Construction And Study On Anti-cml Immunologic Effects Of The Eukaryotic Co-expression Vector Of Bcr/abl Anchored Gpi And Murine IL-12

The origination of chronic myelogenous leukemia(CML) is theformation of BCR/ABL fusional protein with strong tyrosine kinaseencoded by bcr/abl fusional gene owing to t(9；22)(q34；q11). TheBCR/ABL fusional protein is intracellular protein of CML cell and belongsto a kind of tumour specific antigen. How about effective presention of thefusional protein from inter to surface of CML cell and induction ofpersistent cellar immunity as well as to be a target of the specific cytotoxicT lymphocyte (CTL) cell are the research focus of national andinternational. Considering these, this study regarded bcr/abl fusional geneas a target, and used the character of expressing recombined protein in cellembrances of glyeosylphosphatidylinositol (GPI) and the adjuvant ofcytokine interleukine-12(IL-12)to construct the eukaryotic co-expressionvector of bcr/abl anchored GPI and murine IL-12(mIL-12), and to researchits immunogenicity as well as the anti-CML immunologic effects in vivo,which is aimed to explore the feasibility about immunological therapystyategy of CML regarded bcr/abl genes as a target. The study was carried out in four steps as follows:1. Construction of the eukaryotic co-expression plasmid of bcr/ablanchored GPI and murine IL-12and detection of their expressed genesand proteins in COS-7cells:The gene fragment encoding bcr/abl was amplified by PCR using theplasmid containing the DNA sequence of P210as template and wasinserted into eukaryotic expression vector pBudCE4.1. The constructedrecombinant plasmid pBudCE4.1-bcr/abl (PBB) was identified byrestriction analysis and DNA sequencing. The lymphocytes from humanperipheral blood were separated and extracted the total RNA. The genefragment encoding GPI was amplified by RT-PCR using the RNA astemplate and was inserted into the constructed plasmid pBudCE4.1-bcr/abl,and anchored GPI and carboxylic site of bcr/abl fragment to constructrecombinant plasmid pBudCE4.1-bcr/abl-GPI (PBBG). The mIL-12genefragment was also amplified by PCR using the mIL-12plasmid astemplate and was subcloned in restriction enzyme sites of anotherpromoter in the constructed plasmid pBudCE4.1-bcr/abl-GPI to constructrecombinant plasmid pBudCE4.1-bcr/abl-GPI-mIL12(PBBGI). Theconstructed recombinant plasmid pBudCE4.1-bcr/abl-GPI-mIL12wastransfected into COS-7cells with liposome. Expression of bcr/abl genesand BCR/ABL proteins in transfected cells were analyzed by RT-PCR andWestern blot, and the expressed mIL-12was detected by ELISA. The results of restriction analysis, PCR and DNA sequencing proved thatbcr/abl gene fragment anchored GPI and mIL-12gene fragment were allcorrectly inserted into vector pBudCE4.1. The expression of bcr/abl geneand BCR/ABL protein were identified in transfected COS-7cells and theirembrances, the expression of mIL-12was also proved in culturesupernatant of transfected COS-7cells.2. Construction of SP2/0target cells expressing BCR/ABL proteinsin their embrances:The eukaryotic expression vector of bcr/abl anchored GPI (PBBG)was transfected into SP2/0cells with liposome. After screening of Zeocinantibiotics and clonal culture, two positive clonal SP2/0cells werereceived. The expression of BCR/ABL proteins in positive clonal SP2/0cell membranes was analyzed by Western blot. So the SP2/0cellsexpressing BCR/ABL proteins in their membranes were designated as thetarget cells of specific CTL killing test in vitro.3. Inducdtion of specific cellular immune response in the host micewith the eukaryotic co-expression vector of bcr/abl anchored GPI andmurine IL-12(PBBGI):BALB/C female mice were divided into five groups at random:①pBudCE4.1vector group,②PBB recombinant plasmid group,③PBBG recombinant plasmid group,④PBBGI recombinant plasmid group,⑤PBS control group. Mice were individually immunized with above-mentioned plasmids by intramuscular injection, injectioned onetime at10days intervals and continued3times in all. At30days after lastimmunization, the spleen lymphocytes of mice were separated andcultured with ConA in vitro. The index stimulation and the cytotoxicactivity of the spleen lymphocytes were detected by MTT assay. Theexpression of IL-2and IFN-γ in the culture supernatants of the spleenlymphocytes was also tested by ELISA. The results showed that the indexstimulation, expression of IL-2,IFN-γ and the specific cytotoxic activityof the spleen lymphocytes in immunized mice with recombinant plasmidPBBG and PBBGI were statistically higher than PBB, pBudCE4.1immunized group and PBS control group(P<0.05); compared withPBBG immunized group, the expression of IL-2,IFN-γ and the cytotoxicactivity of CTL in PBBGI immunized mice were also markedly higher(P<0.05), but there were no statistically difference in SI.4. Study on anti-CML immunologic effects of the eukaryoticco-expression vector of bcr/abl anchored GPI and murine IL-12(PBBGI)in vitro:The bcr/abl retroviral vector-mediated mice chronic myeloidleukemia model was constructed and identified by Wen-li FENG professorand Wen-ping ZHANG master of our task group. The constructed andidentified bcr/abl retroviral vector-mediated mice CML model wereindividually immunized with pBudCE4.1vector, recombinant plasmid PBB, PBBG, PBBGI and PBS control by intramuscular injection,injectioned one time at10days intervals and continued3times in all. At30days after last immunization, the marrow, spleen and peripheral blood offive immunized group mices and BALB/C mice were individuallycollected. The expression of bcr/abl genes and BCR/ABL proteins inmarrow cells were analyzed by RT-PCR and Western blot, theencroachment of CML cells in spleen were detected by pathologicalsection, and WBC amount, the ratio of neutrophilic granulocyte as well asimmatured granulocytes from peripheral blood were also counted by WBCamount and sort. Compared with PBS control and other plasmidsimmunized groups of chronic myeloid leukemia model mice, theexpression of bcr/abl genes and BCR/ABL proteins in marrow cells andthe encroachment of CML cells in spleen were all weakened in PBBGIimmunized CML model mice, and the quantity of WBC, the ratio ofneutrophilic granulocyte and immatured granulocytes from peripheralblood in PBBGI immunized CML model mice were also showedstatistically downtrend(P<0.05).Conclusions:1. The reombinant vector pBudCE4.1-bcr/abl-GPI-mIL12wassuccessfully constructed and expressed in eukaryotic cells, which laid abasis for further study on tumour specific cellar immunity induced bybcr/abl fusional gene vaccine. 2. The SP2/0target cell expressing BCR/ABL in its embrance wassuccessfully constructed, which provided a research facility on specificCTL detection in vitro induced by bcr/abl fusional gene.3. Immunization of the eukaryotic co-expression vector of bcr/ablanchored GPI and murine IL-12could induce a better cellular immunity inBALB/C mice,especially a specific anti-bcr/abl CTL immune response.4. Immunization of the eukaryotic co-expression vector of bcr/ablanchored GPI and murine IL-12could relieve CML symptom, inhibit thegrowth of CML cells and reduce expression of the cancerigenic genes inCML model mice, which provided feasibility on immunological therapystyategy of CML regarded bcr/abl genes as a target.Generally, the study regarded bcr/abl fusional gene as a target ofCML immunological therapy, the eukaryotic co-expression vector ofbcr/abl anchored GPI and murine IL-12was successfully constructed, anda better cellular immune response was also induced by synergic effects ofGPI anchoring and cytokine IL-12. Simultaneously, the eukaryoticco-expression vector also showed potential anti-CML immunologic effectsby inhibiting the growth of CML cells and reducing expression of thecancerigenic genes.