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Recombinant protein is a protein product produced by recombinant DNA technology. It is composed of recombinant DNA molecules, which are artificial DNA made from two or more different sources. A foreign DNA fragment (gene) of interest is introduced into a vector DNA molecule, which can replicate within a host cell. The most commonly used DNA vector is plasmid of Escherichia coli (E.coli). The plasmids are small, circular and double-stranded DNA molecules that separate from the bacterial chromosome and can replicate within the cell. They contain one or only a few genes encoding antibiotic resistance and the signal for replication. Moreover, different recognition sites for restriction enzymes are located throughout the plasmid. The restriction enzyme breaks a circular plasmid and cuts a linear double-stranded DNA with two sticky ends at its recognition sites for joining fragment of DNA of interest by DNA ligase. After the gene of interest inserts into the plasmid, the recombinant DNA is formed and introduced into host cell. The plasmid containing the inserted gene is replicated along with the vector in the bacteria. Many copies of inserted the gene are produced with the same genetic materials. (Bolsover, Hyams, Shephard, White, Wiedemann, 2004, p.129-181) The transcription process takes place in the host cell that one strand of recombinant DNA is used as mRNA template for synthesis of a new complementary DNA (cDNA) by RNA polymerase. The mRNA is then copied into new DNA strand by enzyme called reverse transcriptase. The newly synthesized DNA is complementary in sequence to the mRNA template. The enzyme ribonuclease H cleaves phosphodiester links to the mRNA strand. DNA polymerase is then added so that it can replace ribonucleotides with deoxyribonucleotides. Lastly, DNA ligase is used to catalyze the formation of phosphodiester links and a double-stranded DNA is generated by the replacement of RNA strand with DNA strand. After transcription, translation occurs in the cytoplasm and decodes the mRNA in order to produce a specific polypeptide for synthesis of protein. Lastly, the conformation of the polypeptide chain is a result from post-translational modification and protein folding. (Lodish, et al. 2004, p.68-p.130) The protein may be analyzed and purified by gel-filtration chromatography. So, large-scale production of desired recombinant protein product can be produced and wide applied in medicine and industry. (Bolsover et al., 2004, p.129-181)
The recombinant proteins can be expressed in bacteria, yeast, insect, or mammalian cells. This protein engineering technique alters the amino acid sequence of protein and can generate new proteins as tools for scientific research, academic, medical and industrial purposes. For instance, insulin was the first human protein to be expressed from recombinant technology for the treatment of diabetes and has now replaced insulin from pigs and cattle. The production of recombinant protein is often used to produce drugs, vaccines, hormones, enzymes, antibodies and antigens. (Bolsover, et al 2004, p.129-181) The commonly therapeutic application of the recombinant protein is the development of vaccines against seasonal or pandemic influenza virus infection. It is the more effective way than the traditional egg-based vaccine approach.
Influenza A virus is enveloped, negative-strand RNA virus which has antigenic properties of glycoproteins. (Horthongkhame et al., 2007) Influenza A virus can be classified into 16 hemagglutinin and 9 neuraminidase subtypes. (Fiers et al., 2001, p.1961-1963) The outbreak of highly pathogenic avian influenza H5N1 occurred in 1997 and continued to spread in poultry and human. It causes a major public health threat as it is transmitted from infected poultry to humans with high mortality rate. (Wei et al., 2008, p.6200-6208) Therefore, the potent vaccines can effective control of seasonal outbreaks and to pandemic preparedness. (Song et al., 2008) It is very important for control measure by reducing the transmission of bird-to-bird as well as bird-to-human. (Cornelissen et al., 2010) The avian influenza H5N1 composes of several genes: haemagglutinin, neuraminidase, matrix and non-structural gene. (Horthongkhame et al., 2007) The hemaglutinin gene is the best use for development of recombinant vaccines to protect against highly pathogenic avian influenza H5N1. (Cornelissen et al., 2010) The neuraminidase gene is a second glycoprotein on the viral surface which can be applied to produce a soluble neuraminidase-based recombinant vaccine in tetrameric form. (Friers, W. et al, 2001) The matrix gene (M gene) is the third virus protein that has only 23 amino acids on the outer membrane surface. It encodes two proteins: the capsid protein (M1) and the ion channel protein (M2). The extracellular part of M2, M2e, can make it a vaccine target for antibody-based immunity. (Tompkins,S.M. et al, 2007)
Recombinant protein expression system for Influenza vaccine
The prokaryotic cell (e.g. E.coli), mammalian cell and Baculovirus-insect cell can be used as a vector for the expression cell system. The avian influenza virus H5N1 is cultured in viral cell line and its viral RNA is extracted from culture supernatant. Then the cDNA can be amplified by RT-PCR. So, the desired gene (e.g. haemagglutinin) will be amplified with forward primer and reverse primer. (Horthongkhame et al., 2007)
In the prokaryotic expression system, amplified hemagglutinin product are cloned into a plasmid vector and transformed into competent E.coli cells. All colonies of E.coli containing inserted fragment of plasmid DNA can be recognized by restriction enzymes. The colonies of competent E.coli cells are harvested to induce the production of polyhistidine tagged protein. (Horthongkhame et al., 2007) As a result, the hemagglutinin protein needs the multiple post-modification in glycosylation and disulfide bond formation for proper folding and trimerization. (Cornelissen et al., 2010)
In order to construct the mammalian expression vector, the hemagglutinin genes in E.coli plasmid system is used for the protein expression in mammalian cell line. The recombinant gene is cloned to mammalian cell with selector gene and going on the transcription process. The transgene is transfected into mammalian cell line. (Wurm, F. M. 2004) The transient transfections in the mammalian system can efficient produce the transgenic protein by post-transcriptional mechanism and proper post-translation modification. (Kwaks & Otte, 2006) Refinement of vector construction, types of selectable markers and improvements in gene-targeting can make recombinant cell lines with high specific productivities. (Andersen, D. C., Krummen, L., 2002)
Baculovirus-insect cell expression system can be used as transfer vector. It cloned with hemagglutunin gene of avian influenza and infected into insect cells under monolayer and suspension culture condition. The expressed hemagglutinin protein was purified and analyzed by SDS-PAGE and immunoblotting. The majority recombinant hemagglutinin presented as a dimeric species that is insoluble and free in the infected culture medium. ( Nwe et al., 2006 )
Detection method of Recombinant protein Vaccine
The recombinant protein produced in E.coli, in mammalian cell or in baculovirus/ insect cell system can be extracted and purified by metal affinity chromatography and then confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and finally analyzed by Western blot hybridization analysis with anti-His tag or anti-hemagglutinin antibodies. Then the recombinant protein can be purified in monomeric, trimeric, or oligomeric forms.
The immunogenicity of different recombinant proteins is determined by measuring the potent neutralizing antibody response after immunization with the recombinant protein vaccine in mice. The specific antibody against avian influenza virus can be measured by enzyme-linked immunosorbent assay (ELISA) and neutralization antibody assay. The monomeric form of recombinant protein is less immunogenic than the trimer/oligomer forms of the same protein. (Horthongkhame et al., 2007)
Factors affecting the quality of recombinant protein vaccine
Selecting an appropriate expression method is very important for the desired yields and quality of a recombinant protein. The bacteria E.coli system is a rapid and simple method and can shorten doubling time. The growth of media culture is cheap and so easily to scale-up bioproducion. The recombinant protein can direct to cytoplasm that is the most efficiently expressed and yields a high density biomass. The accumulation of large, aggregated and insoluble proteins forms inclusion bodies in the cytoplasm after a prolonged incubation. The inclusion bodies are resistance to proteolysis, easy to concentrate by centrifugation and can be refolded to form active, soluble, functional proteins in the optimal condition. To maximize the formation of functional protein and minimize the formation of inclusion body, lower the temperature during the expression period can slow the rate of transcription, translation and refolding. (Brondyk, 2009,p.131-147) Chaperones in the cytoplasm can be used for promoting protein folding and solubility. (Bolsover et al., 2004, p.68) The correct disulfide bonds formation in the cytoplasm can promote the formation of recombinant protein. The capacity for posttranslation modifications in E.coli will affect N-linked and O-linked glycosylation. Furthermore, pH, redox conditions, protein concentration, temperature, the presence of aggregation suppressors, and host cell contaminants may affect protein aggregation. (Brondyk, 2009, p.131-147)
The use of recombinant vaccine
To develop an effective recombinant protein vaccine for influenza virus, the application of genetic engineering must be well developed. As a result, the vaccine can be highly purified, safe and tailor-made for specific diseases in both animals and humans. It can reduce the potential side effects and offer a protective immunity. The recombination technology can avoid highly pathogenic virus cultivation in eggs. It would shorten the time for strain identification and vaccine preparation, to overcome sudden antigenic change in influenza virus.