CN101838611B - Recombinant plasmodium for expressing exogenous gene and application thereof - Google Patents
Recombinant plasmodium for expressing exogenous gene and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant plasmodium for expressing an exogenous gene, wherein the exogenous gene in the recombinant plasmodium comprises an antigen gene, a therapeutic agent gene, an immune regulator gene or a peptide gene. In the invention, the recombinant plasmodium is also applied to biological therapy and vaccine design, in particular to the biological therapy of tumors and the design of HIV vaccine. The recombinant plasmodium of the invention can be used for expressing the exogenous gene comprising any molecules hoping to be transferred to animals and humans or cells thereof, such as an antigen, a therapeutic agent, an immune regulator, peptide and the like, provides a new method for introducing and expressing heterologous genes in the animals and the humans or the cells thereof and stimulating or exciting immune responses and is particularly applied to the aspects of the biological therapy of the tumors and the design of the HIV vaccine.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of recombinant plasmodium and application thereof of expression alien gene.
Background technology
Existing expression vector especially for the expression vector of Biotherapeutics and vaccine design, mostly is bacteria carrier or viral vector.These carriers are less because of himself genome, therefore capacity that can expression alien gene is also just less.As expression vector, particularly as the expression vector of vaccine, the advantage of a lot of self uniqueness is arranged: 1, induce strong innate immune reaction with plasmodium; 2, induce strong Th1 type immunoreation, stimulate the T lymphopoiesis, stimulate T lymphocytic emiocytosis IFN-γ, and can stimulate the maturation of dendritic cell, promote the submission of antigen, as a kind of immunological adjuvant; 3, the capacity of exogenous gene is large, plasmodium not only can be expressed large exogenous gene, and can hold simultaneously, expresses a plurality of exogenous genes, thereby can stimulate simultaneously generation for the immunoreation of plurality of antigens or express immune cofactor, inducing to produce efficient immunoprotection; 4, the exogenous gene expression level is high.
But before this, as the vector expression foreign protein, still rest on the phase of basic research of construction expression system with plasmodium.The plasmodium gene of reporting reporter genes such as adopting plasmodium expressing green fluorescent protein (GFP), luciferase (Luciferase) and chloramphenicol acetyltransferase (CAT) and xenogenesis is arranged, only limit to the basic research scope.
Tumor is the chronic disease of serious threat human health, and the treatment of tumor remains the important problem of present facing mankind.Present Therapeutic Method is mainly with chemotherapy, and radiotherapy and operative treatment are main, but Biotherapeutics is more and more noticeable, becomes gradually a kind of important Therapeutic Method, and is applied to oncotherapy.Biotherapeutics refers to a kind of method of body being treated by biological response modifier (BRM).The Main Function of biotherapy is the general immunity function that improves the patient, especially in oncotherapy, has now become one of important treatment means the most rising after surgery, radiation and chemotherapy.The main feature of Biotherapeutics is: 1, bioactive functions is many, has antitumor, antiviral and immunoregulatory activity.2, sphere of action is wide, almost all tumor cells is had depression effect at external these biological preparation.3, the immunologic function of body there is adjusting potentiation.
As noted earlier, be one of key character of Biotherapeutics to the immunoloregulation function of body, and Infected With Plasmodium just in time also have this feature.Once successfully be applied in history treat nerve syphilis as a kind of biotherapy (malarialization) with malaria itself, and invented the Austrian doctor WagnerJauregg of this therapy thereby obtain the nineteen twenty-seven Nobel Prize in medicine.The therapeutic vaccine of tumor is as the extensive use in laboratory of one of the method for knubble biological therapy, and promotes gradually in clinical.Existing bibliographical information Infected With Plasmodium can directly suppress the tumor growth of tumor-bearing mice.But show that on evidence enhancing body suppresses the effect of tumor to the more effective reinforcement of identification energy of tumour-specific or related antigen, the recombinant plasmodium of therefore imagining expressing tumor specificity or related antigen is more effective for oncotherapy.
Acquired immune deficiency syndrome (AIDS) is the serious infectious diseases that directly threaten human health.The infected more than 95% lives in developing country.Up to now, communication and education and only can play the effect that slows down spread speed to high-risk group's behavior intervention, and can't stop that it is popular.Can not reach the purpose of thorough removing body inner virus although highly active antiretroviral therapy is effective, thereby need to take medicine all the life, price is too expensive concerning most of the infecteds, and toxic and side effects is larger, drug resistance easily occurs.AIDS vaccine is identified as prevention and controls the most effective weapon of acquired immune deficiency syndrome (AIDS).But the development of AIDS vaccine faces serious challenge, because innumerable failed in clinical trial by the AIDS vaccine of traditional method development.In recent years, the research of live vector vaccine more and more comes into one's own.Live vector vaccine is in the expression vector that the gene of coding virus protein insert is lived, and by expression vector, exogenous gene is expressed in the host, stimulates thus body to produce stronger specific immune response.This may be one of most promising AIDS vaccine.
Summary of the invention
The invention provides a kind of recombinant plasmodium of expression alien gene, and this recombinant plasmodium is applied in Biotherapeutics and vaccine design, especially the design of the Biotherapeutics of tumor and HIV vaccine.
Concrete technical scheme of the present invention is as follows:
The recombinant plasmodium of a kind of expression alien gene of the present invention, its exogenous gene comprise antigen gene, therapeutic agent gene, immunomodulator gene or peptide gene.
Above-mentioned recombinant plasmodium is applied in Biotherapeutics.
Preferably, described Biotherapeutics comprises oncotherapy.
Preferably, above-mentioned oncotherapy is: 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 is in plasmodium, obtain the recombinant plasmodium of expressing tumor related antigen, last immune tumor organism.
Above-mentioned tumor associated antigen gene comprises MUC1.
Above-mentioned recombinant plasmodium is applied in vaccine design.
Preferably, described vaccine design comprises the HIV-1 vaccine design.
Preferably, described HIV-1 vaccine design is: 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 is in plasmodium, obtain expressing the recombinant plasmodium of the encoding gene of HIV-1, last immune body.
Preferably, the encoding gene of described HIV-1 comprises the gag gene.
The present invention has following beneficial effect:
A kind of recombinant plasmodium provided by the invention can be used for expression alien gene, comprising: the hope such as antigen, therapeutic agent, immunomodulator, peptide are delivered to any molecule of animal and human or its cell; For introducing and expression of heterologous genes, stimulating or excite immunne response that a kind of new method is provided, particularly be applied to the Biotherapeutics of tumor and the design aspect of HIV vaccine in animal and human or its cell.
The invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the structure chart of pL0015-gag recombiant plasmid;
Fig. 2-A is the figure as a result of a preferred embodiment of the blood smear experiment of the NK65-gag recombinant plasmodium strain that obtains of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain;
Fig. 2-B is the figure as a result of a preferred embodiment of the western-blot experiment of the NK65-gag recombinant plasmodium strain that obtains of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain;
Fig. 2-C is the figure as a result of the preferred embodiment of the NK65-gag recombinant plasmodium strain PCR that obtains of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain;
Fig. 3 is the experimental result picture of the preferred embodiment that after recombinant plasmodium strain NK65-gag immune mouse, ELISPOT detects;
Fig. 4 is the experimental result picture of the preferred embodiment that after recombinant plasmodium strain NK65-gag immune mouse, ELISA detects;
After Fig. 5 was recombinant plasmodium strain NK65-gag immune mouse, Brdu ELISA detected the experimental result picture for a value-added preferred embodiment of HIV-1gag peptide;
After Fig. 6 is Mus recombinant plasmodium strain NK65-gag immune mouse, with the poxvirus vaccinia-gag counteracting toxic substances of recombinant expressed HIV-1 gag, detect the experimental result picture of a preferred embodiment of protectiveness;
Fig. 7 is the structure chart of pL0015-MUC1 recombiant plasmid;
Fig. 8 is the figure as a result of a preferred embodiment of the western-blot experiment of the NK65-MUC1 recombinant plasmodium strain that obtains of pL0015-MUC1 Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain;
Fig. 9 is a preferred embodiment experimental result picture of the tumor growth curve of simple tumor group (T group) and NK65-MUC1+T group;
Figure 10 is the experimental result picture of T group and NK65-MUC1+T group preferred embodiment of secretion of the 27th day flow cytometer detection IFN-γ after inoculated tumour and plasmodium;
Figure 11 is the experimental result picture of T group and NK65-MUC1+T group preferred embodiment of secretion of the 27th day elispot detection granzymeB after inoculated tumour and plasmodium.
The specific embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.
Embodiment 1: the gag gene take the strain of Mus Bai Shi plasmodium NK65 worm as vector expression 1 type human immunodeficiency virus (HIV-1), and with the conceptual vaccine immune mouse of this restructuring worm strain as a kind of HIV, detect mice for the immunoreation of gag with dot enzyme-linked immuno experiment (ELISPOT), enzyme linked immunological experiment (ELISA) and rat bromine deoxynucleoside uracil enzyme linked immunological experiment methods such as (Brdu ELISA).Last again with the poxvirus vaccinia-gag counteracting toxic substances of expressing Gag albumen, observe the immune protective effect to mice.
The structure of pL0015-gag recombiant plasmid: the gag gene clone of HIV-1 to the expression plasmid pL0015 of Mus Bai Shi plasmodium NK65 worm strain, is obtained the pL0015-gag recombiant plasmid.Be specially and design two ends with the gag primer of BamH1 restriction enzyme site, amplify the gag gene with PCR method afterwards, with pL0015 plasmid and this gag product BamH1 enzyme action, then cutting glue reclaims, connect, be transformed into XL-1blue competence antibacterial coated plate, select transformant and identify through bacterium colony PCR, enzyme action identifies, the order-checking direction is identified the pL0015-gag plasmid that obtains recombinating after all correct.The pL0015-gag plasmid structure of restructuring is seen Fig. 1.
The strain of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm: with the conversion of conservation the escherichia coli XL-1blue bacterial strain recovery of pL0015-gag, copy rear extraction and purification in escherichia coli XL-1blue bacterial strain.The a large amount of cultivation extracted pL0015-gag, with the pL0015-gag plasmid transfection Mus Bai Shi plasmodium that extracts, begins pyrimethamine to mice oral 30mg/kg every day after 24 hours with this worm strain Mice Inoculated, continuous four days.Filter out the Mus recombinant plasmodium strain NK65-gag of recombinant expressed gag with pyrimethamine, see Fig. 2.Fig. 2-A is the blood smear figure as a result of the recombinant plasmodium strain NK65-gag that obtains of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain, and this figure shows that the strain of NK65-gag recombinant plasmodium is positive.Fig. 2-B is the western-blot experimental result picture of the recombinant plasmodium strain NK65-gag that obtains of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain, take the strain of NK65 worm as blank, with the positive contrast of pVAX-gag, as seen from the figure, the strain of NK65-gag recombinant plasmodium is the strain of gag positive expression.Fig. 2-C is the figure as a result of the NK65-gag recombinant plasmodium strain PCR that obtains of pL0015-gag Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain; Swimming lane 1-6 is respectively DNA molecular amount standard (DNAmaker), NK65 genomic DNA, NK65-gag genomic DNA, NK65-gag genomic DNA, NK65-gag genomic DNA and pVAX1-gag plasmid.
Gets afterwards tail vein 10 μ l and be dissolved in the 0.2ml normal saline, then intraperitoneal inoculation is in a new Mice Body, this mice again every day oral pyrimethamine 30mg/kg, continuous four days.Reach 5% when above until the protozoan infection rate, the eye socket blood sampling, conservation, and extract respectively protozoon DNA and albumen is identified.After Gag albumen being detected and in protozoon, expression being arranged, again the worm strain of conservation is recovered, and then monoclonal, the worm strain after monoclonal use the same method again identify the Gag protein expression is arranged after, with this can stably express Gag albumen restructuring worm strain called after pbGAGcon.With same method, empty plasmid pL0015 is transfected into the restructuring worm strain called after pbEMPTYcon that the strain of Mus Bai Shi plasmodium NK65 worm obtains.With pbGAGcon and pbEMPTYcon difference immune mouse.
After Mus recombinant plasmodium strain NK65-gag immune mouse, ELISPOT detects: recombinate worm strain immune mouse after 7 days with NK65-gag, killing Mus gets spleen and tells splenocyte, the gag peptide of external use HIV-1 stimulates, and the secretion for the specificity IFN-γ of the gag of HIV-1 detected, sees Fig. 3.
After Mus recombinant plasmodium strain NK65-gag immune mouse, ELISA detects: after 30 days, serum is isolated in the eye socket blood sampling, detects the titre of gag antibody in serum with the ELISA method, sees Fig. 4 with NK65-gag restructuring worm strain immune mouse.Fig. 4 shows, with the gag antibody titer in mice after the pbGAGcon immune mouse apparently higher than with after the pbEMPTYcon immune mouse and do not have the immunity mice in the gag antibody titer, illustrate that this recombinant plasmodium worm strain pbGAGcon can stimulate body to the immunoreation of gag preferably.
After Mus recombinant plasmodium strain NK65-gag immune mouse, Brdu ELISA detects the increment for the HIV-1gag peptide: recombinate worm strain immune mouse after 21 days with NK65-gag, killing Mus gets spleen and tells splenocyte, the gag peptide of external use HIV-1 stimulates, the specificity Proliferation of lymphocytes for the gag peptide stimulation of HIV-1 detected, see Fig. 5.In Fig. 5, absorbance represents splenocyte propagation situation, and * represents that the splenocyte that the gag peptide stimulates breeds obviously compared with the control, and difference has significance statistically.
After Mus recombinant plasmodium strain NK65-gag immune mouse; poxvirus vaccinia-gag counteracting toxic substances with recombinant expressed HIV-1gag; detect protectiveness: recombinate worm strain immune mouse after 30 days with NK65-gag; intraperitoneal inoculation vaccinia-gag poxvirus; after 4 days; put to death mice and get ovary; Ultrasonic Pulverization; grind ovary; detect the virus titer of vaccinia-gag in mouse ovarian; detect after Mus recombinant plasmodium strain NK65-gag immunity, mouse ovarian inner virus carrying capacity is low than matched group in immune group, sees Fig. 6.In Fig. 6, * represent the to recombinate mice of plasmodium immunity of gag is compared with the mice of empty carrier plasmodium immunity, and the virus titer after the counteracting toxic substances experiment of vaccinia-gag is had statistically the significance of difference.
Embodiment 2: be expression vector with the strain of Mus Bai Shi plasmodium NK65 worm, and be applied to the design of tumor vaccine.Tumor associated antigen muc1 gene clone to the expression plasmid pL0015 of Mus Bai Shi plasmodium NK65 worm strain, is obtained the pL0015-muc1 recombiant plasmid, and the pL0015-MUC1 plasmid structure of restructuring is seen Fig. 7.Through identify in the right direction after, copy rear extraction and purification in escherichia coli XL-1blue bacterial strain.The recombiant plasmid pL0015-muc1 that extracts is transfected in the strain of Mus Bai Shi plasmodium NK65 worm, begins after 24 hours to mice oral pyrimethamine 30mg/kg every day, continuous four days with this worm strain Mice Inoculated.Gets afterwards tail vein 10 μ l and be dissolved in the 0.2ml normal saline, then intraperitoneal inoculation is in a new Mice Body, this mice again every day oral pyrimethamine 30mg/kg, continuous four days.Reach 5% when above until the protozoan infection rate, the eye socket blood sampling, conservation, and extract respectively protozoon DNA and albumen is identified.With same method, empty plasmid pL0015 is transfected into the restructuring worm strain called after NK65-PL0015 that the strain of Mus Bai Shi plasmodium NK65 worm obtains.Fig. 8 is the western-blot experimental result picture of the recombinant plasmodium strain NK65-MUC1 that obtains of pL0015-MUC1 Transfected Recombinant Plasmid Mus Bai Shi plasmodium NK65 worm strain, take the strain of NK65 wild type worm as blank, with the negative contrast of NK65-PL0015 worm strain, as seen from the figure, the strain of NK65-MUC1 recombinant plasmodium is the strain of MUC1 positive expression.After MUC1 albumen being detected and in protozoon, expression being arranged, again the worm strain of conservation is recovered, and then monoclonal, worm strain after monoclonal uses the same method and identifies that immune mouse after the MUC1 protein expression is arranged, and observes tumor growth inhibition and the tumor-bearing mice life span of tumor-bearing mice that inoculation is had the tumor of transfection muc1 gene.
The Lewis lung cancer cells system (LLC-MUC1) of Mus recombinant plasmodium strain NK65-MUC1 and expression MUC1 when tumor is grown to visible size, surveys tumor size in the 0th day Mice Inoculated every other day.The results are shown in Figure 9; Can find out that from the tumor growth curve of two groups a group of NK65-MUC1 infection is arranged, slow down than tumor growth is obvious with simple tumor group T group, after the 26th day, tumor size difference all has obvious statistical significance.
Killing Mus after inoculated malaria protozoon and tumor on the 27th day gets spleen and tells splenocyte, external use respectively OVA peptide (non-specific) and MUC1 peptide (special) to stimulate after, the secretion (Figure 10) of flow cytometer detection IFN-γ, elispot detects the secretion (Figure 11) of granzymeB.
Result from Figure 10 IFN-γ secretion: the secretion of CD8IFN-γ has a group slightly higher than simple tumor group of NK65-MUC1 infection under the stimulation of specific peptide MUC1, and a group of having NK65-MUC1 to infect, specific peptide MUC1 stimulates the secretion level of lower CD8IFN-γ obviously to stimulate the secretion level of lower CD8IFN-γ high than non-specific peptide OVA, showed significantly the enhancing for the specific reaction of MUC1, and simple tumor group shows in this not obviously.
Result from Figure 11 granzymeB secretion: have the secretion level of one group of granzymeB that NK65-MUC1 infects higher than simple tumor group on the whole, and this species diversity has significant statistical significance.Infer that plasmodial existence has activated the immunoreation in Mice Body on the whole, thus the secretion that has improved on the whole granzymeB.
Should be noted that at last; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment, the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution 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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| CN106687592B (en) * | 2016-12-26 | 2020-03-24 | 广州中科蓝华生物科技有限公司 | Recombinant plasmid, recombinant plasmodium constructed by recombinant plasmid and application of recombinant plasmid |
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