Rabbit - Human Chimeric Anti-hev Igm Quality Control Material And Resistant To Rnase Virus-like Particles In Double Plasmid Expression

Background The aim of this study was to conduct synthetic rabbit-human antibodies conjugate as controls in immunoassays that measure specific IgM to hepatitis E virus.Methods Two New Zealand white rabbits were injected with the HEV recombinant protein NE2 with ORF2 immunodominant epitope as antigen,and the titer of antibody was detected by ELISA.Rabbit IgG was isolated from immune sera by HiTrap affinity columns chromatography on protein A-Sepharose.The purified IgG was quantified by eppendorf biophotometer,and then evaluated by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis(SDS-PAGE).Cross-linker 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride)(EDC) was used to conjugate IgG fraction and human IgM.To determine if the conjugate was similar to human samples, conjugate and positive control serum from real patients were all detected by anti-HEV IgM ELISA and the end point titers were compared.Finally,the stability of antibody conjugate was examined.The chimeric antibodies were diluted,the stock solution were then incubated,in duplicate,at different temperature for different times.Then samples were removed at each time point and were stored at -70℃until the completion of the examination.All samples were tested by ELISA using a HEV IgM Diagnostic Kit.Results After immunization a reasonable titer 1:100,000 of IgG antibodies was being built up in these antisera.By SDS-PAGE analysis,the major stained bands were the heavy and light chains of IgG,50KD and 25KD respectively.No other minor bands were found in sample.The concentration of IgG was about 10mg/ml.The end point titers of antibody conjugate and positive serum were all 1:160,therefore,no significant difference in the end point titer between the serum-derived and the antibody conjugate, Stability of the antibody conjugate indicated that antibody conjugate were not completely stable at temperature below 4℃,room temperature was the better temperature to keep the antibody conjugate stability.Conclusion Rabbit-human antibodies conjugate against HEV described in this study offers an alternative method to prepare positive control instead of traditional serum positive control.This is the first demonstration of the chemically conjugated method to construct anti-HEV IgM positive control.According to the established method,it could also construct a series of positive controls applicable to any immunoassays,such as ELISA,immunoblot and IFA,to detect the presence of IgM specific for a given antigen. Baekgroud This study changed affinity,number and position of MS2-RNA 19mer stem-loop(pac site) in exougenous RNA and constructed two kinds of ribonuclease resistant virus-like particles by two-plasmid system to demonstrate whether larger size RNA could be encapsulated to produce virus-like particles by increasing the amount and affinity of the pac site.Methods Two kind of two-plasmid expression systems were constructed using vectors pET-28b and pACYCDuet-1.One plasmid was plasmid pET-MC constructed by our lab with 1.7kb maturase and coat protein gene of MS2.Another two plasmids were constructed as follows:(1) HIV gag sequence was amplified using sense and reverse primers containing PacI restriction sites.The C-variant of wild-type MS2 RNA stem loop was inserted into the reverse primer.The PCR-amplified DNA fragments was ligated to pACYC-3V vector(three parts of SARS-CoV genes,C-variant,one part of HCV,two parts of H5N1)to generate recombinant plasmid pACYC-3V-gag.(2) HIV gag sequence was amplified using sense and reverse primers contained PacI restriction sites.The C-variant of wild-type MS2 RNA stem loop was inserted into the reverse primer.The PCR-amplified DNA fragment was ligated to pACYC-pol vector(C-variant,HIV-pol) to generate recombinant plasmid pACYC-pol-gag.Then both plasmids were co-transformed into E.coli strain BL21(DE3),Expression was induced by adding IPTG. The cells were sonicated and centrifuged in order to pellet the cell debris.RNase A and DNase 1 were added in order to eliminate E.coli RNA and DNA.VLP were purified by CsCl gradient.After centrifugation,the virus-like particles band is pulled and then dialyzed against sonication buffer to move CsCl.The nuclease treated crude extract of virus-like particles may be also purified by gel exclusion chromatography using a resin such as Sephacryl S-200.Electron microscopy may be used to count the virus-like particles directly.RT-PCR was carried out using the down stream primer to verify the VLPs.Results Two expression vectors pACYC-3V-gag and pACYC-pol-gag had been constructed.Two kinds of virus-like particles were expressed in E.coli BL21(DE3).The particles were purified by Sephacryl S-200.The OD₂₆₀ was higher than OD₂₈₀.Electron microscopy was used to observe the VLP directly.The RT-PCR results of the first VLP showed that the 2,698bp target RNA was packaged into VLP.The RT-PCR results of the second VLP showed that parts of HIV pol(600bp) and gag(450bp) were amplified,and the 3,250bb target RNA could be packaged into VLP.Conclusion By using two C-virants of wild-type stem loop and changing their position in exogenous RNA,the virus-like particles with 2.7～3.2kb exogenous RNA were constructed using two-plasmid system.Therefore,increasing the number of the C-virant of wild-type stem loop and chaging its position could improve the length of exogenous RNA packaged into virus-like particles.