Molecular Cloning, Expression And Antigenicity Analysis Of Artificial SARS-CAG Gene

ObjectiveSevere acute respiratory syndrome ( SARS) , a newly emerging infectious disease, is caused by SARS - associated coronavirus ( SARS - CoV) , which may originate from some wild animals. New SARS cases were reported in 2004,seriously threatening public health worldwide. SARS - CoV is an enveloped, positive -strand RNA virus. Its genome is about 29.7kb. The viral proteins include the major proteins of spike (S) , matrix ( M) , envelope (E) glycoproteins and the nucleocapsid protein ( N). Among them the spike (S) protein is a major virion structural protein and the main virulence factor . It is a large transmembrane glycoprotein, responsible for receptor binding , membrane fusion and inducing neutralizing antibodies. It is highly imperative to develop effective and safe vaccines to prevent and control new SARS epidemic. Currently, a number of SARS vaccine candidates,including inactivated SARS — CoV vaccines, DNA vaccines and peptide vaccines, are being developed. In our research, we designed epitope peptides of S protein after predicting and analyzing the relational parameters of the antigenicity of S protein with online sharing software. A chimeric gene including ten antigenic epitopes of S protein was synthesized and expressed in vitro. Rabbits were immunized with the recombinant protein, which could provide the basis for developing a successful genetic engineering vaccine again SARS infection.MethodsThe antigenic parameters of the spike protein were predicted and analyzedby bio informatics and ten antigenic epitopes peptides coded 1274bp were selected and designed. The chimeric gene encoding these epitopes was synthesized and tested in Dalian TaKaRa Inc.CAG gene was amplified by PCR from pBluescript SK H /CAG plasmid and cloned into the prokaryotic expression vector pGEX -6p -1 fused with GST tag to prepare the pGEX -6p - 1/CAG. then transformed into E. Coli JM109. The strains containing the recombinant plasmids were cultured in 2 X YTA medium and the target protein was expressed followed by induction with IPTG and detected by 10% SDS - PAGE. The cell ly sates included recombinant protein and its positive control were loaded into 10% SDS - PAGE and analysed by western blot. The GST — tagged recombinant protein were purified using gel elusion.Rabbits were immunized with 500 fxg purified fusion protein induced in the presence of Freund& complete adjuvant ( FCA) ,and boosted two times with freshly prepared emulsion of 500 fAg immunogen and Freund's incomplete adjuvant ( FIA) at 4 — week intervals. Pre - immune sera were collected before starting the immunization and antisera were collected 10 days after the last boost. Sera were kept at 41 before use. then specific antibody of antisera was detected by Blot Dot assay, in general, purified protein was spot onto nitrocellulose,blocking and incubating with anti - rabbit IgG labeled with HRP, then adding DAB solution and color display, meanwhile, ELIS A assay was performed according to instruction of SARS antibody detection Kit.ResultsAfter CAG gene was subcloned and expressed, recombinant plasmids were confirmed by restriction enzyme mapping and DNA sequencing analysis, two -fragments could be released from it,and their size is corresponding to the vector and target gene,respectively. Sequence analysis indicated that CAG gene was completely correct. These evidences showed that GST - CAG recombinant plasmid was constructed successfully.GST - CAG fusion protein could be expressed by IPTG induction , Western blot analysis indicated that the fusion protein could specifically bind to anti -GST - Ab and its size was about 71KDa contained 26KDa GST tag. the recombi-nant protein were quantitated by means of hydroxybenzene reagent and its concentration is 567jxg/ml.After Dot blot and ELISA,the highest titre of antibody was 1/1024 and showed prominent difference compared to control. These results showed that CAG recombinant protein had favourable antigenicity and could effectively induce neutralizing antibodies to elicit protective humoral immunity.ConclusionThe recombinant plasmid pGEX — 6p — 1/CAG was successfully constructed , and the artificial chimeric gene was proved to be expressed by prokaryocyte efficiently , and purified recombinant protein could induce specific antibodies production in rabbits. These results suggested that our multi - epitope based genetic engineering vaccine is a good candidate for further SARS vaccine development.