Preliminary Study Of Transgenic Attenuated Malaria Parasite Or Toxoplasma Gondii Stably Expressing Exogenous Proteins To Treat Human Diseases

Many diseases are caused by the lack or mutation of expected proteins,so the diseases could be treated by supplying with these target proteins or peptides.For example,the administration of insulin is commonly used to treat diabetes.However,only several proteins and polypeptides candidates are successfully applied in clinic.There are several reasons why most proteins or peptides couldn’t be moved into clinical stage.(1)Proteins or peptides are unstable,and strict conditions are needed to avoid to be deactivated.(2)The half-life of proteins and polypeptides is very short in the body(about a few minutes).Although the modification of amino acid sequence or supplement of sustained releaser could prolong the half-life,it may also reduce the activity of protein and peptides.(3)Most of the protein and peptides needed to be repeatly administrated through intravenous or subcutaneous injection to prolong the half-life,but it could also lead to many side effects.Therefore,it is urgent to find a novel way to safely deliver the protein and polypeptide in natural confirmation.Parasites have coevolved with humans.In order to adapt to the environment in the human body,they have developed a variety of immune evasion and inhibition mechanisms to survive in the host.Therefore,some parasites could persist in human body for a long time.In addition,with the rapid development of genetically modified technology,some protozoa could be attenuated by knocking out some of the key genes.The infection of attenuated protozoa is safe because its growth could be well controlled.Furthermore,the parasite could secrete a variety of proteins and polypeptides into the blood of host.Furthermore,comparerelative to the prokaryotic bacteria,protozoa was a eukaryote and can correctly express exogenous proteins and polypeptides in natural confirmation.Therefore,protozoa could persistly express expected proteins or peptides if target genes could be genetically inserted into protozoa genome.Thus,genetically attenuated protozoa with target genes might be a promising strategy to safely treat human diseses.This study aims to test this concept of principle by the following two experiments.(1)Investigate whether infection of plasmodium yoelii 17 XNL genetically stably expressing leptin gene could significantly reduce the body weight of mice or not.(2)Attenuated Toxoplasma gondii stably expressing melanoma antigen gp100 peptide was constructed,and we would like to investigate whether it can resist melanoma through induction of robust gp100-specific CD8+ T cellular immune responsein the future.A: The body weight of mice was significantly reduced after infection of plasmodium yoelii expressing leptin.1.Leptin was successfully fused to MIF of P.yoelii 17XNL: Firstly,a recombinant plasmid p YC/leptin containing sg RNA specific for MIF()gene of P.yoelii 17 XNL and homologous arms of Leptin gene was successfully constructed.Then the recombinant plasmid was electroporated into P.y 17 XNL mature schizonts,and a P.yoelii 17 XNL clone inserted with leptin gene was successfully selected under pyrimidine pressure.2.The genetically modified P.yoelii 17 XNL could stably express Leptin: RT-PCR was firstly performed to observe whether the genectically modified P.y 17 XNL could successfully transcribe MIF-Leptin;Then,the expression of leptin in the genectically modified parasite was examined by using indirect immunofluorescence staining assay.Both RT-PCR and indirect immunofluorescence staining assay demonstrated that the genetically modified parasites could successfully express leptin.3.The body weight of mice was significantly lost after infection of the genetically modified parasite:After mice were infected with WT or genetically modified parasites,the parasitemia and body weight of mice were measured every days.As a result,the body weight of mice infected with genetically modified parasites was significantly reduced as compared to that of mice infected with WT parasiteB: The genetically attenuated toxoplasma express melanoma antigen gp100 was successfully constructed.A recombinant CRISPR Cas9 plasmid containing sg RNA specific for CPS II(carbamoyl phosphate synthetase II),homologous arms of CPS II and GRA2 promoter plus gp100 CD8+ T peptides between CPS II arms was successfully constructed.Then,the recombinant plasmid was electroporated into toxoplasma,and an attenuated parasite clone expressing gp100 peptides was successfully selected under pyrimidine pressure.Take together,we preliminarily demonstrated that genectically engineering protozoa could explored as a verctor to stably deliver exogenous protein to to treat human diseases.