Expression Of Recombinant HHSP110 In Pichia Pastoris And Its Large-scale Fermentation

In 1986, Srivastave reported that heat shock protein (HSP) preparations derived from tumor cells had ability to induce protective immune reaction against tumor cells. Since then, isolated HSP from tumor cells as a vaccine has been tested in mice with different tumors, results showed that tumor size was reduction in all, some even complete regression.RhHSP110 was noncovalently bound to a Synthesized one CTL epi-position of HER2/neu protein. This HSP110-peptide vaccine elicited CD8+ T-cell response. The immuno response elicited by HSP110-peptide was more evidence than that of peptide alone. In vivo depletion studies revealed that the CD8+ T-cell response was independent of CD4+ T-cell help. No CD8+T cell or antibody response was detected against HSP 110. The use of recombinant HSP110 to form natural chaperone complex with protein antigens represents a new and powerful approach for the design of protein-targeted cancer vaccines. This kind of recombinant vaccine is more convient, because availability of a surgical specimen of sufficient quantity for purification of the HSP is diffcult.Some works were done as follows: ?Constructed expression vector pPICZα-hHSP110 and transforme it into Pichia pastoris via electroporation. The rhHSP110 secreted into the culture supernatant at a high level and Picia pastoris was screened. (2) Study the large-scale fermentation process of rhHSP110 in the New Brunswick Scientific bioflow 5000 fermentor. We explored the optimizing parameters such as pH,the percent of DO,the supply speed of methanol on fermentation and creat a new method to purify rhHSP110.â‘¡Synthesize one CTL epi-position of HER2/neu protein and bind the peptide with rhHSP110 in vitro to form rhHSP110-peptide complex. The peptide complex was used in the study of induction of specific CTL and tumor suppression activity in BALB/C mice with different cytokine as adjuvant.1. Construction of Pichia pastoris expression system for rhHSP110:The rhHSP110 expression vector pPICZα/hhsp110 was construction by performing PCR with specific primers with Xhoâ… and Kpnâ… target sites and a part ofα-mating factor signal peptide. After identification by endonuclease digestation assay and sequencing, linearize the expression vector pPICZα/hhsp110 and transform it into Pichia pastoris via electroporation. The transformed Pichia pastoris were cultured in medium with zeocin resistant at 28℃for 72 h. Screening of the transformed Pichia pastoris: Extract the genomic DNA of the transformed yeasts to perform PCR using the expression primers. The supernatant of fermentation was identified by SDS-PAGE and western blot. The results indicated that rhHSPl 10 was identical with the native hHSP110.2. Studies on large-scale fermentation and purification process of rhHSP110:We also explored the large-scale fermentation process of rhHSP110. Studies were focused on pH value, culture medium, resolved oxygen, methanol feed speed, initial biomass etc. We found that the best pH is pH4.2, DO between 25%～30% and the supply speed of methanol is 11ml/h/L initial fermentation volume. The concentration of rhHSPl 10 in the broth can reached 500mg·L^-1. The fermentation supernatant was purified by SP Sepharose XL on pH3.8. The purity could be more than 80% and the yield coefficient was higher than 70%. 3. Experimental studies on rhHSP110-peptide complex to induce the specific immune reaction:(1)Peptide synthesis: One of the CTL epitope of HER2/neu protein was synthesised with solid phase peptide synthesis method. The synthesized peptide was purified with C18 RPLC. The fraction interested was confirmed via measuring the molecular weight with EMS. The purified peptide was binded uncovalently with rhHSP110 to form the rhHSP110-peptide complex.(2)Study on induction of specific CTL in vivo by rhHSP110-peptide complex vaccine immunizationDigested D2F2 cells with trypsine and EDTA using PBS to regulate the cell density to 1×10⁸·ml^-1. BALB /c mice were inoculated with D2F2 cells 0.1ml (1×10⁷cells) ,then were divided into 4 groups, 12 mice per group. Every group was injected subcutaneously with PBS 200μl and 20μg rhHSP110,GM-CSF+P_369-377,rhHSP110-P_369-377 respectively and weekly, two weeks.â‘ Sacrificed 4 mice and separated the lymphocyte from the spleens from every group seven days after the last immunization,. The results showed that the numbers of T cells secreting IFN-γare lower in PBS group than the others, the numbers of T cells secreting IFN-γin rhHSP110-P_369-377 groups are more than those in and GM- CSF+P_369-377 markedly (P＜0.01).â‘¡The specific CTL of PBS group was much less than that of GM-CSF +P_369-377,rhHSP110-P_369-377,rhHSP110 groups. The killing abilities to the target cells of rhHSP110-P_369-377 groups are stronger than that of GM-CSF+P_369-377 group (P＜0.01).(3) Study on the tumor suppression of rhHSP110-peptide complex vaccineâ‘ Sacrificed all of the mice, measured the weight of the tumors and calculated the volumes and suppression ratio of tumors twenty-eight days after the last immunization. Results showed that the tumor's volumes and weights of GM-CSF+P_369-377 and rhHSP110-P_369-377 groups were much smaller than the PBS and rhHSP110 groups. The tumor's volumes and weights of GM-CSF+P_369-377 group were much largerer than that of rhHSP110 -P_369-377 group (P＜0.01) .â‘¡Changes on the tumor pathology: There was no area in the tumors of PBS and rhHSP110 group but large necrotic areas in the tumors of rhHSP110-P_369-377 and GM-CSF+P_369-377 group. The inflammatory cells were seen in the areas of necrosis in the tumors of rhHSP110-P_369-377 and GM-CSF+P_369-377 groups. The tissue frame was still there but the cell frame was disappeared. In some cells the nucleolus was dissolved even diappeared.â‘¢Changes on the microstructures of tumor cell: The tumor cell of GM-CSF+P_369-377 and rhHSP110-P_369-377 groups: irregular karyon, sunken karyotheca. There was also cell with smashed karyon, damaged membrane, enlarged clearance around the cell. Agglomerated chromatin and nucleolus were close to the karyotheca, dwindled karyon, swoln organella, many vacuole in the cytoplasm. The tumor cell of PBS group: Many natural tumor cell with big karyon and many nucleolus.The tumor cell of rhHSP110: Natural tumor cell is majority with immuno cells attacking tumor cell in some areas.To sum up, the rhHSP110 was expressed in 80L fermentor with optimizing parameters and the broth was harvested with yield of 500mg·L^-1. We constructed the methods of fermentation of rhHSP110 on large scale and purified of rhHSP110. The rhHSP110-peptide complex could induce specific immune response. Our studies made the foundation of potential to use rhHSP110 as adjuvant to develop vaccines by binding rhHSP110 with antigen polypeptides or proteantigens.