Expression, Purification Of SARS Coronavirus Nucleocapsid Protein And Preparation Of Its Monoclonal Antibody

Severe acute respiratory syndrome (SARS), also known as infectiveatypical pneumonia, is a new type of respiratory syndrome arose first in thesouthern China in the middle of November 2002. After that, the prevalencesoon erupts in the mainland, Hong Kong, Canada, Singapore and Vietnam.In a brief time of several months, it spreads 30 countries, 8,000 people wereinfluenced, and 1,000 people died of it. The prevalence of SARS not onlyseverely threatens the health and life of the human beings, but also hasgreat influence on the society and economy. The medical and scientificworkers did the best to meet the challenge of this unexpected calamity.After the global alarm of SARS by the world health organization, the causeof SARS has been identified as a novel corona virus( or corona virusvariant)-SARS-CoV by the corporation of 13 top laboratories of 11countries and regions within a month. The preliminary genome remark ofhas been finished on the basis of open reading frames analysis throughsequence similarity to the known corona viruses. According to the genecoding, the anticipating protein includes RNA polymerase protein, the spike(S) protein, the small envelope (E) protein, the matrix (M) protein and thenucleocapsid (N) protein.The exploitations of SARS vaccine and diagnostic kit based on virusinactivation are great breakthroughs. But this technology need to extractvirus from cultured cells, and can only be carried on in P3 stage lab. So itmay have the limitation of reproducibility and standardization. Themethods based on antigen recombination can be more repetitive, easier tobe controlled and has higher biology security.The former researches of animal coronavirus structural protein findthat N protein of coronavirus has significant effects in virus replication andthe pathologic reaction; it has the strongest antigenicity among the severalstructural proteins. Our research plan to express recombinant SARS Nproteins with procaryon E coli system, achieve a great quantity of purifiedrecombinant antigen, and detect serum anti N protein antibody of SARSpatients. We also try to prepare the anti rat SARS-N protein monoclonalantibody.Our work includes the following parts:1. Obtain The SARS-N GeneWe synthesize the gene encoding the nucleocapsid protein (N protein)according to the published SARS corona virus CUHK-W1 completesequence with DNA synthesizer of TaKaRa Company. We use this methodto avoid the problem of biological security of handling virus.2. Construct the Recombinant Expression Vector pET28a-chap10We express the interest protein in E coli cell, we first select thecommon prokaryotic expression carrier pET28a, but the expression of theinterest protein is very low. Many documents reported that the molecularchaperones can help to correct the protein folding, improve the expression,and the expression products usually are soluble. Our research uses the BCGgenome as the template, design the primer, and obtain the molecularchaperones chap10 gene, which belongs to the HSP family. The result hasbeen proven to be correct according to the sequencing. We clone the geneon pET28a prokaryotic expression vector; construct the recombinantexpression vector pET28a-chap10. The product is proven to be correctaccording to the digestion restriction enzyme.3. Obtain The chap10-N Fusion ProteinWe clone the SARS-N gene fragment on the downstream of chap10gene of the recombinant expression vector; construct the recombinantexpression vector pET28a-chap10. The product is proven to be correctaccording to the digestion restriction enzyme. Then transform E. coli BL21(DE3)with the recombinant plasmid, after the IPTG induction; we obtainthe soluble highly expressed fusion protein. The result of SDS-PAGEelectrophoresis show that the molecular mass of the fusion protein is about60KD, agree with the anticipation.4. Western Blot Appraisement of Recombinant ProteinSDS-PAGE electrophoresis of the whole expressed bacteria before andafter induction. Transfer the protein on nitrocellulose filter with transferringelectrophoresis, block it with confining liquid containing 10% calf serum.Then we use the convalescent serum of SARS patient as monoantigen, theHRP marked anti human goat IgG as diploantigen to react with the proteinseparately. The coloration solution were colored. The result showed that thefusion protein whose molecular weight is about 60KD can specially bindingto SARS patients'serum. All confirm the expressed recombinant protein isSARS-N protein, and with well antigenicity.5. Obtain Abundant Recombinant Proteins With FermentationCultureIn order to get enough recombinant antigen for further research, weuse 30L fermentation tank to culture recombinant SARS-N protein in massculture. We make the preliminary exploration to optimize the fermentationcondition. We observed that the alteration of induction temperature,dissolved oxygen and IPTG concentration could influence the expressionand solubility of the protein. The results show that the OD600 can attain 10when the protein cultured for 8h at 37°C before induction. Induce theexpression for 4h; control the dissolved oxygen not below 30%. Theexpression of the interest protein is high and the expression product issoluble. When the concentration of IPTG between 0.5-1.5mmol/L, theinduction effect has no significant deviation. We also try to use lactose toreplace the IPTG induction, the effect is similar to IPTG induction, it hintthat lactose can be used in engineering bacteria fermentation culture as acheap, asepsis, and harmless inductor.6. Purifying the Recombinant ProteinWith deeper exploration, we suspense some fermentation culturedengineering bacteria in 20mmol/L Tris.Cl buffer at pH8.0, break thebacteria with hypersound, and collect the supernatant withcentrifugalization.First, the solution pass through the DEAE Sepharose FF negioncolumn, under this condition, the objective protein directly flow out withoutsticking, and many hybridprotein can stick on it, so it can be purifiedpreliminarily.Then the flow with the objective protein pass through the SPSepharose FF powerful cathodolyte column, the protein can be eluted under500mmol/L NaCl with NaCl gradient elution. Most hybridprotein has beenremoved, hydrophobic chromatography and molecular sieve were both noteffective with the hybridprotein that can not be removed with ion exchangechromatography.At last, we use the BLUE Sepharose CL-6B column to effectivelyseparate the objective protein and the hybridprotein with their difference ofnonspecific binding capability with the medium.The purity can above 95% with the SDS-PAGE running gel scananalysis. The concentration of purified recombinant N protein can achieve0.58mg/ml with Lowery method.7. ELISA Detection of purifying recombinant SARS-N ProteinThe patients'serums were proved to be masculine with RT-PCR. Theserum of both patients and devotees were diluted in 1:50, the ratios ofSARS patients'A492 and the devotees'A492 is above 2.1, demonstrate thesignificant difference between the two. It implicates that the expressedpurified recombinant SARS-N protein can be used in SARS patients'serological diagnosis.8. Preparing the monoclonal antibodyWe immune the Babl/c mouse with purified recombinant N protein,the result of sepharose double diffusion experiment indicate that the fusionprotein can stimulate the body to produce corresponding antibody. Afterthat, we use hybridoma fusion method to successfully prepare a mousemonoclonal antibody after 5 cloning screening. The result of Western blotanalysis demonstrates that the prepared monoclonal antibody can speciallyreact with SARS-N protein, the controlled SARS-S2 protein not have thisreaction. It proves that the screened monoclonal antibody is anti SARS-Nprotein monoclonal antibody.