type Ⅱ GCRV.