CN101838611B - Recombinant plasmodium for expressing exogenous gene and application thereof - Google Patents

Abstract

The invention discloses a recombinant malaria parasite expressing exogenous gene, and the exogenous gene in the recombinant malaria parasite includes antigen gene, therapeutic agent gene, immune regulator gene or peptide gene. The present invention also applies the recombinant plasmodium to biological treatment and vaccine design, especially the biological treatment of tumors and the design of HIV vaccine. The recombinant Plasmodium of the present invention can be used to express foreign genes, including: antigens, therapeutic agents, immunomodulators, peptides, etc., any molecule that is expected to be delivered to animals and humans or their cells; for animals, humans or their cells Introducing and expressing heterologous genes, stimulating or eliciting immune responses provides a new method, especially in the biological treatment of tumors and the design of HIV vaccines.

Description

A recombinant malaria parasite expressing exogenous gene and its application

technical field

The invention relates to the field of biotechnology, in particular to a recombinant malaria parasite expressing foreign genes and its application.

Background technique

Existing expression vectors, especially those used for biological therapy and vaccine design, are mostly bacterial vectors or viral vectors. Because of their small genomes, these vectors have a small capacity to express foreign genes. Using Plasmodium as an expression vector, especially as a vaccine expression vector, has many unique advantages: 1. Induce a strong natural immune response; 2. Induce a strong Th1 type immune response, stimulate T lymphocyte proliferation, and stimulate T lymphocytes. The cells secrete IFN-γ, which can stimulate the maturation of dendritic cells, promote the presentation of antigens, and act as an immune adjuvant; 3. The capacity of foreign genes is large, and malaria parasites can not only express large foreign genes, but also It can accommodate and express multiple exogenous genes at the same time, thereby simultaneously stimulating the generation of immune responses against multiple antigens or expressing immune cofactors to induce efficient immune protection; 4. The expression level of exogenous genes is high.

However, the expression of foreign proteins using Plasmodium as a vector is still in the basic research stage of constructing an expression system. It has been reported that Plasmodium expresses reporter genes such as green fluorescent protein (GFP), luciferase (Luciferase) and chloramphenicol acetyltransferase (CAT) and heterogeneous Plasmodium genes, which are limited to the scope of basic research.

Tumor is a chronic disease that seriously threatens human health, and the treatment of tumor is still an important problem facing human beings. The current treatment methods are mainly chemotherapy, radiotherapy and surgery, but biological therapy is getting more and more attention, gradually becoming an important treatment method, and applied to tumor treatment. Biological therapy refers to a method of treating the body through biological response modifiers (BRM). The main function of biological therapy is to improve the systemic immune function of patients, especially in the treatment of tumors. It has become one of the most promising and important treatments after surgery, radiotherapy and chemotherapy. The main features of biological therapy are: 1. Multiple biological activities, with anti-tumor, anti-viral and immune-modulating activities. 2. Wide range of action, these biological agents have inhibitory effects on almost all tumor cells in vitro. 3. It can regulate and enhance the immune function of the body.

As mentioned earlier, the immune regulation function of the body is one of the important characteristics of biological therapy, and Plasmodium infection happens to have this characteristic. Using malaria itself as a biological therapy (malaria therapy) has been successfully applied to the treatment of neurosyphilis in history, and the Austrian doctor Wagner Jauregg who invented the therapy won the 1927 Nobel Prize in Medicine. Tumor therapeutic vaccines are widely used in the laboratory as one of the methods of tumor biotherapy, and are gradually promoted in the clinic. It has been reported that Plasmodium infection can directly inhibit tumor growth in tumor-bearing mice. However, evidence has shown that enhancing the body's recognition of tumor-specific or related antigens can more effectively strengthen the effect of tumor suppression, so it is more effective to imagine that recombinant malaria parasites expressing tumor-specific or related antigens are used for tumor treatment.

AIDS is a major infectious disease that directly threatens human health. More than 95% of those infected live in developing countries. So far, publicity and education and behavioral interventions for high-risk groups can only slow down the spread, but cannot stop the epidemic. Although highly effective antiretroviral therapy is effective, it cannot achieve the purpose of completely eliminating the virus in the body, so it needs to be taken for life. For most infected people, the price is too expensive, the side effects are relatively large, and drug resistance is prone to appear. AIDS vaccine is recognized as the most effective weapon to prevent and control AIDS. However, the development of AIDS vaccines is facing serious challenges, because AIDS vaccines developed by traditional methods have repeatedly failed in clinical trials. In recent years, more and more attention has been paid to the research of live vector vaccines. Live-vector vaccines insert genes encoding viral proteins into live expression vectors, and express foreign genes in the host through the expression vectors, thereby stimulating the body to produce a stronger specific immune response. This may be one of the most promising AIDS vaccines.

Contents of the invention

The invention provides a recombinant malaria parasite expressing exogenous gene, and applies the recombinant malaria parasite in biological treatment and vaccine design, especially tumor biological treatment and HIV vaccine design.

Concrete technical scheme of the present invention is as follows:

A recombinant malaria parasite expressing exogenous genes of the present invention, the exogenous genes include antigen genes, therapeutic agent genes, immune regulator genes or peptide genes.

The above-mentioned recombinant plasmodium is used in biological therapy.

Preferably, the biological therapy includes tumor therapy.

Preferably, the above tumor treatment is as follows: cloning tumor-associated antigen genes into expression plasmids to obtain recombinant plasmids, extracting and purifying them after replication in Escherichia coli, transfecting the extracted recombinant plasmids into Plasmodium, and obtaining tumor-associated Antigens of recombinant Plasmodium, and finally immune tumor organisms.

The aforementioned tumor-associated antigen genes include MUC1.

The recombinant Plasmodium described above is used in vaccine design.

Preferably, said vaccine design includes HIV-1 vaccine design.

Preferably, the HIV-1 vaccine is designed as follows: clone the HIV-1 coding gene into an expression plasmid to obtain a recombinant plasmid, extract and purify it after replication in Escherichia coli, and transfect the extracted recombinant plasmid into malaria In the parasite, the recombinant malaria parasite expressing the coding gene of HIV-1 is obtained, and finally the body is immunized.

Preferably, the HIV-1 coding gene includes gag gene.

The present invention has following beneficial effect:

A recombinant malaria parasite provided by the present invention can be used to express foreign genes, including: antigens, therapeutic agents, immunomodulators, peptides, etc., any molecules that are expected to be delivered to animals and humans or their cells; Introducing and expressing heterologous genes in cells, stimulating or eliciting immune responses provides a new method, especially in the biological treatment of tumors and the design of HIV vaccines.

The present invention will be further described below in conjunction with drawings and embodiments.

Description of drawings

Fig. 1 is the structural diagram of pL0015-gag recombinant plasmid;

Fig. 2-A is the result figure of a preferred embodiment of the blood smear experiment of the NK65-gag recombinant Plasmodium strain obtained by pL0015-gag recombinant plasmid transfection Plasmodium berghei NK65 strain;

Fig. 2-B is the result figure of a preferred embodiment of the western-blot experiment of the NK65-gag recombinant Plasmodium strain obtained by pL0015-gag recombinant plasmid transfection Plasmodium berghei NK65 strain;

Fig. 2-C is the result figure of a preferred embodiment of the NK65-gag recombinant Plasmodium strain PCR obtained by transfection of the pL0015-gag recombinant plasmid into the Plasmodium berghei NK65 strain;

Fig. 3 is the experimental result figure of a preferred embodiment of ELISPOT detection after recombinant malaria parasite strain NK65-gag immunization mouse;

Fig. 4 is the experimental result figure of a preferred embodiment of ELISA detection after recombinant malaria parasite strain NK65-gag immunization mouse;

Fig. 5 is a graph showing the experimental results of a preferred embodiment of Brdu ELISA detection of HIV-1 gag peptide proliferation after recombinant Plasmodium strain NK65-gag immunized mice;

Fig. 6 is after mouse recombinant Plasmodium strain NK65-gag immunizes mice, is challenged with the poxvirus vaccinia-gag expressing HIV-1 gag recombinantly, the experimental result diagram of a preferred embodiment of detection protection;

Figure 7 is a structural diagram of the pL0015-MUC1 recombinant plasmid;

Fig. 8 is a result figure of a preferred embodiment of the western-blot experiment of the NK65-MUC1 recombinant Plasmodium strain obtained by transfecting the Plasmodium berghei NK65 strain with the pL0015-MUC1 recombinant plasmid;

Fig. 9 is a graph showing the experimental results of a preferred embodiment of the tumor growth curves of the simple tumor group (T group) and the NK65-MUC1+T group;

Figure 10 is a graph showing the experimental results of a preferred embodiment of the flow cytometric detection of the secretion of IFN-γ on the 27th day after inoculation of tumors and malaria parasites in the T group and the NK65-MUC1+T group;

Fig. 11 is a graph showing the experimental results of a preferred example of elispot detection of the secretion of granzymeB in the T group and the NK65-MUC1+T group on the 27th day after inoculation of tumors and malaria parasites.

Detailed ways

In order to make the present invention easier to understand, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

Embodiment 1: take Plasmodium berghei NK65 worm strain as the carrier to express the gag gene of type 1 human immunodeficiency virus (HIV-1), and use this recombinant worm strain as a conceptual vaccine of HIV to immunize mice, use The immune response of mice against gag was detected by spot enzyme-linked immunosorbent assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA) and rat bromodeoxynucleoside uracil enzyme-linked immunosorbent assay (Brdu ELISA). Finally, the poxvirus vaccinia-gag expressing Gag protein was used to challenge the virus, and the immune protection effect on mice was observed.

Construction of the pL0015-gag recombinant plasmid: the gag gene of HIV-1 was cloned into the expression plasmid pL0015 of Plasmodium berghei NK65 strain to obtain the pL0015-gag recombinant plasmid. Specifically, we designed gag primers with BamH1 restriction sites at both ends, and then amplified the gag gene by PCR. The pL0015 plasmid and the gag product were digested with BamH1, then recovered by cutting the gel, ligated, and transformed into XL-1blue Competent bacteria were plated, and the transformants were selected to be identified by colony PCR, enzyme digestion, and sequencing direction identification to obtain the recombinant pL0015-gag plasmid. The structure of the recombinant pL0015-gag plasmid is shown in Figure 1.

The pL0015-gag recombinant plasmid was transfected into the Plasmodium berghei NK65 strain: the preserved Escherichia coli XL-1blue strain transformed with pL0015-gag was recovered, replicated in the Escherichia coli XL-1blue strain, extracted and purified. Mass culture, extract pL0015-gag, transfect mouse Plasmodium berghei with the extracted pL0015-gag plasmid, inoculate mice with this strain 24 hours later, start to give mice 30mg/kg of pyrimethamine orally every day for four consecutive days . The murine recombinant Plasmodium strain NK65-gag expressing gag was selected by pyrimethamine, as shown in FIG. 2 . Fig. 2-A is a blood smear result diagram of the recombinant Plasmodium strain NK65-gag obtained by transfection of the pL0015-gag recombinant plasmid into the murine Plasmodium berghei NK65 strain, which shows that the NK65-gag recombinant Plasmodium strain is positive. Figure 2-B is a western-blot experiment result diagram of the recombinant Plasmodium NK65-gag obtained by transfecting the mouse Plasmodium berghei NK65 strain with the pL0015-gag recombinant plasmid, with the NK65 strain as the blank control and pVAX-gag as the As a positive control, it can be seen from the figure that the NK65-gag recombinant Plasmodium strain is a gag-positive expression strain. Fig. 2-C is the result figure of NK65-gag recombinant Plasmodium strain PCR that pL0015-gag recombinant plasmid is transfected mouse Plasmodium berghei NK65 worm strain; , NK65-gag genomic DNA, NK65-gag genomic DNA, NK65-gag genomic DNA and pVAX1-gag plasmid.

Afterwards, 10 μl of tail vein blood was taken and dissolved in 0.2 ml of normal saline, and then intraperitoneally inoculated into a new mouse. The mouse was then orally administered pyrimethamine 30 mg/kg every day for four consecutive days. When the infection rate of the protozoa reached above 5%, blood was collected from the eye sockets to preserve the species, and the DNA and protein of the protozoa were extracted for identification. When the expression of Gag protein in the protozoan is detected, the preserved strain is revived, and then monoclonalized. After the monoclonal strain is identified with the expression of Gag protein by the same method, this can be stabilized. The recombinant strain expressing Gag protein was named pbGAGcon. The recombinant strain obtained by transfecting the empty plasmid pL0015 into Plasmodium berghei NK65 strain in the same way was named pbEMPTYcon. Mice were immunized with pbGAGcon and pbEMPTYcon respectively.

ELISPOT detection after immunizing mice with recombinant Plasmodium strain NK65-gag: 7 days after immunizing mice with NK65-gag recombinant strain, the mice were killed and the spleen was taken to separate splenic lymphocytes, stimulated with HIV-1 gag peptide in vitro, and detected Specific IFN-γ secretion against gag of HIV-1, see FIG. 3 .

ELISA detection after immunization of mice with recombinant Plasmodium strain NK65-gag: 30 days after mice were immunized with recombinant Plasmodium strain NK65-gag, blood was collected from the orbit to separate serum, and the titer of gag antibody in serum was detected by ELISA, as shown in Figure 4. Figure 4 shows that the gag antibody titer in mice after immunizing mice with pbGAGcon is significantly higher than the gag antibody titers in mice immunized with pbEMPTYcon and in mice without immunization, indicating that the recombinant Plasmodium strain pbGAGcon can compare Good to stimulate the body's immune response to gag.

After the mice were immunized with the recombinant Plasmodium strain NK65-gag, Brdu ELISA was used to detect the proliferation of HIV-1 gag peptide: 21 days after the mice were immunized with the NK65-gag recombinant strain, the mice were killed and the spleen was taken to separate the splenic lymphocytes, and the HIV-1 gag peptide was used in vitro -1 gag peptide stimulation, detect the proliferation of specific lymphocytes stimulated by the gag peptide against HIV-1, see Figure 5. The absorbance in Fig. 5 indicates the splenocyte proliferation, and * indicates that the gag peptide-stimulated splenocytes proliferate significantly compared with the control, and the difference is statistically significant.

After the mice were immunized with the recombinant Plasmodium strain NK65-gag, the mice were challenged with the recombinant poxvirus vaccinia-gag expressing HIV-1gag, and the protection was detected: 30 days after the mice were immunized with the recombinant NK65-gag strain, the mice were inoculated intraperitoneally with vaccinia-gag Pox virus, after 4 days, the mice were sacrificed to take the ovaries, ultrasonically crushed, and the ovaries were ground to detect the virus titer of vaccinia-gag in the mouse ovaries. After immunization with the mouse recombinant Plasmodium strain NK65-gag, the virus in the mouse ovaries The load in the immune group was lower than that in the control group, as shown in Figure 6. In Figure 6, * indicates that there is a statistically significant difference in the virus titer after the challenge experiment of vaccinia-gag between the recombinant gag Plasmodium immunized mice and the empty vector Plasmodium immunized mice.

Example 2: Using murine Plasmodium berghei NK65 strain as an expression vector and applying it to the design of tumor vaccines. The tumor-associated antigen muc1 gene was cloned into the expression plasmid pL0015 of the murine Plasmodium berghei NK65 strain to obtain the pL0015-muc1 recombinant plasmid. The structure of the recombinant pL0015-MUC1 plasmid is shown in FIG. 7 . After being identified in the correct direction, it was replicated in Escherichia coli XL-1blue strain and then extracted and purified. The extracted recombinant plasmid pL0015-muc1 was transfected into the murine Plasmodium berghei NK65 strain, and the mice were inoculated with pyrimethamine 30 mg/kg daily for four consecutive days after 24 hours of inoculation with the strain. Afterwards, 10 μl of tail vein blood was taken and dissolved in 0.2 ml of normal saline, and then intraperitoneally inoculated into a new mouse. The mouse was then orally administered pyrimethamine 30 mg/kg every day for four consecutive days. When the infection rate of the protozoa reached above 5%, blood was collected from the eye sockets to preserve the species, and the DNA and protein of the protozoa were extracted for identification. The recombinant strain obtained by transfecting the empty plasmid pL0015 into Plasmodium berghei NK65 strain in the same way was named NK65-PL0015. Figure 8 is a diagram of the western-blot experiment results of the recombinant Plasmodium strain NK65-MUC1 obtained by transfecting the mouse Plasmodium berghei NK65 strain with the pL0015-MUC1 recombinant plasmid. The NK65 wild-type strain was used as a blank control, and the NK65-PL0015 worm was used as a blank control. The strain is a negative control, and it can be seen from the figure that the NK65-MUC1 recombinant Plasmodium strain is a MUC1 positive expression strain. When the expression of MUC1 protein in the protozoan is detected, the preserved strain is revived, and then monocloned, and the monoclonal strain is identified by the same method as having MUC1 protein expression, and then immunized mice. Observation Tumor growth inhibitory effect and survival time of tumor-bearing mice inoculated with tumors transfected with muc1 gene.

Mouse recombinant Plasmodium strain NK65-MUC1 and mouse Lewis lung cancer cell line expressing MUC1 (LLC-MUC1) were inoculated into mice on day 0. When the tumor grew to a visible size, the tumor size was measured the next day. The results are shown in Figure 9; from the tumor growth curves of the two groups, it can be seen that the growth of the tumor in the group infected with NK65-MUC1 was significantly slower than that in the T group of the simple tumor group, and the difference in tumor size was statistically significant after the 26th day learning meaning.

On the 27th day after the inoculation of Plasmodium and tumor, the mice were killed and the spleen was taken to separate the splenocytes. After being stimulated with OVA peptide (non-specific) and MUC1 peptide (specific) in vitro, the secretion of IFN-γ was detected by flow cytometry (Figure 10). Elispot detected the secretion of granzymeB (Figure 11).

From the results of IFN-γ secretion in Figure 10: under the stimulation of the specific peptide MUC1, the secretion of CD8IFN-γ was slightly higher in the group infected with NK65-MUC1 than in the simple tumor group, and in the group infected with NK65-MUC1, the specific The secretion level of CD8IFN-γ stimulated by the sex peptide MUC1 was significantly higher than the secretion level of CD8IFN-γ stimulated by the non-specific peptide OVA, showing an obvious enhancement of the specific response to MUC1, while the simple tumor group showed no obvious.

From the results of the secretion of granzymeB in Figure 11: the secretion level of granzymeB in the group infected with NK65-MUC1 was higher than that in the tumor group alone, and the difference was statistically significant. It is speculated that the presence of Plasmodium generally activates the immune response in mice, thereby increasing the secretion of granzymeB as a whole.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that Modifications or equivalent replacements are made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (6)

1. the recombinant plasmodium of expression alien gene is as the application of carrier in preparation HIV-1 vaccine, and described exogenous gene is the encoding gene of HIV-1.

2. application according to claim 1, it is characterized in that, described application is specially: the encoding gene of HIV-1 is cloned on expression plasmid, obtain recombiant plasmid, extract and purification after copying in escherichia coli, the Transfected Recombinant Plasmid that extraction is obtained obtains expressing the recombinant plasmodium of the encoding gene of HIV-1, as the HIV-1 vaccine in plasmodium.

3. application according to claim 1, is characterized in that, the encoding gene of described HIV-1 is the gag gene.

4. the recombinant plasmodium of expression alien gene is as the application of carrier in the preparation anti-tumor medicine, and described exogenous gene is the tumor associated antigen gene.

5. application according to claim 4, it is characterized in that, described application is specially: with the tumor associated antigen gene clone to expression plasmid, obtain recombiant plasmid, extract and purification after copying in escherichia coli, the Transfected Recombinant Plasmid that extraction is obtained obtains the recombinant plasmodium of expressing tumor related antigen, as anti-tumor medicine in plasmodium.

6. application according to claim 4, is characterized in that, described tumor associated antigen gene is the MUC1 gene.

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