Experimental Studies Of Recombinant Calreticulin In Anti-tumor Immunotherapy

Objective To clone the ORF of mouse calreticulin (CRT) gene and acquire recombinant calreticulin in E. Coli. After painted with purified recombinant CRT, apoptotic tumor cells were inoculated into mouse as immunogenic cells and then the effect of anti-tumor immune response irritated by these immunogenic cells was analyzed. The main aim of this research is to explore a new way for anti-tumor immunotherapy.Methods The mouse CRT cDNA is cloned by RT-PCR (Reverse-transcription PCR). Mouse recombinant calreticulin (rCRT) is prepared by cloning CRT cDNA into pET15b vector and expressing in E. Coli. and then purified by Ni-NTA resin. MTT method is used to select the appropriate concentrations and acting times for apoptosis-inducing drugs. DNA fragmentation electrophoretic assay is used to detect cell apoptosis; real time PCR and Western blotting assay are used to detect the change of CRT expression level in B16-F1 cells after drug treatment. Immune cell fluorescence chemistry (ICC) is used to detect the sub-cellular distribution of CRT in B16-F1 cells. Three different types of apoptotic cells were used as immunogenic cells to inoculate Balb/c mouse, they are: BENS-treated B16-F1, mitoxantrone-treated B16-F1 and BENS-treated/CRT-painted B16-F1. And then the inhibition effects of immuno-inoculation on the growth of B16-F1 live cells inoculated latterly were observed. LDH release assay is used to detect the kill rate of mouse splenocytes against live B16-F1 cells and mouse H22 cells.Results 1) The cDNA that contains CRT ORF was cloned successfully from the KM Mouse. The rCRT were expressed in and purified from E. coli. 2) MTT assay showed that proliferation of B16-F1 cells was inhibited by BENS or mitoxantrone in a time- and concentration-dependent way. DNA fragmentation electrophoresis assay showed that both BENS and mitoxantrone had ability to induce apoptosis in B16-F1 cells. 3) According to the results of Real time PCR and Western blotting assay, the expression level of calreticulin gene was not changed in B16-F1 cells by BENS or mitoxantrone treatment. However, ICC showed that mitoxantrone treatment resulted in the translocation of CRT from cytoplasm to cell membrane. No obvious CRT translocation was observed in B16-F1 cells treated with BENS. 4) Animal experiment showed that, inoculating Balb/c mouse with cells that treated by mitoxantrone or BENS/CRT-painted B16-F1 cells resulted in growth inhibition of B16-F1 live cells inoculated latterly. 5) LDH release assay proved that the two groups of mouse described above not only have a lower tumor incidence, but also their splenocytes have a higher kill rate against B16-F1 live cells than that of control group. No difference of the kill rate was observed when mouse H22 cells were used in place of B16-F1 in this experiment.Conclusion Apoptotic B16-F1 cells painted with recombinant CRT can be used as antigen to initiate a specific anti-tumor immune response in Balb/C mouse. This research suggests the potential value of CRT in clinic cancer prevention and therapy.