| The severe acute respiratory syndrome (SARS), also called infectious atypical pneumonia, is a new type of respiratory system syndrome first occurrenced in the south of China in Jan. 2002. Soon after then, SARS broke out severely in mainland of China, Hongkong, Canada, Singapore and Vietnam. The cause of SARS has been identified as a novel coronavirus (SARS-CoV). During a month , the genome sequencings for the coronaviruses from different SARS patients have finished, which have been deposited in the GenBank already (http://www ncbi.nlm.nih.gov/). After the appearance of SARS, it caused the global including Chinese scientists'attention. The epidemiology, etiology, diagnositic and therapeutic method on SARS has been deeply studied and investigated. The organization and structure of SARS-CoV genome has no difference with general coronaviruses, including the coding gene of replicase, spike(S) protein, envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein in line from 5'to 3'of the cDNA.Coronaviruses infect the host cells by binding to cellular surface virus receptor with the spike protein on the virus surface, which mediate the adherency between the virus and the cell and cause the viral RNA to enter into the host cells. According to the study of other human and animal coronaviruses infection, people found that the spike protein of coronaviruses contain two sections of S1 and S2. The top globular structure of the S protein is composed of S1, which is the major section of CoV recognizing cellular receptor. Research shows that S1 segment is a SARS-CoV cell receptor binding sites. In addition, the rate of SARS-CoV S gene mutation is relatively higher,expecially the high percentage of sense mutations. This means that S protein may be the major antigen protein which leads viruses inducing organism generating neutralizing antibody, and causes cytotoxic T cell producing immune responses. Based on the above,we believe that the research on S1 protein of the SARS virus is very important. Therefore, in this study, we chose the 143-448 S1 fragment of 304 amino acid length expression in Escherichia coli. This segment has a strong activity. The high-performance expression and purification of recombinant SARS virus S1 protein is the important foundation of human single-chain antibody screening and treatment of SARS in future, and meanwhile, is also necessary for the research on the SARS virus protein structure and function.The cloned genes can be expressed in different host cells, include Escherichia coli, Bacillus subtilis, yeast, insect cells, cultured mammalian cells, as well as the whole animal. With the different expression systems, it is necessary to build a different expression vectors. The probability that we succeed to express cloning gene in different gene depends on how much we know about the rules of gene expressing regulation in these system After long-term research on E. coli,People have understood its genetic and properties very clearly. The expression system of E.coli have many advantages, such as simple culture,rapid growth reproduction,and low price. People have more than 20 years of accumulated experience in utilizing E coli to express exogenous gene. The expression level of the exogenous in E coli is much higher than other systems. The Expression quantity of interest protein can be even more than 80% of the total bacterial protein. Thus the protein expression system of E coli have been widely used.According to the different positions of the E. coli which the foreign proteins express in,the target protein expression in E. coli, can be divided into two categories: expression in the cytoplasm and expression in the periplasm. The expression of interest proteins in E. coli cytoplasm often forms inclusion bodies. Although the target protein of this form easily separated from the cells and can be protected from the protease degradation, and will not harm the host cell, but it is not the form of biological activity. Although, in vitro refolding can be obtained biological activity, the process is too complicated. In this study, the expression vector pPelB we used, have a signal peptide,which can guide soluble expression, make the interest protein secreted into the periplasm cavity of Escherichia coli and express a soluble S1 protein.There are many advantages of the expression in the periplasm, firstly,it can be effectively concentrated and purified; secondly, The oxidative environment of periplasm is propitious to protein folding by the right way, in the progress of the protein transferring to the cytoplasm, it is easily to occur signal peptide cleavage in vivo and form the correct N-terminal interest protein; finally, protein degradation in the periplasm is not as widespread as in the cytoplasm. So, both prokaryotic and eukaryotic foreign genes have successfully achieved an effective expression in E. coli periplasm.The protein expression in this study is a histidine-tagged protein. So we wanted to use immobilized Ni2+ chelation chromatography to purify histidine-tagged protein. Comparing with other affinity chromatography, immobilized metal affinity chromatography (IMAC) has these advantages: high stability ligand is not easily removed; metal ion-low prices, low-cost renewable; operating at high salt concentrations, thus save a desalination pretreatment step and can reduce non-specific adsorption; easier protein elution, lower pH or competitive substances such as EDTA or imidazole can be used to desorb down adhesion protein. Because of these advantages of IMAC, it has been widely used in protein separation and purification. The ideal object of protein purification is to obtain the maximum output rate in the least separation steps, while ensuring the purity of protein products.We have carried out these works:1. We select the part sequence (base pairs from 691 to 1608 in SARS-CoV genome sequence) of spike protein gene of SARS Coronavirus, and insert it into pPelB plasmid.2. The recombinant plasmid, p-S1, was transformed into E. coli Rosetta. The expression of S1 segment in E coil was performed by induction with IPTG in OD600=0.6. After the expression, the SDS-PAGE and Western blot were performed for initial identification.3. The product was magnanimously expressed after activity verification. The product obtained was purified by immobilized metal affinity chromatography purification methods and was analyzed for immunologic competence test.ResultsThe active segment was inserted into prokaryotic express vector pPelB. The recombinant protein can be expressed in E.coil Rosetta. The results of SDS-PAGE and Western Blot demonstrated that in E.coli Rosetta recombinant S1 protein gene was expressed with the molecular weight about 34kDa,which accord with its theoretical molecular weight. Pure recombinant S1 protein expressed by E.coli Rosetta could be obtained through IMAC.
|
PDF Full Text Request