Construction Of Attenuated Plasmodium Sporozoite Expressing MAGE-A3 And Its Effect And Mechanism Of Against Lung Cancer

Lung cancer is the most common malignancy in the world,and its incidence is increasing year by year.Despite the continuous development of surgery,chemotherapy,radiotherapy,molecular targeted drug therapy,but the efficacy of lung cancer is not ideal,the prognosis is poor.In recent years the rise of immune therapy is mainly through the strengthening of the body's immune response to anti-tumor,with sensitivity,specificity characteristics,is considered the most promising lung cancer treatment strategy.Tumor vaccines can not only prevent the occurrence of cancer,but also can be used for cancer treatment,is one of the important immunotherapy strategy.A large number of studies have confirmed that CD4 / CD8 + T cell immune response is the body's immune system monitoring and inhibition of tumor the most important mechanism of action,it is an important basis for the design of effective tumor vaccine.However,there is no ideal tumor vaccine.The reason may be related to the immune tolerance of the body to inhibit tumor cells to induce a strong CD4 / CD8 + T cell immune response.In view of the malaria parasite(GAS)can overcome the immune tolerance and successfully induce the specific activity of Plasmodium falciparum CD8 + / CD4 + T cells,the project uses the genetic attenuated sporozoites as the carrier to construct the major lung cancer related antigen MAGE-A3,and to investigate whether it can activate MAGE-A3 specific CD4 / CD8 + T cells in order to achieve the effect of anti-lung cancer,and to further explore the mechanism,which can be designed for effective lung cancer and other tumors Vaccine offers new ideas.1.The use of CRISPR/Cas9 technology to construct stable expression of MAGE-A3 Plasmodium attenuated sporozoitesIn order to investigate whether the recombinant genetically attenuated sporozoites expressing lung cancer-associated antigen MAGE-A3 has the effect of anti-lung cancer,this study was to replace the UIS3 protein expression sequence of Plasmodium berghei ANKA strain(Pb ANKA)by CRIPSR-cas9(GAS-MAGE-A3),which is capable of expressing hepatocellular carcinoma-associated antigen MAGE-A3,which can successfully invade hepatocytes but can not continue to proliferate in vivo.The CRIPSR-cas9 recombinant plasmid(PYC plasmid)was designed and constructed for editing the genome of Plasmodium falciparum according to the experimental method reported by Professor Yuan Jing,Xiamen University.And then through the electric transfer,drug screening,etc.screening of positive monoclonal recombinant sporozoites.1.1 PYC / MAGE-A3 recombinant plasmid was successfully constructed by CRISPR/Cas9 techniqueThe sgRNA sequence was designed and synthesized according to the PYC plasmid and the UIS3 gene sequence of P.b ANKA strain,and then successfully ligated to the Bsm BI site of PYC plasmid by annealing.The 5'UTR and 3'UTR sequences of UIS3 gene were amplified from the genome of P.berghei ANKA strain.The lung cancer-associated antigen MAGE-A3 sequence was amplified from A549 human lung adenocarcinoma cell line.Three fragments were then amplified by overlapping PCR,And cloning were inserted into the multiple cloning sites of the PYC plasmid.The results of the final digestion and sequencing showed that all fragments were inserted into the correct position and no base mutation was found.1.2 Through the electric transfer,drug screening,etc.successfully obtained GAS / MAGE-A3 recombinant attenuated sporozoitesPlasmodium falciparum and PYC plasmids were mixed and electrokultured to the mice by tail vein injection.After 24 h,the drug was screened with 8 ug / mL pyrimethamine solution.The drug was continuously reappeared to protozoa.Monoclonal screening,according to each mouse inoculated with a malaria parasite inoculation,after the sixth day of inoculation began to protozoa blood disease,30 mice,a total of 5 protozoanemia,the emergence of protozoa blood monoclonal mice The extracted g DNA was identified by PCR and sequencing.The results showed that two of them were positive monoclonal malaria parasites.1.3 GAS / MAGE-A3 recombinant attenuated sporozoites successfully transcri bed and translated MAGE-A3 recombinant proteinIn order to verify whether the transgenic Plasmodium can transcribe and translate the lung cancer antigen MAGE-A3,we extracted the recombinant sporozoite sporozoite RNA and then amplified it by reverse transcription.The amplified fragment of the MAGE-A3 sequence was amplified by 950 bp The MAGE-A3 gene in GAS / MAGE-A3 sporozoites was successfully transcribed.HepG2 cells were then transfected into Hep G2 cells by immunofluorescence.The expression of MAGE-A3 protein in HepG2 cells was observed by indirect immunofluorescence.The results showed that the MAGE-A3 protein appeared in the vacuoles of the parasitoid sporozoites,while the wild type malaria parasites did not express the MAGE-A3 protein,indicating that the MAGE-A3 gene in GAS / MAGE-A3 sporozoites was successfully expressed.2.The expression of MAGE-A3 Plasmodium attenuated sporozoites anti-lung cancer immune effect and mechanism.In order to investigate whether recombinant recombinant adenocarcinoma expressing lung cancer-associated antigen MAGE-A3 can induce CD4 + / CD8 + T cell responses in mice to achieve anti-lung cancer effect.In this study,immunized HLA-A2 mice were injected with PBS,GAS,GAS / MAGE-A3,and the CD4 + / CD8 + T cells in peripheral blood and spleen were detected in 7 days after the last immunization.Elispot detected MAGE-A3-specific CD8 + T cells secreting IFN-?.Then,CD8 + T cells from the spleen of the immunized mice were collected and subcutaneously in nude mice bearing A549 subcutaneously to observe the effect of anti-lung cancer.2.1 GAS / MAGE-A3 recombinant genetically attenuated sporozoites successfully induced CD4 + / CD8 + T cell responses in mice.The frequencies of CD4 + / CD8 + T cells in peripheral blood and spleen were detected by FACS at 7 days after the last immunization.The results showed that compared with PBS group,GAS / MAGE-A3 group and GAS group could significantly induce peripheral blood Spleen CD4 + / CD8 + T cells respond,and CD8 + T cell activation is more pronounced.MAGE-A3-specific CD8 + T cells were detected by Elispot detection.The results showed that GAS + MAGE-A3 group significantly activated MAGE-A3 specific CD8 + T cells,while GAS group and PBS group were not Find.2.2 GAS / MAGE-A3 Recombinant sporozoite-induced CD8 + T cells have a significant effect on lung cancer.In order to investigate whether the CD8 + T cells induced by recombination of GAS / MAGE-A3 recombinant sporozoites had anti-lung cancer,CD8 + T cells were isolated from the spleen of the mouse 7 days after the last immunization,and then the cells were subcutaneously inoculated with A549 Cells in nude mice,according to 5 * 106 / only,once every 1 week,a total of three injections to observe whether the inhibition of A549 subcutaneous tumor proliferation,to achieve the effect of anti-lung cancer.The results showed that GAS / MAGE-A3 group could effectively inhibit the proliferation of A549 subcutaneous tumor and prolong the survival time of mice,which indicated that MAGE-A3-specific CD8 + T cells played a major role in inhibiting A549 proliferation.2.3 GAS / MAGE-A3 recombinant genetically attenuated sporozoites by inhibiting tumor cell proliferation,promoting apoptosis and inhibiting angiogenesis induced antitumor effects.After tumor growth for 60 days,the tumor was removed and stained with parafor maldehyde.Paraffin-embedded sections were used to detect the proliferation,apoptosis and angiogenesis of Ki-67 antibody,Tunel apoptosis detection kit and CD31 antibody respectively.The results showed that GAS / MAGE-A3 recombinant biosynthesis significantly inhibited tumor proliferation and angiogenesis,and promoted the apoptosis of tumor cells.In summary,compared with the traditional gene editing technology,CRISPR/Cas9 technology in the malaria parasite gene editing showed high efficiency,simple and so on.GAS / MAGE-A3 recombinant genetically attenuated sporozoites can not only induce CD4 + / CD8 + T cell responses,but also activate MAGE-A3 specific CD8 + T cells.GAS / MAGE-A3-induced CD8 + T cells can effectively inhibit the proliferation of A549 cells and prolong the survival of mice,in which MAGE-A3-specific CD8 + T cells play a major role.