Sequencing The Full Genome, Cloning And Expressing Of Structural Protein And The Related Research In SARS-CoV

Severe acute respiratory syndrome is called a novel serious and fatal infectious disease mostly transmitted through respiratory track, and many characteristics of it are still unknown presently. We utilized the molecular biological method to research the infection to various human tissues on SARS-associated coronavirus(SARS-CoV), and studied the genome sequence of the virus at the initial stage of SARS epidemic. The total genome RNA was extracted with TRIZOL reagent from various tissue specimens of three fatal cases who died of SARS, and cDNA was obtained through RT-PCR. PCR was performed using SARS-CoV specific primers to amplify the various gene fragments of SARS-CoV. A whole virus genome sequence was mensurated using lung tissue specimen from one fatal case. The virus gene fragments was amplified and obtained from various tissue specimens of one case, including trachea, lung, kidney, liver and lymphnodes, but was only obtained from lung tissue specimens of two others. The results provide the whole sequence is 29,760bp with typical coronavirus sequence characteristics, which not only contains the gene fragments of polymerase, spike protein, membrane protein, nucleocapsid protein, but also contains other 8 open reading fragments(ORFs). Compared with other whole coronavirus genome sequences on GenBank, it has been shown that the sequence has a more gene fragment including 29 nucleotides besides a small quantity of SNP. The fragment is inserted between ORF10 and ORF11, which completely transforms two ORFs to one ORF, and makes the ending protein translation to continue extending. The result demonstrates that, on the one hand, SARS-CoV is a multiple-tissue tropismvirus; on the other hand, this whole genome sequence could be the original sequence from animal to human.For getting more information about the essence of SARS and immune nosogenesis of SARS-CoV, we set about to construct prokaryotic expression vectors of prime structural proteins of SARS-CoV, and to research each protein expression in E. coli strain and the nosogenesis of SARS-CoV. RNA was extracted from tissues of a fatal case with SARS. Gene fragments encoding spike protein, membrane protein and nucleocapsid protein were obtained by RT-PCR, and were cloned into prokaryotic expression vectors, pET32 and pET28. These vectors were transformed into competent E. coli BL21, respectively. Expression of proteins was induced by IPTG under the different condition, and was tested through the method of SDS-PAGE and WB. In the mid of results, expression vectors of recombinant structural proteins of SARS-CoV were established, including pET32-Sl, pET32-S2, pET32-N, pET32-M, pET28-Sl, pET28-S2 and pET28-N. Gene of N protein is over-expressed in E. coli BL21, and S2 protein takes second place. N protein could be expressed easily in prokaryotic expression system. Transmembrane domains and more cysteines could affect expression of S protein and M protein in vitro. Through WB and ELISA, N protein and S protein were both demonstrated highly antigenic, and the former was better at detecting SARS antibody in early serum for SARS diagnosis.To investigate the variation in the titer of the antibody in convalescent patients, and perfect the immunological data of the patients infected with SARS-CoV, ELISA was preformed to measure the titer of antibody against the SARS-CoV in the sera of the sixth month and twelfth month after onset of the illness in the 23 patients with SARS. At same time, HLA-A2 alleles of those 23 patients were measured through labelling the PBMCs by monoclonal antibody and testing by FACS machine. Of these 23 patients, the highest titer in the sixth month was l:160(+), the lowest was l:10(-)? and the average was 1:57; the highest titer in the twelfth month was l:80(+), the lowest was l:10(-), and the average was 1:27. Statistical difference between titers of the two points was clearly significant(P=0.002). 14 patients with HLA-A2 allele were found in the 23 patients(60.9%). Combined and compared with Prof. Li's report, it was concluded that the titer of SARS special IgG antibody in convalescent patients with SARS has presented a fall tendency with the curse of disease prolonging after 6months, and it's reasonable and feasible for the current criterion of the ELISA to diagnose SARS. HLA-A2-restricted peptide vaccine could prevent from above 50% of infections with SARS-CoV.As a new highly contagious and mortal disease, SARS treatment is still experimental (steroid, interferon), and also no vaccine available. Therefore it will be very important to prevent SARS through early diagnosis, quarantine patients with SARS, and inoculating vaccine to high risk people. The first inactivated vaccine in the world has been investigated in clinical trial I, which will improve the passion for completing SARS vaccine research. But the attenuated vaccine and inactivated vaccine have their own advantage and disadvantage respectively, it is an urgent project for all researchers to develop the safety, effective, convenient and economic recombinant vaccine. The present work in our unit is a project collaborated with MRC of Oxford University in SARS. The aim of the project is to identify the T cell epitopes in SARS-CoV infection and develop synthetic peptides vaccine. Compared with the similar study, the characteristics of our work are as follow.1.About 2000 overlapping synthetic peptides including the whole SARS genome(15-mer overlapped by 10 a.a. residues), 88 peptide pools and matrix IFN-gamma Elispot were designed to identify the T cell epitopes in SARS-CoV infection.2. The cohort samples are more than 300 and have extensive representativeness.3. The technology utilized Elispot, Tetrema staining and FCM in our study was advanced, reliable and stable at present. Now we have obtained some surprising results at first part, and will continue to do the further work.