Construction, Expression And Purification Of A Hepatocellular Carcinoma Therapeutic Vaccine-HBcAg

Despite many developments in the last decades, primary hepatocellular carcinoma (PHCC) still is one of the most malignant tumor and it remains the second most common cause of death in China. Over 130,000 people die of PHCC each year in our country. In these days, the main therapeutics method of PHCC is surgery, supplemented by radiotherapy and chemotherapy. However, the indications for surgical treatment are strict. Various factors, such as the tumor size, liver function, blood vessels invasion by tumor, have limited the surgical treatment in clinical use. Considering the insensitivity and toxicity, the clinical applications of conventional radiotherapy and chemotherapy treatment are very limited. Liver transplantation has been demonstrated to achieve an effective cure. However, the relatively strict indication for liver transplantation, as well as the lack of post-transplant, relapse and so on, makes it impossible to promote liver transplantation widely. The targeted non-surgical treatment emerged as an important potential way in the PHCC clinic theraphy.For the past few years, therapeutic vaccine has been set up and become the new concept of immune therapy. The main idea is to build therapeutic vaccines for viral infections and tumor treatmentand. With the epitope research it has been possible to precise positioning in a few amino acid residues. Molecular weight of epitope peptide is small and vulnerable to intracellular protease degradation, and the immunogenicity of one separate epitope is weak, and is not conducive to the vaccine preparation. Therefore vector or fusion protein-coupled are necessary to cause effective immunization. It is essential to select a good immunological carrier protein and good immune manner.The HBc protein of Hepatitis B virus core antigen (HBcAg) is capable to self-assembly correctly into a natural shape of HBc core particles in the absence of any other viral component. Because of its unique structure and immunological characteristics, it can not only be used as an ideal vaccine vector, but also play the role of adjuvant in the immune response and provide an extensive thought for vaccines designing. As immune carriers hepatitis B core antigen (HBcAg) has three characteristics as follow : (1) As a particulate antigen, it is susceptible to be uptaked, procesed, handled and presented by antigen-presenting cell, and can stimulate the stronger humoral and cellular immune responses; (2) It plays a direct role to activate B cell responses. It can also activate T cell epitope,in an indirect way. Therefore it is either a TD antigen and a TI antigen; (3) HBcAg 78-83 amino acid residues located in Spike of the apex. As a few amino acid residue of the HBc immune advantage are deleted, its own antigenicity may be down-regulated in HBcAg. Also the immunogenicity of insertion sequence may be enhanced. Thus, HBcAg becomes one of the optimal recombinant vaccine vectors.MAGE-A3 is a member of the melanoma antigen MAGE-A family. Which is characterized by such: There antigens expressed in types of tumor, and was highly expressed In hepatocellular carcinoma, but not in normal tissues except testis and placenta.In this study, MAGE-A3 (112-120 aa) was choosen as the insertion sequences. HBcAg vector is prepared as the vehicle particle. Through the prokaryotic expression, purification, procuring getting massive recombinant protein as a vaccine, so that the body generate effective cellular and humoral immune response against the tumor specific antigen epitope of vaccine for the treatment research of primary hepatocellular carcinoma as theoretical foundation. The three parts of our experiment are showed as follow:1. The construction of ptrc-pep prokaryotic expression vectorptrc Plasmid is preserved in our laboratory, it obtains through the following methods :PBc404 plasmid was choosen as a template for PCR to obtain HBcAg epitope gene, While introduce restriction endonuclease sites of NcoⅠand PstⅠ, connecting modified HBcAg into gene pKK233.2 downstream of trc promoter. Using the method of gene mutation generate NheⅠrestriction endonuclease sites at gene e1 district of HbcAg to form the prokaryotic expression vector ptrc. Synthetic MAGE-A3 (112-120aa) epitope genes, At the same time the introduction of restrictive enzyme NheⅠsites at both ends, Inserting MAGE-A3 (112-120 aa) epitope gene into NheⅠrestriction endonuclease sites ptrc digested by NheⅠ,forming the prokaryotic expression vector ptrc-pep.2. DNA identification of ptrc and ptrc-pep prokaryotic expression vectorAs a template Ptrc and ptrc-pep plasmid extracted for PCR amplification, PCR products by agarose gel electrophoresis results showed that, in the 850 bp, 916bp band Office has the purpose. DNA sequencing results showed that the purpose fragment has been connected successfully into the right direction and two tandem T epitope.3. Massive expression and purification of Ptrc-pep fusion proteinInoculated the correct identified ptrc-pep monoclonal into 10ml LB Medium containing ampicillin (Amp 100μg/ml), 37℃200 rpm culture overnight, the next day inoculate it into 50ml LB Medium containing ampicillin (Amp, 100μg/ml) by the proportion of 1:50,37℃, 200 rpm culture to OD≈1.0, and inoculate again it into 1L LB Medium containing ampicillin, induce IPTG when the OD≈0.8, to final conce- ntration of 1.0mol / L, to continue to foster a culture medium to OD600 = 4,then harv- est cells. Crack Bacterial precipitation through repeated freeze-thaw, ultracentrifugati- on and purification the supernatant containing the target protein ptrc-pep cleavaged through sucrose density gradient. Dot blot results show that ptrc-pep fusion protein distributed mainly in the 30-50% sucrose media. merger Samples, through dialysis, and concentration, then cryopreservation standby protein.