Cloning And Expressing Mc Region Of SARS-CoV And Studying On Antigenic Activities Of The Expression Protein

Background and objectiveSevere acute respiratory syndrome coronavirus(SARS-CoV)is a newly emerged pathogenic microbes, which is divided into Nidovirales, Coronaviridae, Coronavirus, and its genome is a single-strand positive sense RNA. Severe acute respiratory syndrome(SARS), which was now known caused by SARS-CoV, is a kind of disease with strong communicability and high mortality rate. The first case was identified in Guangdong Province in southern China in November 2002, and the epidemics spread out to word wide within a short period of time. Now, it is controlled temporarly, but there are so many key problems unsettled in the course of its prevention and therapy, for instance, the mechanism of SARS-CoV invasion the host target cells and its pathogenesis, the development of the specific anti-virus drugs, the researches on its effective vaccine and diagnostic reagent, all of which are unsettled for the present.According to the past immunologically researching results of coronaviruses, we get to know that the spike protein(S protein) and the matrix protein(M protein) of their membrane proteins have immunogenicity and could induce neutralizing antibody response. M protein is the most aboundent structural protein, which transes the membrane of coronavirus 3 times and its short N-terminals reach out of the membrane while the long C-terminals locate inside membrane of virus particle. The long C-terminals consist of 121 amino-acids, and thought to interact with the nucleocapsid proteins.In this research, we have analyzed the mutation of 82 SARS-CoV M genes, as a result, the amino-acids mutation takes place mainly locating at the outside-membrane and the trans-membrane regions but hardly does in the membrane. Followed with this work, the B cell epitopes of M protein were predicted and the results showed that over 85% epitopes were inside of membrane(Mc protein).Another researcher had also predicted that, the T cell epitopes of M protein were predominately located in regions of 81-220aas of M protein, accurately accounting for 84.7% of the total T cell epitopes of M protein. As far as antigenicity is concerned, the Me protein is the representative region for M protein, and except for that, the Me protein is less hydrophobic than M protein, which facilitates the protein expression on a large scale. So the Me protein was selected to be the target protein in this research. After amplifying, cloning and expressing the Me region, we have purified and renatured the fusion Me protein, then the antigenicity activities of Me protein were investigated. Methods1. According to the sequence of SARS-CoV Tor2(AY274119) genome, we designed a pair of primers and amplified M gene, SARS-CoV GD322(isolated by our lab) genome as the template, then we constructed the clone vector pGEM-T-M and identified it by PCR.2. Put the 81 SARS-CoV genomes down loaded from GenBank into Editseq software separately, identified and cut out the M genes and translated them into aminoacids respectively. Compaired the bases and aminoacids of the amplified M gene with those of above 81 M genes by Clustal X, then, Frankfurt 1 and TW5 were selected to be the representatives of the 81 isolates. Predicted the epitopes and a helix of M proteins of GD322, Frankfurt 1 and TW5 individually, then compaired them.3. Me region was selected as the target fragment according to the result of (bases and aminoacids) sequences analysis, we designed a pair of primers using M gene sequence of GD322 isolate as template that contains a EcoR I site in the 5" terminal of the upstream primer while a Xho I site in the 5' terminal of the downstream primer. Then, using pGEM-T-M plasmid as template, we amplified Me region with this primers and cloned it into pGEM-T vector, as a result, a recombinant plasmid of pGEM-T-Mc was obtained.4. Connected pET-32a and pGEM-T-Mc together after their digestion with EcoR I/Xho I, then the fusion expression vector of pET-32a-Mc was constructed and the host strain BL21 was transformed with it. Analyzed the recombinant plasmid of pET-32a-Mc with PCR and digestion before its sequenceing.5. Expressed the pET-32a-Mc and induced it by IPTG while identified the expressed fusion protein (Me protein) with SDS-PAGE and Western-Blot.6. After explored the optimum expression and induction conditions, the fusion protein was largely expressed before the purification. The inclusion bodies(contained fusion protein) were dissolved by 8M denaturant urea and rinsed resin with bending buffer(20mM imidazole) before purification. 100ml solution with pET-32a-Mc/BL21 was dissolved and bended to 1.25ml Ni-IDA chelate affinity chromatography medium, then rinsed hetero-protein with 60mM imidazole, finally rinsing the targetfusion protein with 250mM imidazole at the speed of 10 folds volue of resin per hour.7. The purified protein should be diluted to lOOug/ml before excluding its denaturants gradually, and in order to stabilize the purified protein, 0.02% surfactant should be added to the renatured system in case of the renatured protein was precipitated. In order to form the right disufide bonds by the way of mercapto oxygenolysis among moleculars, the whole experiment should be conducted under the condition of 4°C.8. The rabbits were immunized by the renatured protein and the multicloned antibodies were analyzed with indirect ELISA and Western-Blot. With renatured protein as antigen, the serum of rabbits anti-OC43 and anti-229E, the serum of suspected SARS patients and of the healthy persons were also analyzed by indirect ELISA.9. To investigate the resistance to heat of Me protein, renatured protein was heated to 75 °C for 15mins, The multicloned serum of rabbit anti- renatured protein as antibody and the the heated renatured protein as antigen were analyzed by indirect ELISA.Results1. The M gene and Me region of SARS-CoV GD322 were amplified by RT-PCR and cloned, which demonstrated that the M gene was 666bp in length and showed 99.86% in homology with M gene of SARS-CoV Tor 2. The sequencing result has been loaded on to GenBank whose accession number is AY702026o The amplified Me region, with a terminator codon and 366bp in length, could translate a protein of 121 amino acids and showed 100% in homology with that of SARS-CoV Tor 2. There were only 4 nonsynonymous mutation locations in Me regions compared GD322 with 81 strains of SARS-CoV downloaded from GenBank.2. The epitopes of B cells of GD322> Frankfurtl and TW5 M proteins were predicted by Poly-Kyte-Doolitth Polt-Emini and Jameson-Wolf, but to be surprised, there were over 85% epitopes of B cells in Me regions of above 3 isolates and their distribution were completely same. Their centers of a helix were also same as eachother.3. The fusion protein could be largely expressed in BL21 by pET-32a-Mc recombinant plasmid after optimized its expressing conditions. The fusion protein, accounted for 45% in total protein, was about 34KDa and existed in the form of inclusion bodies in cytoplasm. The protein purification was facilitated for 6-His tags of fusion protein.4. The purity of purified fusion protein was over 95% after regulating the purifying conditions and decreasing the contaminations caused by hetero-proteins.5. An active protein was obtained through a serial of experiments described above.6. Multicloned antibodies could be produced by immunizing rabbits with purified and renatured protein and these protein could react with the acquired multicloned antibodies but not react with those serum of rabbit anti-OC43 and 229E, it could also react with part serum of suspected SARS patients while no reaction with those of healthy ones.7. When water-bathing the renatured protein at 75 °C and lasting for 15mins,the indirect ELISA showed it losted the function of reacting with the multicloned antibodies described above.Conclusions1. The bioinformatics analysis results indicated that the Me region of M protein was a relatively dense region for B cell epitopes.2. The expressing products of pET32a(+)/Mc existed mainly in form of inclusion bodies of fusion protein whose molecular weight was 34KDa or so and the 6*His tag facilitate the protein purification and its elementary identification.3. The creative activities protein was acquired after renaturation and multicloned antibodies could be produced after immunolizing rabbits with those renatured proteins. When the experiment of indirect ELISA was conducted, a antigen-antibody reaction took place between Me protein and its multicloned antibodies, which showed antigenic activities of the renatured protein. The renatured protein didn't react with anti-OC43 and 229E serum which implied there might be no cross-antigen between Me protein and coronavirus 229E and OC43. The renatured protein could react with part serum of suspected SARS patients but not reacted with those of healthy ones which implied that Me protein might be as diagnostic antigen for SARS detecting and maybe SARS-Co V doesn't inapparently infecte healthy persons4. After been heated, Me protein didn't react with multicloned antibodies any longer, which elementarily deduced that Me protein might be conformation-dependant antigen but not be linear-dependant antigen and its antigenic activities might be related to its conformation.