Attenuation system and use thereof

Disclosed are an attenuation system and the use thereof for attenuating plasmodia, specifically the use of an EF1g gene for attenuating plasmodia. The attenuation system regulates the expression or degradation of the EF1g gene by using a regulatory system, thereby controlling the growth of plasmodia and achieving the attenuation of plasmodia.

TECHNICAL FIELD

The present disclosure relates to the field of genetic engineering and, in particular, to an attenuation system and use thereof, especially an attenuation system and use thereof for attenuatingPlasmodium.

BACKGROUND

Malaria, AIDS, and tuberculosis are three major infectious diseases in the world. Malaria is an infectious disease caused by the single-celled protozoanPlasmodiumand spread byAnophelesmosquitoes. ThePlasmodiumliving inside human bodies is divided into four species:P. falciparum, P. malariea, P. vivax, andP. ovale.95% of malaria deaths are caused byP. falciparuminfection and mainly distributed in sub-Saharan Africa. Currently, the main animal models for malaria researches are mouse malaria models and monkey malaria models.Plasmodiumfor mouse can be divided intoP. chaubdi, P. berghei., P. yoelii, andP. vinckei, etc.Plasmodiumfor monkey mainly includesP. knowlesiandP. cynomolgi.

Hosts of humanPlasmodiuminclude humans andAnophelesmosquitoes. HumanPlasmodiumwill reproduce asexually in humans and reproduce sexually inAnophelesmosquitoes. HumanPlasmodiumundergoes two stages: a liver stage and an erythrocytic stage in the human body. In the liver stage, thePlasmodiumundergoes schizogony to form merozoites. In the erythrocytic stage, the merozoites undergo schizogony, part of which form gametocytes which can sexually reproduce. MaturePlasmodiumsporozoites exist in the salivary glands ofAnophelesmosquitoes. When theAnophelesmosquito bites the human body, the sporozoites are injected into the human blood. Through blood circulation, the sporozoites invade hepatocytes and undergo schizogony in the hepatocytes within a few minutes. After the sporozoites develop for ten to twelve days and become mature, the hepatocytes are ruptured by a schizont which releases merozoites into the blood. Some merozoites continue to infect hepatocytes, some merozoites invade erythrocytes and enter the erythrocytic stage, and most of the remaining merozoites are swallowed by phagocytes. The merozoites that invade the erythrocytes continue to develop and go through the stages of rings, macrotrophozoites, immature schizonts, and mature schizonts. The mature schizonts overflow out of erythrocytes. In this stage, the schizonts will not invade the liver, and some schizonts can continue infecting erythrocytes. Some schizonts no longer divide after several times of schizogony and continue to develop into female or male gametocytes. A large number ofPlasmodiumgametocytes exist in the body of a malaria patient. When theAnophelesmosquito bites the malaria patient, the mature male and female gametocytes enter the mosquito's stomach and begin the sexual reproduction. The male and female gametocytes further develop into male and female gametes. The male and female gametes fuse into a zygote which further develops into an ookinete. The ookinete invades the stomach wall of theAnophelesmosquito to form an oocyst which undergoes asexual reproduction in the spore proliferation stage. The spores proliferate to form a large number of sporozoites free from the oocyst, and the sporozoites migrate to the salivary glands of theAnophelesmosquito and enter the next life cycle.

According to the latest estimates released by the World Health Organization in December 2016, there were 212 million malaria cases and 429,000 deaths in 2015. It was found from the statistics in 2015 that about half of the world's population was threatened by malaria which mainly occurred in the Sahara and South Africa and threatened people in Southeast Asia, Latin America, and the Middle East to different degrees. The malaria statistics in 2015 showed that malaria still continuously occurred in 91 countries and regions in the world, and malaria cases in sub-Saharan Africa accounted for 90% of global malaria cases and 92% of the total malaria deaths. Moreover, in these regions where malaria is highly spread, children under the age of 5 are very susceptible to malaria and get sick, and serious ones are even dead. More than 70% of malaria deaths happen to children under the age of 5, and one child dies of malaria every two minutes. Therefore, malaria is the first killer for the children under the age of 5. In addition to the children under the age of 5, infants, pregnant women, and AIDS patients with low immunity are all at a high risk of malaria.

Therefore, a highly effective malaria vaccine is of great significance for protecting humans and eliminating malaria. However, little progress has been made in vaccine research and development due to the complicated life cycle ofPlasmodium, the variable components of antigens, and the imperfect experimental model of vaccine researches, etc. At present, malaria vaccines mainly include the following vaccines: 1. a pre-erythrocytic vaccine, such as RTS, which induces an antibody against circumsporozoite proteins and has relatively good clinical protection effects but low protectivity of only 25-50%, and other pre-erythrocytic subunit protein vaccines and DNA vaccines that have no obvious protective effect; 2. an erythrocytic vaccine that is developed using a merozoite surface antigen, an antigen involved in invading erythrocytes, and an infected erythrocyte surface antigen, among which erythrocytic subunit vaccines developed for MSP1 and AMA1 have no obvious protective effect; 3. a transmission-blocking vaccine that prevents the binding of gametocytes or the development of the zygote using gametocyte or zygote surface antigens so as to block the spread of malaria, while the current transmission-blocking vaccines induce antibodies at a low level and thus have no practical value; 4. a multi-stage multi-antigen vaccine that is developed using a composite antigen, such as SP66, which includes an erythrocytic antigen MSP1 peptide and an intermediate replication region of the circumsporozoite protein (CSP), where current clinical experiments show that the multi-stage multi-antigen vaccine exhibits no protective effect; 5. a whole-Plasmodiumvaccine which is a live attenuated malaria vaccine, including a radiation-attenuated vaccine, a genetically attenuated vaccine, and a drug-attenuated vaccine.

The radiation-attenuated vaccine is generated by irradiatingAnophelesmosquitoes infected withPlasmodiumto mutant DNA of sporozoites such that the sporozoites cannot enter erythrocytic stage, thereby achieving attenuation. The sporozoite vaccines obtained using radiation-attenuatedP. falciparumandP. vivaxhave a protective effect but low protectivity. In addition, radiation attenuation is not controllable and cannot guarantee safety, which limits the application of the radiation-attenuated vaccine.

The drug attenuated vaccine provides immunity by infecting a host with a wild-typePlasmodiumand killingPlasmodiumby administering the host with an antimalarial drug. Early experiments verified that the oral administration of chloroquine to a volunteer bit by anAnophelesmosquito infected withP. falciparumto control erythrocytic infection can induce complete protective effects. However, the volunteer who does not take the antimalarial drug on time after inoculated withP. falciparumwill suffer from parasitemia and adverse reactions, and may spread malaria after bit by theAnophelesmosquito, which has a greater risk and restricts the application of attenuated vaccines.

At present, the genetically attenuated vaccine is mainly to knock out necessary genes in the late liver stage or the pre-erythrocytic stage ofPlasmodiumso thatPlasmodiumcannot enter the erythrocytic stage. Compared with the drug attenuated vaccine, the genetically attenuated vaccine has no risk of spreading malaria and will not cause parasitemia. Moreover, as a whole-Plasmodiumliving vaccine, the genetically attenuated vaccine can induce obvious protective effects and is an excellent malaria vaccine. However, knocking out genes necessary for the development ofPlasmodiumor toxic genes may affect the growth ofPlasmodiumor the expression of surface antigens.

A ubiquitin-proteasome system (UPS) is a non-lysosomal protein degradation pathway in cells.

Ubiquitin is a small molecule globular protein which consists of 76 amino acid residues, is ubiquitous and highly conservative in eukaryotic cells, has a molecular weight of about 8.5 kDa, and can bind to receptor proteins in cells through covalent bonds. Cells can degrade proteins through the UPS pathway, so as to control an expression level of protein produced through constitutive regulation and environmental stimuli. A variety of physiological processes of cells, including cell apoptosis, cell proliferation and differentiation, quality control of endoplasmic reticulum proteins, protein translocation, inflammatory response, antigen presentation, DNA repair, and cellular stress responses are all related to the UPS. In addition, the UPS can degrade abnormal proteins, such as unfolded proteins, damaged proteins, mutated proteins, and incorrectly transcribed proteins. Therefore, the UPS plays an important role in maintaining normal cell functions.

A DHFR degradation domain (DDD) is a regulatory system which regulates a target protein by using the ubiquitin-proteasome system. The DDD regulates the target protein by fusingE. colidihydrofolate reductase (ecDHFR) with the target protein and controlling the addition of a stabilizer. The ecDHFR can be stabilized by trimethoprim (TMP), a DHFR inhibitor. With no TMP added, ecDHFR and the protein fused therewith are labelled with ubiquitin, and recognized and degraded by proteasomes. With TMP added, TMP binds to and stabilizes ecDHFR, so that the protein fused with ecDHFR remains stable and not degraded by ubiquitin, thus the target protein can be expressed normally. The binding of TMP to ecDHFR to stabilize the protein from being degraded is reversible. The addition of TMP can stabilize ecDHFR, and the withdrawal of TMP will cause the degradation of ecDHFR and the protein fused therewith. In addition, the expression level of the target protein can be controlled by controlling the amount of TMP, so it is very convenient to control the expression of the target protein and the expression amount of the target protein through TMP In addition, since the DDD regulatory system controls the expression of the target protein through ubiquitination and degradation, secreted proteins cannot be regulated.

Therefore, there is a need to develop a technology for conditionally regulating the expression of necessary genes ofPlasmodium. The necessary genes are expressed first to makePlasmodiumsurvive, and stopped to be expressed after the immune protection is obtained to achieve attenuation. The regulatory system is required not to express the necessary genes in the absence of a regulatory drug and to express the genes after a regulatory drug is added to makePlasmodiumsurvive, thereby avoiding the spread of malaria caused by the survivingPlasmodium.

SUMMARY

In view of the defects in the existing art and the actual requirements, the present disclosure provides an attenuation system and use thereof. A regulatory system is adopted to regulate the expression or degradation of an EF1g gene, so as to control the growth ofPlasmodiumand attenuatePlasmodium.

To achieve the object, the present disclosure adopts solutions described below.

In a first aspect, the present disclosure provides use of an EF1g gene for attenuatingPlasmodium.

In the present disclosure, the inventors have found that EF1g (PBANKA 1352000 elongation factor 1-gamma) is a necessary gene ofP. berghei, and thus a regulatory element is adopted to regulate its expression, so as to control the survival ofPlasmodiumand attenuatePlasmodium.

According to the present disclosure, the EF1g gene has a name of “elongation factor 1-gamma”, and has a gene identification number of PBANKA 1352000 and a nucleotide sequence as shown in SEQ ID NO. 1, wherein the specific sequence is as follows:

In a second aspect, the present disclosure provides a recombinant vector, where the recombinant plasmid includes an EF1g gene.

The present disclosure constructs a vector for achieving knock-in. A Cas9 knock-in vector is constructed to knock a regulatory element together with a reporter gene in a specific gene in a genome of a host cell.

According to the present disclosure, the vector is any one or a combination of at least two of a plasmid vector, a phage vector, or a viral vector, preferably a plasmid vector.

According to the present disclosure, the recombinant vector further includes a regulatory element located upstream of the EF1g gene.

In the present disclosure, the regulatory element can be knocked in a genome ofPlasmodiumthrough the recombinant vector. The regulatory element is knocked in the upstream of the EF1g gene in the genome, so that the expression of the EF1g gene in the genome can be controlled, and the transcription of the gene or the corresponding protein expressed by the gene can be controlled by a regulatory system.

According to the present disclosure, the regulatory element is any one or a combination of at least two of a dihydrofolate reductase regulatory element (DDD), a tetracycline operon regulatory element, or an FKBP12 regulatory element, preferably a dihydrofolate reductase regulatory element.

In the present disclosure, the inventors have found that when a DDD assembly is used to regulate the necessary gene to control the expression of the necessary gene ofPlasmodiumat a protein expression level so as to control the survival ofPlasmodium, the DDD has a low background and a large regulatory expression range and is convenient for regulation compared with other regulatory elements.

According to the present disclosure, the dihydrofolate reductase regulatory element has a nucleotide sequence as shown in SEQ ID NO. 2, wherein the specific sequence is as follows:

According to the present disclosure, the recombinant vector further includes a reporter gene located between the regulatory element and the EF1g gene.

In the present disclosure, the reporter gene is inserted, so as to observe the regulatory effect of the regulatory element on the EF1g gene.

According to the present disclosure, the reporter gene is selected from, but not limited to, a reporter protein GFPm3, and other reporter genes are also usable and will not be detailed here. Those skilled in the art can select a suitable reporter gene as needed.

According to the present disclosure, the reporter protein GFPm3 has a nucleotide sequence as shown in SEQ ID NO. 3, wherein the specific sequence is as follows:

In a third aspect, the present disclosure provides an attenuation system which inserts a regulatory element upstream of an EF1g gene in a genome ofPlasmodiumthrough the recombinant vector described in the second aspect.

In a fourth aspect, the present disclosure provides a host cell, where a regulatory element is inserted upstream of an EF1g gene in a genome ofPlasmodiumthrough the recombinant vector described in the second aspect.

According to the present disclosure, the host cell isPlasmodium, preferably, any one or a combination of at least two ofP. berghei, P. falciparum, P. vivax, P. malariea, P. ovale, orP. knowlesi, more preferablyP. berghei.

In a fifth aspect, the present disclosure provides a vaccine including the attenuation system described in the third aspect and/or the host cell described in the fourth aspect.

In a sixth aspect, the present disclosure provides a method for attenuatingPlasmodium, including:

infecting an animal with the attenuation system described in the third aspect, the host cell described in the fourth aspect, or the vaccine described in the fifth aspect, and controlling the addition of trimethoprim (TMP) to achieve attenuation.

In the present disclosure, the used regulatory drug TMP can control the growth ofPlasmodium, and can be directly used in human bodies and penetrate the blood-brain barrier and the placental barrier.

In a seventh aspect, the present disclosure provides use of the attenuation system described in the third aspect, the host cell described in the fourth aspect, or the vaccine described in the fifth aspect for preparing a medicament alleviating side effects ofPlasmodiuminfection.

Compared with the existing art, the present disclosure has the following beneficial effects:

(1) the present disclosure has found the necessary EF1g gene ofPlasmodiumfor the first time and regulated the EF1g gene through a regulatory element to control the expression or degradation ofPlasmodiumEF1g protein, thereby controlling the growth ofPlasmodiumand attenuatingPlasmodium; and

(2) as a new and feasiblePlasmodiumattenuation strategy, the present disclosure adopts the DDD to regulate the EF1g gene accurately and controllably with a good regulatory effect, and the DDD regulatory system has a low background, is convenient for regulation, and controls the growth ofPlasmodiumin conjunction with TMP, and can be directly used in the human body to attenuatePlasmodiumafter the human body is infected withPlasmodium.

DETAILED DESCRIPTION

To further elaborate on the technical means adopted and the effects achieved in the present disclosure, the solutions of the present disclosure are further described below with reference to the drawings and embodiments, but the present disclosure is not limited to the scope of the examples.

Example 1 Construction of a Strain that Adopts DDD to Regulate the EF1g Gene in P.bANKA

In this example, a Cas9 knock-in vector pBC-DHFR-GFPm3-EF1g-Tar was constructed. The schematic diagram of the vector is shown inFIG.1. The vector had Amp resistance and contained a gene encoding Cas9 protein and a hDHFR gene conferringPlasmodiumpyrimethamine resistance which were expressed in tandem, where pbeef1aa was used as a promoter, the Cas9 gene and the hDHFR gene were linked by a 2A peptide, and 3′Pb dhfr/ts was used as a terminator. In addition, the vector further included a fusion expression cassette that included successive sequences of an EF1g homologous arm 1, a regulatory element DHFR, a reporter protein GFP, and an EF1g downstream homologous arm, with pbeef1aa as the promoter and 3′Pb dhfr/ts as the terminator. The vector further included a P.b U6 promoter to express sgRNA.

Homologous arms of the EF1g gene are shown in SEQ ID NO. 4 and SEQ ID NO. 5, and sgRNA primers are shown in SEQ ID NO. 6 and SEQ ID NO. 7. The specific sequences are shown in Table 1.

Plasmid was extracted and linearized, and Pb ANKA was transfected with the plasmid. After electroporation, mixed TMP/pyrimethamine was administrated. The manner for administering to mice infected withPlasmodiumafter electroporation is described below.

Pyrimethamine solution: Pyrimethamine powder was dissolved in DMSO and prepared as a mother liquor with a final concentration of 7 mg/mL (shook and mixed uniformly), and the mother liquor was stored at 4° C. The mother liquor was diluted by a factor of 100 with distilled water, and adjusted to a pH within a range of 3.5 to 5.0 to prepare a working solution which was replaced every seven days.

Administration of mixed TMP/pyrimethamine: 100 mg of TMP was dissolved in 2 mL of DMSO and then added with 1 mL of pyrimethamine mother liquor, and the volume was adjusted to be 100 ml with distilled water, the pH was adjusted to be within a range of 3.5 to 5.0, and the mixed solution was replaced every three days.

Balb/c (8w, female) mice were inoculated withP. bergheielectroporated with the plasmid, so that the strain P.bANKA/pBC-DHFR-GFPm3-EF1g-Tar (simply referred to as a DDD-EF1g strain) was successfully obtained. After electroporation, the strain was observed with a fluorescence microscope, and the results are shown inFIG.2. The strain underwent drug withdrawal experiments (administration of mixed TMP/pyrimethamine, followed by administration of only pyrimethamine once the infection rate exceeded 10%). The change of the infection rate of the strain was observed, and the results are shown inFIG.3. The transferred second-passage strain underwent TMP withdrawal experiments (administration of mixed TMP/pyrimethamine, followed by administration of only pyrimethamine oncePlasmodiumwas found through microscopic examination), and the results are shown inFIG.4.

It can be seen fromFIG.2that GFP initiated by the promoter of EF1g has obvious fluorescence whose positions are consistent with those ofPlasmodiumnuclei labelled with Hoechst, and it is determined that GFP was correctly expressed. It can be seen fromFIG.3that the infection rate decreased from 32.6% to 0.09% after 120 h of withdrawal, and became 0 after 144 h of withdrawal. It can be seen fromFIG.4that the infection rate increased slightly after 24 h of withdrawal due to residual TMP and decreased to 0 after TMP withdrawal.

It is found from the results of the two experiments thatPlasmodiumcan survive only when TMP is administered, which proves that the DDD regulatory system can control the expression of the necessary gene ofPlasmodiumby administering TMP or not to control the survival ofPlasmodiumand adjust the toxicity ofPlasmodium.

Example 2 Verification of the Effect of DDD in Regulating EF1g Gene in P.bANKA

Two Balb/c (8w, female) mice were inoculated with the P.bNAKA/pBC-DHFR-GFPm3-EF1g-Tar strain, and one mouse was inoculated withP. bergheiwhose non-necessary gene NT1 was knocked out by using a CRISPR-Cas9 system as a control group. Mixed TMP/pyrimethamine was initially administrated, and TMP withdrawal experiments were carried out after thePlasmodiuminfection rate exceeded 1%. After TMP withdrawal, blood was collected from mice and smears were prepared to calculate the infection rate. The results are shown inFIG.5.

It can be seen fromFIG.5that the mouse in the control group NT1 died 7 days after TMP withdrawal, while the infection rates of all mice in DDD-EF1g group decreased to 0; after TMP withdrawal (administration of mixed TMP/pyrimethamine at first, followed by administration of pyrimethamine after TMP withdrawal), the infection rate of the mouse in the control group NT1 continued increasing and the mouse died 5 days later, while the infection rates of the two mice in DDD-EF1g group decreased to 0.5 days after TMP withdrawal, and the mice survived.

It can be seen that after TMP withdrawal,Plasmodiumin the mice infected with the DDD-EF1g strain died, indicating that the DDD can regulate the expression of the EF1g gene, control the survival ofPlasmodium, and attenuatePlasmodium.

Example 3 Effect of DDD in Regulating EF1g Gene in P.bANKA

In this example, a Balb/c (8w, female) mouse was inoculated with a DDD-GFP strain constructed by our company (the DDD regulates GFP expression and does not regulate any necessary gene) to verify whether TMP remains after TMP withdrawal. In addition, 6 Balb/c (8w, female) mice were inoculated with the DDD-EF1g strain. The administration method for mice is shown in Table 2.

TABLE 2Administration of mice in groupsStrainDDD-GFPDDD-EF1gGroup No.G1G2G3AdministrationAdministration of TMPContinuousAdministration of TMPfollowed by withdrawaladministration offollowed by withdrawalTMPNumber of mice122PurposeDetermine whether TMPDetermine anDetermine whetherremains after withdrawaleffect of TMPEF1g is necessaryAdministrationDescription (administration by drinking water with a pH of 3.5 to 5)Administration of1 mg of TMP and 0.07 mg of pyrimethamine/TMP followed bymL of water→infection rate reaching 1%→0.07 mgwithdrawalof pyrimethamine/mL of waterContinuous1 mg of TMP and 0.07 mg of pyrimethamine/mL of wateradministration ofTMP

After thePlasmodiuminfection rate exceeded 1%, TMP was withdrawn for groups G1 and G3. Before TMP withdrawal, GFP fluorescence of the DDD-EF1g strain was observed, and the results are shown inFIG.6. After the TMP withdrawal, the fluorescence of G1 was observed, and the results are shown inFIG.7. The results of the infection rate and the survival rate of the mice in all groups are shown inFIG.8(A) andFIG.8(B).

It can be seen fromFIG.6that the administration of TMP can stimulate the fluorescence of the strain in G1 before TMP withdrawal, which proves that TMP works. It can be seen fromFIG.7that the fluorescence of the DDD-GFP strain in G1 increased on the third day after withdrawal, and no GFP fluorescence was detected on the fifth day and the seventh day after withdrawal. It is considered that TMP remained in the mouse on the third day after withdrawal, and TMP was consumed on the fifth day after withdrawal.

It can be seen fromFIG.8(A) andFIG.8(B) that except those in G3, all mice in G1 and G2 died, the infection rate of one of the two mice in G3 decreased to 0, and the infection rate of the other mouse remained 50% to 60%, but no mice died; all mice in G2 died, which proved that the continuous administration of TMP cannot cause the death of the DDD-EF1g strain, and the withdrawal of TMP is a key factor affecting the death of the DDD-EF1 g strain; the death of the mouse in G1 proved that the strain where the DDD regulated a non-necessary gene would not die after withdrawal of TMP following TMP administration, and only the strain whose necessary gene was regulated by the DDD was affected by regulation of TMP administration.

To conclude, this example proves that the growth of the DDD-EF1g strain is affected by regulating TMP, and that the regulation of DDD in the expression of the EF1g gene ofPlasmodiumis an effective means to achieve the survival ofPlasmodiumand the attenuation ofPlasmodiumthrough external regulation.

Example 4 Verification of the Effect of a DDD-Regulated Attenuated Vaccine in PreventingPlasmodiumInfection

Balb/c (female, 8w) mice were inoculated with the P.bNAKA/pBC-DHFR-GFPm3-EF1g-Tar strain constructed in Example 1 (experimental group) and a wild-type P.bANKA strain (control group). The mice were administrated with TMP (1 mg of TMP/mL of water, administration by drinking water) for 3 days before inoculated withPlasmodium(8 mice in the experimental group and 6 mice in the control group), and then administered with TMP/pyrimethamine (1 mg of TMP and 0.07 mg of pyrimethamine/mL of water with a pH of 3.5 to 5, administration by drinking water). After thePlasmodiuminfection rate exceeded 1%, TMP was withdrawn (0.07 mg of pyrimethamine/mL of water with a pH of 3.5 to 5, administration by drinking water). ThePlasmodiuminfection rate of the experimental group decreased to 0. The mice were inoculated with 1×105P.bANKA one month later for challenge experiments. The mice in the experimental group and the control group were administered and inoculated according to the process inFIG.9. The change curves of the infection rates of the mice in the experimental group and the control group are shown inFIGS.10to12.

It can be seen fromFIGS.10to12thatPlasmodiumin all mice in the experimental group died and the infection rate decreased to 0 after TMP withdrawal. 8 mice in the experimental group and the mice in the control group were inoculated with 1×105P.bANKA, and thePlasmodiuminfection rates were calculated. After inoculation with 1×105P.bANKA, no growth ofPlasmodiumwas observed in the mice in the experimental group and all the mice survived (FIGS.10and12), while the mice in the control group all died from a high infection rate 22 days after inoculation (FIGS.11and12) and all showedPlasmodiumgrowth. It is proved that thePlasmodiumvaccine where the necessary gene is controlled using the DDD regulatory system has obvious preventive and protective effects, and can effectively prevent the vaccinated mice from being infected byPlasmodiumand is valuable for serving as aPlasmodiumvaccine.

To conclude, as a new and feasiblePlasmodiumattenuation strategy, the present disclosure adopts the DDD to regulate the EF1g gene accurately and controllably with a good regulatory effect, and the DDD regulatory system has a low background, is convenient for regulation, and controls the growth ofPlasmodiumin conjunction with TMP, and can be directly used in the human body to attenuatePlasmodiumafter the human body is infected withPlasmodium.

The applicant has stated that although the detailed method of the present disclosure is described through the examples described above, the present disclosure is not limited to the detailed method described above, which means that the implementation of the present disclosure does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present disclosure, equivalent replacements of various raw materials of the product, the addition of adjuvant ingredients, and the selection of specific manners, etc. in the present disclosure all fall within the protection scope and the scope of disclosure of the present disclosure.