| Identifying the appropriate drug targets for the development of a novel anti-tumorimmunotherapy has currently aroused common attention in the medicial field. We haveidentified a hematopoietic cell-restricted serine/threonine kinase, hematopoietic progenitorkinase1（HPK1）, as a possible target for therapeutic intervention. Targeted disruption ofHPK1alleles confers T cells with an elevated Th1cytokine production in response to TCRengagement. HPK1-/-T cells proliferate more rapidly than the haplotype-matched wild-typecounterpart and are resistant to prostaglandin E2（PGE2）-mediated suppression. Moststrikingly, mice that received adoptive transfer of HPK1-/-T cells became resistant to lungtumor growth. Also, the loss of HPK1from dendritic cells （DCs） endows them withsuperior antigen presentation ability, enabling HPK1-/-DCs to elicit a more potent anti-tumorimmune response when used as cancer vaccine. It is probable that blocking the HPK1kinaseactivity with a small molecule inhibitor may activate the superior anti-tumor activity of bothcell types, resulting in a synergistic amplification of anti-tumor potential. Given that HPK1isnot expressed in any major organs, it is less likely that an inhibitor of HPK1kinase activitywould cause any serious side effects.Major research results:At the beginning, HPK1plasmid chosen from CHO Cell line is electric transfect, whichcan be divided into wild type （WT） and Mutant type （Mutation）. Then compare this twotypes of gene plasmid in CHO cell protein expression by using Western blot andimmunohistochemistry. The result is that CHO cells after electrotransfection, regardless of itstype, has higher protein expression than CHO cells before electrotransfection. In addition, theprotein expression of both types are about the same. After that, HPK1plasmid is electrictransfect again. Culture the cells overnight for24hours. Induce CHO cells afterelectrotransfection and cells without electrotransfection by using tetracycline. Then compare this two types of gene plasmid in CHO cell protein expression by using Western blot andimmunohistochemistry. The result is that induced CHO cells after HPK1electrotransfectionhave higher protein expression than the ones which are not induced, both wild type andMutant type. In addition, induced CHO cells after HPK1electrotransfection and itscounterpart have almost the same protein expression.To establish stable transfect HPK1CHO cell line, we use Lipofectamine2000liquidtransfect method. Then with the help of flat plate clone technology and Zeocin selectiveculture medium, we select monoclonal transfect HPK1CHO cells. Culture the cells for aboutone month. When the cells develop steadily, we select healthy HPK1CHO cells with largercell clusters under the microscope lens. Transfer the cells to24-well Plate then continueculturing them in selective medium. When we culture enough cells, we use exactly onemillion cells for each monoclonal cell. After inducing them with tetracycline, cultivate thecells overnight for24hours. Compare all the monoclonal transfect HPK1cells proteinexpression with Western blot. Select cells with higher HPK1protein expression for furtherresearch.After identifying wild type and Mutant type HPK1protein expression, we need topurify the HPK1protein that we have extracted by using Glutathione Sepharose4B GSTBeads. Cleanse Glutathione Sepharose4B GST Beads preserved in alcohol for three timeswith1×PBS. Then blend them with protein extracted from cell lysis for about one to twohours. Centrifuge the Glutathione Sepharose4B GST Beads in low speed and then collectsupernatant. Try not to suck away Glutathione Sepharose4B GST Beads. After that, CleanseGlutathione Sepharose4B GST Beads again for three times with1×PBS. At last, elute proteinfrom Glutathione Sepharose4B GST Beads with elute buffer. Elute them for three times,15minutes every time. Suspend the Glutathione Sepharose4B GST Beads at fixed period duringthe elution so that it will blend with eluent better. Centrifuge in low speed and collect thesupernatant eluent. Test the purified HPK1protein with Western blot and compare the proteinexpression of the wild type, Mutant type, before purification and after purification samples.The result shows that the protein after purification has no impurity. HPK1with molecular much difference in the aspect of protein expression. The result of purified protein is ideal.The research before has identified protein expression of HPK1, which is high proteinexpression for induced ones. Our next goal is to identify whether the protein expressionstability changes as we further prolong the period of culture. We melt the transfect HPK1CHO cell which is before under cryopreservation. After cell lysis we extract the protein.Compare the protein expression with Western blot before and after the test. The result showsthat, after half a year of continuous culture, HPK1protein expression and the HPK1CHOcells before under cryopreservation show no sign of attenuation. This proves that protein ofthe HPK1CHO cells we transfect is stable.After identifying the protein expression and its stability, the next step is to detect theconcentration of the protein that we purified, with the method of BCA. We compare theprotein concentration expression the groups of wild type, Mutant type,. The result shows thatthe protein expression after purification is ideal. The concentration of wild type one andMutant one are almost the same. The protein expression of cryopreservation one andcontinuous cultured one are almost the same.After the research above, we draw the conclusion that transect HPK1CHO cells havestable protein expression. But further research is needed to identify whether the proteinextracted has activity, or whether there is activity difference between the wild type one andMutant type one. We test the activity expression of extracted protein by radioactivity32P-γ-ATP. The result shows that under the condition of same protein concentration, same amount ofprotein and the same doses, the protein activities of the groups are opposite. HPK1wild typehas high protein activity. Conversely, HPK1Mutant type group has little protein activity.To ensure the accuracy of the protein we have extracted and its activity, we sent thepurified protein to the University of Yale for amino acid sequencing. The result shows that nochanges occurred in amino acid sequencing and HPK1nucleotide sequence, with also no genemutation occurred. This proves the accuracy of our research.Conclusion:Through the research of this subject, we prove the expression of hematopoietic progenitor kinase1（HPK1） in CHO Cell Line. By using Lipofectamine2000liquidtransfect method and tetracycline inducement, we promote the CHO cells target gene proteinexpression. By examining the inducement time and dose difference, different culture time, weidentify that transfect HPK1in CHO Cell Line has stable and high protein expression. Thenwe identify one of the gene locus of HPK1, make it Mutant and the HPK1protein loses itsactivity. By32P-γ-ATP. test we can identify that the Mutant type has no activity. Bysequencing amino acid from which we extract protein, we prove that HPK1genes, both wildtype and Mutant type, do not change after a long period of culture.Our research reveals that changing one gene locus of HPK1can make it lose its activity.And if the protein expression has not changed, this means the Mutant HPK1has lost activityand thus no side effect for human tissues and organs. We transfer Hpk1to the cells withoutHpk1to create the stable cancer cell line, on this research level, we can seek to HPK1inhibitor. In the future resreach, we may inhibit HPK1activity without killing immune T cells,and promote immune system to inhibit tumor via PGE2. This may provide new treatmenttarget for the treatment of intracranial tumor and man-made damage caused by surgery. |
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