Expression Of HHSP70 In Pichia Pastoris And Study On Its Large-scale Fermentation And Purification Process

Malicious tumors are the enemy to health of the humankind, and the incidencerate and mortality rate of them are growing up over the world. With the deepenedresearch of tumor, it is known that cell immunity is the most important way to clearthe abnormal cells in body, and that some tumor epitopes of CTL can bind with theMHC I molecular to induce cell immunity without the processing of APC. Butbecause of their low molecular weigh, weak immunogenicity and short half-life,it's hard to induce specific CTL by these small polypeptides in practical use. Soresearchers tried some molecular carriers or adjuvants to use with these CTLepitopes, but hardly obtained satisfying results. However HSPs brought new lighton this field in 1980s'. It was found that HSP70 could bind with polypeptides, andthen submit them to MHC I to induce CTL through cross-primming. At the sametime, HSP70 can activate T-cell, mainly γ/δT lymphocyte, and produce memoryT-cells directly. HSP70 also can boost NK to kill the target cells. Oligopeptides orproteantigens can bind with HSP70 uncovalently in vitro to form complexes andthey can provoke immune system to induce specific immune reactions. Because theHSP70-proteantigens complexes can circumvent the MHC barrier, it is veryconvenient to make multivalent vaccine without MHC limits. To date, hHSP70 was extracted from tissues/cells or expressed in E.colithrough gene engineering. But these methods have some insurmountable flaws. Inorder to improve these situations, studies were done as follows: ①Constuctexpression vector pPICZα-hHSP70 and transforme it into Pichia pastoris viaelectroporation. Obtain rhHSP70 secreted into the culture supernatant by the Pichiapastoris, then perform SDS-PAGE, western blot and amino acid sequencing usingthe fermentation broth to identify the recombinant protein; ②Synthesize two CTLepi-positions of HER2/neu protein and bind these two peptides with rhHSP70 invitro to form rhHSP70-peptides complexes. Use BALB/c mice as experimentalanimal to confirm the immunological activities of rhHSP70; ③Study thelarge-scale fermentation process of rhHSP70 in the New Brunswick Scientificbioflow 5000 fermentor; ④Creat a new method to purify rhHSP70. 1. Construction of Pichia pastoris expression system for rhHSP70: (1)Construction of rhHSP70 expression vector pPICZα/hhsp70:Perform PCRusing specific cloning primers to obtain hhsp70 gene, then insert it into T-vector.After identification by endonuclease digestation assay, perform PCR usingexpression primers with Xho I and Xba I target sites and insert the PCR productsinto pPICZ α. After confirmation by endonuclease digestation assay andsequencing, linearize the expression vector pPICZα/hhsp70 and transform it intoPichia pastoris via electroporation. (2)Screening of the transformed Pichia pastoris: Extract the genomic DNA ofthe transformed yeasts to perform PCR using the expression primers. (3)Expression and assay of rhHSP70: Proliferate the PCR tested positively yeastclones, then induced the expression of rhHSP70 with methanol. Perform SDS-PAGE,Western blot and amino acid sequencing to identify rhHSP70 and purify it by ATP-agaroseand SP Sepharose XL. The results indicated that rhHSP70 was identical with the nativehHSP70. 2. Experimental studies on rhHSP-peptides complexes to induce the specific immunereaction: (1)Peptides synthesis: The two CTL epitopes of HER2/neu protein were synthesizedwith Fmoc-strategy and solid phase peptide synthesis method, and were purified with C18RPLC. The fraction interested was confirmed via measuring the molecular weight withEMS. Then they were binded with rhHSP70 uncovalently to form rhHSP70-peptidescomplexes. (2)Immunization with rhHSP70-peptides complexes to induce the specific immunereaction: BALB /c mice were inoculated with mouse breast tumor cell line D2F2(whichwas transfected with full cDNA of human HER2/neu), then were divided into 6 groups, 12mice per group. Every group was injected subcutaneously with PBS 200μl and 20μgrhHSP70 ,GM-CSF+P106-114 ,GM-CSF+P369-377 ,rhHSP70-P106-114 ,rhHSP70-P369-377respectively and weekly,two weeks. ①Seven days after the last immunization, sacrificed 4 mice from every group andseparated the lymphocyte from the spleens. Detected the numbers of T cells secreting IFN-γand found that they are lower in PBS group than the others, at the same time, thenumbers of T cells secreting IFN-γin rhHSP70-P106-114 and rhHSP70-P369-377 groups aremore than those in GM-CSF+P106-114 and GM-CSF+P369-377 markedly(P<0.01). ②Compared with the PBS group, the ability of the specific CTL to kill D2F2cells is much stronger in GM-CSF+P106-114,GM-CSF+P369-377,rhHSP70-P106-114 andrhHSP70-P369-377 groups. The killing abilities to the target cells of rhHSP70-P106-114and rhHSP70-P369-377 groups are stronger than that of GM-CSF+P106-114 andGM-CSF+P369-377 groups(P<0.01). ③Twenty-eight days after the last immunization, sacrificed all the mice,measured the weight of the tumors and calculated the volumes and suppressionratio of tumors. The results showed that compared with the PBS group, thetumor's volumes and weights of GM-CSF+P106-114,GM-CSF+P369-377,rhHSP70-P106-114and rhHSP70-P369-377 groups were much smaller. At the same time, thetumor's volumes and weights of rhHSP70-P106-114 and rhHSP70-P369-377 groupswere much smaller than those of GM-CSF+P106-114 and GM-CSF+P369-377 groups(P<0.01). ④Changes on the tumor pathology: There were large necrotic areas in thetumors of rhHSP70-P106-114 and rhHSP70-P369-377 groups. The necrosis occurred notonly in the center of the tumors but also occurred on the periphery of the tumors.This result indicated that the necrosis weren't caused by ischemia. The necrosisalso occurred in GM-CSF+P106-114 and GM-CSF+P369-377 groups, but the areas anddegrees were smaller than those of rhHSP70-P106-114 and rhHSP70-P369-377 groups. ⑤Changes on the microstructures of tumor cell: The tumor cells ofGM-CSF+P106-114,GM-CSF+P369-377,rhHSP70-P106-114 and rhHSP70-P369-377 groupsappeared agglomerated chromatin and nucleolus were closed to the karyotheca, andthere were also cells with smashed karyon, damaged membrane, enlarged clearancearound the cells, which showed the characters of apoptosis and necrosis. 3. Studies on large-scale fermentation and purification process of rhHSP70: (1)Studies on large-scale fermentation process of rhHSP70: Pichia pastoris hasmany advantages as a kind of expression host, and it's very suitable for large-scaleexpression of the extraneous proteins. So after identifying the bioactivities of the...